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Metabolic Therapy for Age-Dependent ImpairedWound HealingShannon Lynn Kesl

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Scholar Commons CitationKesl, Shannon Lynn, "Metabolic Therapy for Age-Dependent Impaired Wound Healing" (2016). Graduate Theses and Dissertations.http://scholarcommons.usf.edu/etd/6104

Metabolic Therapy for Age-Dependent Impaired Wound Healing

by

Shannon Lynn Kesl

A dissertation submitted in partial fulfillment of the requirements for the degree of

Doctor of Philosophy Department of Molecular Physiology and Pharmacology

Morsani College of Medicine University of South Florida

Co-Major Professor: Dominic D’Agostino, Ph.D. Co-Major Professor: Mack Wu, M.D.

Lisa Gould, M.D., Ph.D. Thomas Taylor-Clark, Ph.D.

Paula Bickford, Ph.D. Kenneth Ugen, Ph.D.

Date of Approval: December 09, 2015

Keywords: Aging, Chronic Wounds, Ketosis, Exogenous Ketone Supplementation

Copyright © 2016, Shannon Lynn Kesl

ACKNOWLEDGEMENTS

The research included in this dissertation was made possible by support from our funding

sources, including the James A. Haley Veterans Hospital’s Merit Review, the Department of

Molecular Pharmacology and Physiology, Scivation Inc., and the Office of Naval Research

(ONR).

I would like to express my heartfelt gratitude to everyone who has supported me in my

pursuit of my doctoral degree. Firstly, I want to acknowledge the contribution from all of the

ratticans that have sacrificed their lives to further scientific research.

To my mentor, Dr. Dominic D’Agostino thank you for your relentless encouragement,

guidance, and always going above and beyond for me no matter what was going on around you.

You are one of the hardest working people I know. Thank you for always being just an email or

phone call away, and always working diligently through the night for the betterment of our lab,

my career, and my life. Thank you for instilling in me your passion and drive for helping others

through our research and for helping me to realize why our job as scientists is so important. To

my Co-mentor, Dr. Mack Wu, thank you for investing in me when I needed someone and for

your continuous direction and support. To Dr. Lisa Gould, thank you for believing in me as your

first Ph.D. student, for always being there even when we are far apart, for giving me an

opportunity to see the clinical aspect of our research, and especially for challenging me and

making me a better scientist.

To the members of my dissertation committee, Dr. Thomas Taylor-Clark, Dr. Paula

Bickford, Dr. Kenneth Ugen, Dr. Patrick Bradshaw for their scientific guidance and support over

the past five plus years. To my outside chair, Dr. Stanley Stevens Jr., thank you for stepping in

last minute and handling everything efficiently. I wish I had gotten to know you earlier in my

career, but am hopeful for a continued relationship in the future.

To my lab family, Carol Landon, Dr. Jay Dean, Geoffrey Ciarlone, Jacob Sherwood, Dr.

Christopher Rogers, Dr. Csilla Ari, Andrew Koutnik, and Nathan Ward, thank you for making

work such an entertaining but reassuring environment even when I needed to order something

last minute, we have had a midnight time point, or when I have forced you to listen to musicals. I

will be forever grateful for the time we have shared. To Dr. Andrea Trujillo for your continued

guidance and support, thank you for teaching me so much. I want to give a special recognition

and thank you to my wonderful friend and colleague, Dr. Angela Poff, from my whole heart I

wish to thank you for your unwavering love, support, and encouragement, none of this would

have been possible without you.

To my school family, Dr. Franklin Poff, Randi McCallian, Dr. Chase Lambert, and Dr.

Jillian Whelan, who have always been there with wine, excellent dinners, wine, relaxing

company, wine, ridiculous conversation, and wine to ease the stress of this journey. To Shelby

Evans and Marilyn Rodriguez, who always listened when I gushed about science even when they

didn’t understand and thought it was boring, thank you for being there whether near or far. To

Myshell (Michelle) Jung, thank you for being a source of constant inspiration and love even

when we didn’t get to see each other every day or get to braid hair during incubation times. To

my friends and family who have always loved and encouraged me throughout my life, Beth

Webster, Joshua Smith, Katie Rader, Chad Rader, Harper Rader, June Guy, Merle Guy, John

Fry, Michelle Adkins, Kyle Adkins, Travis Adkins, Amanda Fox, James Fox, Keith Waldron,

Jeanette Kesl, and Robert Kesl. To my Presley Rader for always being enthusiastic to learn about

science, watching your face light up when you looked into a microscope for the first time

restored my wonderment for science. To my hippo and chicken, thank you for your

unconditional love and for always being there to melt my stress away with snuggles on the

couch.

To my best friend, my heart, my husband, Jason, I know it has been hard being apart for

the majority of my dissertation work and prep, but know that I felt your unconditional love and

support even from Germany. Thank you for the countless times of reassurance, encouragement,

laughter, and inspiration. I look forward to many more years of our life together. Last but not

least to my mother, Jennifer Fry, thank you for always believing in me and pushing me to

achieve my dreams. None of this would have been possible without your continual words of

encouragement, your unconditional love, and your unwavering support no matter where life has

taken me or how long it has taken me to get there. I am forever grateful.

i

TABLE OF CONTENTS

List of Tables ................................................................................................................................. iv List of Figures ..................................................................................................................................v List of Abbreviations .................................................................................................................... vii Abstract ........................................................................................................................................ xiii Chapter 1: Wound Healing Physiology ...........................................................................................1

1.1. Chapter Synopsis .....................................................................................................1 1.2. Skin Morphology .....................................................................................................2 1.3. Wound Classification and Closure Types ................................................................2 1.4. Acute Wound Healing Cascade ...............................................................................4

1.4.1. Hemostasis ...................................................................................................5 1.4.2. Inflammation ................................................................................................5 1.4.3. Proliferation .................................................................................................7 1.4.4. Maturation ..................................................................................................10

1.5. Cellular Energy Metabolism ..................................................................................10 1.5.1. Catabolism .................................................................................................11 1.5.2. Anabolism ..................................................................................................13

1.6. Wound Healing Metabolism ..................................................................................14 1.7. Nutrition and Wound Healing ................................................................................15 1.8. Chronic Wounds ....................................................................................................17

1.8.1. Classification of Chronic Wounds .............................................................18 1.8.2. Current Standard of Care, Therapies and Their Disadvantages .................20

1.8.2.1. TIME and Amputation .................................................................21 1.8.2.2. Adjunctive Therapies ...................................................................22

1.9. Age Influence on Chronic Wounds ........................................................................26 1.9.1. Aged Related Changes in Skin Morphology .............................................26 1.9.2. Age Related Changes in the Wound Healing Cascade ..............................27

1.10. Closing Remarks ....................................................................................................30 1.11. References for Chapter 1 .......................................................................................31

Chapter 2: Oral Ketone Supplementation as a Novel Therapy for Age-Dependent Delay Wound

Healing .........................................................................................................................46 2.1. Chapter Synopsis ...................................................................................................46

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2.2. Ketone Body Synthesis and Metabolism ...............................................................47 2.3. Review of Ketogenic Diet and Therapeutic Uses ..................................................48

2.3.1. Potential Concerns and Side Effects ..........................................................49 2.4. Exogenous Ketone Supplementation .....................................................................50

2.4.1. 1,3-Butanediol ............................................................................................51 2.4.2. MCT Oil .....................................................................................................52 2.4.3. βHB Salt .....................................................................................................54 2.4.4. Combination BMS+MCT ..........................................................................55 2.4.5. Ketone Esters .............................................................................................56 2.4.6. Potential Concerns and Side Effects: Ketoacidosis ...................................57

2.5. Potential Physiology Affected to Enhance Wound Healing ..................................58 2.5.1. Ketones and Inflammation .........................................................................59 2.5.2. Ketones and ROS .......................................................................................60 2.5.3. Ketones and Blood Flow and Angiogenesis ..............................................62 2.5.4. Ketones and Metabolism ............................................................................63 2.5.5. Ketones Suppress Blood Glucose via Increasing Insulin Sensitivity ........64

2.6. Ischemic Wound Healing Model ...........................................................................66 2.7. Use of Primary Human Dermal Fibroblasts in vitro ..............................................69 2.8. Central Hypothesis and Project Goals ...................................................................70 2.9. Closing Remarks ....................................................................................................71 2.10. References for Chapter 2 .......................................................................................72

Chapter 3: Establishing Therapeutic Ketosis (Metabolic Therapy) with Exogenous Ketone

Supplementation ..........................................................................................................86 3.1. Chapter Synopsis ...................................................................................................86 3.2. Effects of Exogenous Ketone Supplementation on Blood Ketones, Glucose,

Triglycerides, and Lipoprotein Levels in Sprague-Dawley Rats ...........................87 3.2.1. Ketone supplementation causes little to no change in triglycerides and

lipoproteins ................................................................................................88 3.2.2. Therapeutic levels of hyperketonemia suppress blood glucose levels .......90 3.2.3. Effects of ketone supplementation on organ weight and body weight

percentage ..................................................................................................92 3.3. Effect of Sustaining Dietary Ketosis Through Exogenous Ketone

Supplementation on the Hippocampal and Serum Metabolome of Sprague-Dawley Rats .........................................................................................................103 3.3.1. Oral administration of ketone supplements elevates blood ketone levels,

increases Krebs cycle intermediates, MCFAs, antioxidants, and adenosine in serum and hippocampal tissue and implications for wound healing ...103

3.4. References for Chapter 3 .....................................................................................113

Chapter 4: Enhancing Wound Healing with Metabolic Therapy ................................................123 4.1. Chapter Synopsis .................................................................................................123 4.2. Dietary Ketone Supplementation Increases Blood Flow and Wound Closure in an

Ischemic Wound Model in Young and Aged Fischer Rats ..................................124

iii

4.2.1. Aged rats metabolize exogenous ketone supplements differently than young rats .................................................................................................125

4.2.2. Hypoglycemic effect of hyperketonemia attenuated with age .................127 4.2.3. Ketone supplementation increases blood flow in both young and aged

rats ............................................................................................................128 4.2.4. Ketone supplementation accelerates wound closure in young and aged

rats ............................................................................................................129 4.2.5. Food- integrated ketone supplementation did not elicit weight loss in

young and aged rats .................................................................................131 4.3. Closing Remarks ..................................................................................................133 4.4. References for Chapter 4 .....................................................................................139

Chapter 5: Potential Mechanisms for Exogenous Ketone Supplementation to Enhance Wound

Healing .......................................................................................................................144 5.1. Chapter Synopsis .................................................................................................144 5.2. Potential Mechanisms of Action for Exogenous Ketone Enhancement of Ischemic

Wound Healing in Young and Aged Fisher Rats .................................................145 5.2.1. Effects of ketone supplementation on inflammation ...............................146 5.2.2. Effects of ketone supplementation on ROS production ...........................150 5.2.3. Effects of ketone supplementation on angiogenesis ................................152 5.2.4. Effects of ketone supplementation on metabolism ..................................153

5.3. Closing Remarks ..................................................................................................154 5.4. References for Chapter 5 .....................................................................................165

Chapter 6: Discussion: Implications for Wound Healing ............................................................172 6.1. Chapter Synopsis .................................................................................................172 6.2. Implications for Wound Healing and Future Directions ......................................172 6.3. References for Chapter 6 .....................................................................................176

Appendix A: Methods and Materials ...........................................................................................179 Appendix B: Copyright Permissions ...........................................................................................198 Appendix C: Published Manuscripts ...........................................................................................202 About the Author ............................................................................................................... End Page

iv

LIST OF TABLES

Table 3.1 Global metabolism profile of serum and hippocampal tissue following chronic

administration of KE and BMS+MCT .................................................................109 Table A.1 Caloric density of standard rodent chow and dose of ketone supplements .........181

v

LIST OF FIGURES

Figure 3.1 Effects of ketone supplementation on triglycerides and lipoproteins ....................96 Figure 3.2 Effects of ketone supplementation on blood βHB .................................................... 97 Figure 3.3 Effects of ketone supplementation on blood glucose ................................................... 98 Figure 3.4 Relationship between blood ketone and glucose levels ............................................... 99 Figure 3.5 Effects of ketone supplementation on organ weight ..............................................100 Figure 3.6 Effects of ketone supplementation on body weight .................................................... 101 Figure 3.7 Effects of ketone supplementation on basal blood ketone and basal blood glucose

levels ....................................................................................................................102 Figure 3.8 Ketone supplementation elevates blood ketone levels in serum and hippocampal

tissues ...................................................................................................................110 Figure 3.9 Ketone supplementation increases medium chain fatty acids in serum and

hippocampal tissue ...............................................................................................110 Figure 3.10 Ketone supplementation increases Kreb’s cycle intermediates ...........................111 Figure 3.11 Ketone supplementation increases antioxidants ..................................................112 Figure 3.12 Ketone supplementation increase Adenosine ......................................................112 Figure 4.1 Effects of ketone supplementation on blood ketone and blood glucose levels ....134 Figure 4.2 Relationship between blood ketone and blood glucose levels attenuated with age ........................................................................................................................134 Figure 4.3 Ketone supplementation increases blood flow in both young and aged rats .......135 Figure 4.4 Ketone supplementation enhances wound closure time in young and aged rats .......................................................................................................................136

vi

Figure 4.5 Ketone supplementation elevates blood ketones levels but does not affect blood glucose levels .......................................................................................................137

Figure 4.6 Body and Spleen Weights ....................................................................................138 Figure 5.1 Heterogeneity of Aged Ischemic Wound Healing ...............................................155 Figure 5.2 Effects of ketone supplementation on pro-inflammatory and anti-inflammatory

cytokines ..............................................................................................................156 Figure 5.3 Effects of ketone supplementation on growth factors and leptin .........................157 Figure 5.4 Ketone supplementation does not affect M1 to M2 ratio of day 7 ischemic wounds

in aged rats ...........................................................................................................158 Figure 5.5 Ketone supplementation decreases ROS production ...........................................159 Figure 5.6 Ketone supplementation does not affect Antioxidants SOD2 and NQO1 in vitro

and ex vivo ............................................................................................................160 Figure 5.7 Ketone supplementation does not significantly increase blood vessel density in

day 7 aged ischemic wounds ................................................................................161 Figure 5.8 Ketone supplementation increases migration in young and aged HDFs .............162 Figure 5.9 Ketone supplementation increases proliferation in young and aged HDFs .........163 Figure 5.10 Ketone supplementation decreases lactate levels in young and aged HDFs .......164

vii

LIST OF ABBREVIATIONS

α-SMA ................................................................................................... Alpha-smooth muscle actin βHB ................................................................................................................ Beta-hydroxybutyrate AcAc ............................................................................................................................. Acetoacetate AD ..................................................................................................................... Alzheimer’s disease ADH .......................................................................................................... Aldehyde dehydrogenase ADP ............................................................................................................. Adenosine diphosphate ALS .................................................................................................... Amyotrophic lateral sclerosis AMP ....................................................................................................... Adenosine monophosphate ARE.......................................................................... Antioxidant or electrophilic response element ATP .............................................................................................................. Adenosine triphosphate BD ................................................................................................................... R, S-1, 3- Butanediol BMI ....................................................................................................................... Body Mass Index BMS ....................................................................................................................... Na+/K+ βHB Salt BMS+MCT ....................................................... Na+/K+ βHB Salt: Medium Chain Triglyceride Oil CAT...................................................................................................................................... Catalase CD31 ..................................................................................................... Cluster of differentiation 31 CD68 ..................................................................................................... Cluster of differentiation 68 CD206 ................................................................................................. Cluster of differentiation 206

viii

CMS .............................................................................. Centers for Medicare & Medicaid Services CNS ............................................................................................................ Central Nervous System CRP .................................................................................................................. Cysteine-rich protein CVD .............................................................................................................. Cardiovascular disease DHE ....................................................................................................................... Dihydroethidium DMEM .................................................................................... Dulbecco’s Modified Eagle Medium ECM .................................................................................................................. Extracellular matrix EGF .......................................................................................................... Endothelial growth factor EMT ........................................................................................................... Electromagnetic therapy eNOS ............................................................................................. Endothelial nitric oxide synthase ET-1 .............................................................................................................................. Endothelin-1 ETC ............................................................................................................. Electron transport chain FADH2 .................................................................................................. Flavin adenine dinucleotide FDA ........................................................................................... US Food and Drug Administration FGF ............................................................................................................. Fibroblast growth factor FOXO3A .............................................................................................................. Forkhead box O3a G6P .................................................................................................................. Glucose-6-phosphate G6PDH .................................................................................... Glucose-6-phosphate dehydrogenase GC/MS ............................................................................ Gas chromatography - mass spectrometry GI ............................................................................................................................. Gastrointestinal GLUT ................................................................................................................. Glucose transporter GNG ....................................................................................................................... Gluconeogenesis GSH .................................................................................................................. Reduced glutathione

ix

GSSG ............................................................................................................... Oxidized glutathione H2O2 .................................................................................................................... Hydrogen peroxide HBOT ..................................................................................................... Hyperbaric oxygen therapy HDAC ................................................................................................................ Histone deacetylase HDACI ................................................................................................ Histone deacetylase inhibitor HDF ............................................................................................. Primary human dermal fibroblast HDL ........................................................................................................... High density lipoprotein HKE ............................................................................................................ High Dose Ketone Ester I-CAM ............................................................................................. Intercellular adhesion molecule IFN-γ ..................................................................................................................... Interferon gamma IGF-1 ..................................................................................................... Insulin like growth factor-1 IL-1 ............................................................................................................................... Interleukin-1 IL-1β ..................................................................................................................... Interleukin-1 beta IL-4 ............................................................................................................................... Interleukin-4 IL-6 ............................................................................................................................... Interleukin-6 IL-8 ............................................................................................................................... Interleukin-8 IL-10 ........................................................................................................................... Interleukin-10 IL-13 ........................................................................................................................... Interleukin-13 IL-18 ........................................................................................................................... Interleukin-18 IRF-5 .................................................................................................. Interferon regulatory factor-5 KD .............................................................................................................................. Ketogenic diet KE ......................................................................... R, S-1, 3-butanediol diacetoacetate ketone ester KEAP-1 ................................................................................. Kelch-like ECH-associated protein -1

x

LBM ......................................................................................................................... Lean body mass LC-MS/MS .............................................. Liquid chromatography with tandem mass spectrometry LCFA .............................................................................................................. Long chain fatty acid LDH .............................................................................................................. Lactate dehydrogenase LDL ............................................................................................................. Low density lipoprotein LKE ................................................................................................................ Low dose ketone ester LPS ..................................................................................................................... Lipopolysaccharide M1 .................................................................................................... Pro-inflammatory macrophage M2 ................................................................................................... Anti-inflammatory macrophage MCFA ........................................................................................................ Medium chain fatty acid MCT ........................................................................................................ Medium chain triglyceride MCP-1 ..................................................................................... Monocyte chemoattractant protein-1 MDA ...................................................................................................................... Malondialdehyde Mg-ATP .................................................................................... Magnesium adenosine triphosphate MMP ........................................................................................................... Matrix metallopeptidase MnSOD ........................................................................................ Manganese superoxide dismutase MRC ................................................................................................ Mitochondrial respiratory chain MT2....................................................................................................................... Metallothionein-2 mtDNA .............................................................................................................. Mitochondrial DNA NAD+ .......................................................................... Oxidized nicotinamide adenine dinucleotide NADH ......................................................................... Reduced nicotinamide adenine dinucleotide NADPH .................................................................... Nicotinamide adenine dinucleotide phosphate NLRP3 ............................................................................... NLR family, pyrin domain containing 3

xi

NO .................................................................................................................................. Nitric oxide NOS ................................................................................................................. Nitric oxide synthase NPWT ....................................................................................... Negative Pressure Wound Therapy NQO1 .................................................................................. NAD(P)H dehydrogenase [Quinone]-1 Nrf2 ................................................................................ Nuclear factor (erythroid-derived 2)-like 2 O2

- ......................................................................................................................... Superoxide anion PAI-1 ............................................................................................ Plasminogen activator inhibitor-1 PCOS .................................................................................................... Poly cystic ovary syndrome PDGF ................................................................................................. Platelet-derived growth factor PDH ........................................................................................................... Pyruvate dehydrogenase PEM ...................................................................................................... Protein energy malnutrition PHMB ............................................................................................... Polyhexamethylene Biguanide PO2 .......................................................................................................... Partial pressure of oxygen PMN ................................................................................................. Polymorphonuclear leukocytes PPP ........................................................................................................ Pentose phosphate pathway QUICKI ........................................................................ Quantitative insulin-sensitivity check index RANTES ........................................ Regulated on activation, normal T cell expressed and secreted ROS ............................................................................................................ Reactive oxygen species S-BHB .............................................................................. Na+/Ca+2-β-hydroxybutyrate mineral salt S/MCT ........................ Na+/Ca+2-β-hydroxybutyrate mineral salt: Medium Chain Triglyceride Oil SAD ............................................................................................................. Standard American diet SD ................................................................................................................................ Standard diet SOD ............................................................................................................... Superoxide dismutase

xii

SS ............................................................................................................................. Skin substitutes T2D ............................................................................................................ Type 2 diabetes mellitus TBOOH .............................................................................................. Tert-butyl hydrogen peroxide TBI ................................................................................................................ Traumatic brain injury TGF-β ............................................................................................ Transforming growth factor-beta TIME ........................................................................... Tissue, infection, moisture, and wound edge TNF-α ................................................................................................... Tumor necrosis factor-alpha VCO ..................................................................................................................... Virgin coconut oil VEGF ......................................................................................... Vascular endothelial growth factor

xiii

ABSTRACT

Chronic wounds represent an under-acknowledged socioeconomic epidemic, affecting 1.8

million new patients per year and costing the US health care system upwards of $25 billion

annually. This substantial cost is rapidly growing due to a disproportionate occurrence in the

ever-aging population. Key features associated with age-related impairment of wound healing

include limited energy and nutrient exchange, unremitting inflammations, increased reactive

oxygen species (ROS), and diminished blood flow. Most chronic wound therapies target specific

molecular mechanisms; however, there are often multiple mitigating factors that prevent normal

wound closure. This is likely one reason most wound therapies are minimally effective. In the

standard American diet, carbohydrates are broken down for fuel (glucose). While fasting,

starvation, and calorie or carbohydrate restriction, beta-oxidation of stored fats in the liver

produces ketone bodies (primarily acetoacetate (AcAc) and β-hydroxybutyrate (βHB) to serve as

energy metabolites for extra-hepatic tissues. In addition to enhancing metabolic physiology,

ketone bodies have recently been discovered to have signaling properties that are independent of

their function as energy metabolites. Here we present the evidence for a novel method of

inducing therapeutic ketosis via exogenous ketone supplementation to promote enhanced

ischemic wound healing in young and aged Fischer 344 rats. Preliminary mechanistic studies

demonstrated that exogenous ketone supplementation enhanced wound healing via increasing

proliferation and migration, decreasing lactate production, and decreasing ROS production as

xiv

well as affecting inflammatory cytokines and growth factors. We conclude that exogenous

ketone supplementation will be an effective, cost efficient, low toxicity therapy to promote

enhancement of wound healing in an aged population.

1

CHAPTER 1: WOUND HEALING PHYSIOLOGY

1.1. Chapter Synopsis

The following is a brief review of skin morphology, wound classification and types of

wound healing, and the classic wound healing cascade. It is important to understand the

complexity of the orchestration in conjunction with the magnitude of participants and the vast

number of interactions it takes to heal a wound effectively. Even though most of the time wound

healing proceeds to completion without complication, the majority of this chapter addresses what

happens when things do not follow the standard progression. The current topical approach to

wound healing treatments has failed not only in reversing chronic wound stagnancies but has

cost the US alone billions of dollars in health care costs. There is a dire need to discover novel

and effective treatments for chronic wounds. The aged population has a high prevalence of

chronic wounds due to comorbidities in combination with physiological changes of aging.

Increasing evidence shows that unresolved inflammation, increased reactive oxygen species

(ROS), diminished blood flow, and decreased metabolism and ATP production are key

physiological features associated with age-related impairment of chronic wound healing. In this

chapter, we concentrate on the importance of these factors as well as how age affects them in a

wound-healing environment. Additionally, these are the four physiological features that we will

seek to target in our proposed treatment, which will be discussed in later chapters. A good

understanding of the information presented in this chapter will be critical to interpreting the

2

rationale behind our proposed therapies and the data represented in the subsequent chapters of

this dissertation.

1.2. Skin Morphology

The skin is the largest organ in the human body, comprising up to 12-15% of total body

weight and covering 1.5-2m2 of surface area [1]. There are three distinct layers of the skin: the

epidermis, the dermis, and the hypodermis. The epidermis is the avascular outermost layer,

primarily populated by keratinocytes, which regulates body temperature, absorbs nutrients, and

defends against pathogens. The dermis, the thicker layer deep to the epidermis, contains an

extracellular matrix (ECM) that provides tensile strength and flexibility. It also includes

fibroblasts, nerve endings, hair follicles, sweat glands, sebaceous glands, apocrine glands,

lymphatic vessels, and blood vessels. Though they are only present in the dermis, the blood

vessels supply nutrients to both the dermis and the epidermis [2-4]. The deepest layer, the

hypodermis, is mainly made up of adipose tissue and serves as an anchor, permitting most areas

of the skin to move freely over the deeper tissues [1]. These distinct layers each play a unique

role in wound healing and add to the heterogeneity of wound classification.

1.3. Wound Classification and Closure Types

Wound classification takes into account the nature and the severity of the wound to

determine an appropriate treatment plan. First, the wound is classified as acute or chronic. Acute

wounds achieve sustained restoration of structure and function in an orderly and timely

3

reparative manner. In contrast, a chronic wound does not proceed through the healing process in

a timely fashion and does not advance to closure [5]. Discussion of the classification of chronic

wounds and treatment protocols will occur later in this chapter. The rest of this section will focus

mostly on acute wound closure.

Initially, acute wounds are classified as being open or closed. Open or penetrating

wounds are those with exposed underlying tissue and/or organs. These include stab, gunshot, and

surgical wounds. Closed or non-penetrating wounds do not break through the skin. These include

abrasions, lacerations, contusions, and concussions. There are also some wounds that do not fit

into either of these categories; these include thermal (burns or frostbite) and chemical wounds

[6]. Each type of wound has an additional classification for severity. For example, open surgical

wounds are further classified into four different classes based on surgical location and level of

contamination [6].

Wound healing, or cicatrization, is the process by which tissues restore anatomical

structure and function after an injury [7, 8]. Factors that affect the wound closure process include

the type of wound, size, depth, location, the age of wound, presence of infection, condition of the

patient, and urgency of closure [9]. Wound depth is classified as partial thickness or full

thickness. Partial thickness wounds are shallow and involve epidermal loss and partial loss of

the dermal layer. Full thickness wounds involve a total loss of the epidermal and dermal layers

and extend to at least the subcutaneous tissue layer and possibly as deep as the fascia, muscle

layer, and the bone. Partial thickness wounds heal primarily by re-epithelialization from the

remaining dermis with minimal scar formation. There are three methods of open wound closure:

primary, secondary, and tertiary repair [5, 7, 9]. In primary closure or first intention, the wounds

are sealed immediately with simple suturing, skin graft placement, or flap closure. Wound edges

4

are brought close together, and the wound heals spontaneously with scar formation oriented

along the Langer’s lines (collagen fiber alignment within the dermis) [5]. Closure by secondary

or spontaneous intention involves no active intent to seal the wound. This type of healing is

associated with large, highly contaminated wounds, coupled with extensive tissue loss. They

close by the natural wound-healing cascade, which results in contraction and increased scar

formation. Failure to heal within a month can lead to a chronic state [5]. Wound closure by

tertiary intention or delayed primary closure occurs when wounds are infected or contain foreign

debris and cannot be closed until the complications are resolved. A contaminated wound is

initially treated by various methods for several days to control infection. Once the wound is

ready for closure, surgical intervention allows the wound to heal by first intention. Again, failure

to heal within a month can lead to a chronic state. Primary or tertiary wound healing is preferred

to secondary since secondary involves a more severe wound contraction and scar formation.

1.4. Acute Wound Healing Cascade

All wounds undergo the same basic steps of repair regardless of tissue type or nature of

the injury. Full thickness wounds damage many structures within in the skin. These include

epidermal keratinocytes and appendages (sweat glands, sebaceous glands, and hair follicles), the

basement membrane, and dermal fibroblasts and appendages (ECM, nerves, and blood vessels)

[10]. To heal the gamut of damaged structures, the classic wound-healing model consists of four

distinct but overlapping phases: hemostasis, inflammation, proliferation, and maturation [11-14].

5

1.4.1. Hemostasis

Hemostasis or the coagulation phase is the initiator of healing and occurs immediately

following an injury [15, 16]. Ruptured cells and damaged blood vessels activate the blood-

clotting cascade, which promotes platelet activation, adhesion, and aggregation at the site of

injury to form a fibrin clot. This fibrin clot prevents exsanguination, creates a protective layer to

minimize infection and further injury, and provides a provisional matrix of support as the wound

heals [3, 7, 16-18]. Activated platelets are a major source of growth factors that play a significant

role in the subsequent phases of wound healing [10].

1.4.2. Inflammation

Inflammation, the next phase of wound healing, begins within the first 24 hours of injury

and can last up to two weeks in the normal wound healing cascade [16]. Physically,

inflammation is associated with signs of redness, swelling, heat, and pain caused by the release

of histamine and other active amines by mast cells [16]. The priority of the inflammatory phase

is to neutralize infection and prepare the wound bed for repair; therefore, it is not the active stage

of wound closure. Nevertheless, it is the most critical phase of the wound-healing cascade.

Successful wound repair requires effective and timely resolution of inflammatory responses to

activate subsequent steps of the wound healing process. Incomplete resolution of the

inflammatory phase leads to a chronic state [8, 19].

The initial vasoconstriction needed to prevent excess blood loss is closely followed by

vasodilation and increased capillary permeability. The increased capillary permeability and

6

chemotactic factors facilitate diapedesis of neutrophils into the wound bed. Likewise, there is an

influx of pro-inflammatory cytokines (IL-1, IL-6, IL-8, and TNF-α) and growth factors (PDGF,

TGF-β, IGF-1, and FGF) into the wound bed [16]. Polymorphonuclear neutrophils (PMNs) are

the most abundant cells in the wound site during the first two days post initial wounding [8].

They generate a respiratory burst to destroy phagocytized bacteria by the release of reactive

oxygen species (ROS) [2, 10, 20, 21]. Electrons donated by the reduced form of nicotinamide

adenine dinucleotide phosphate (NADPH) are transported across the membrane into lysosomes,

and superoxide anion (O2-) is produced [22]. O2- is bactericidal but is also toxic to neutrophils

and surrounding viable tissue if not stringently regulated. In healthy young cells, downstream

antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase, and catalase

mitigate ROS production by catalyzing the formation of H2O2 [23-25]. Neutrophils utilize

myeloperoxidase to combine H2O2 with Cl- to form hypochlorite, which plays a supplementary

role in eliminating bacteria [3]. Additionally, neutrophils release high levels of proteases

(elastase, neutrophil collagenase, and neutrophil collagenase MMP-8) and remove necrotic

debris and foreign material [16]. Once the neutrophils complete their function, usually after 2-3

days, they are extruded in the eschar (the crust containing dead cells and degraded products of

the wound) or phagocytized by macrophages.

Monocytes from the bloodstream infiltrate into avascular hypoxic areas of the wound site

and mature into macrophages by stimulation from pro-inflammatory cytokines produced by the

neutrophils [3, 26]. Macrophages in the wound bed can exhibit two distinct functional

phenotypes: M1 (classically activated, pro-inflammatory) and M2 (alternatively activated, anti-

inflammatory). Early during the inflammation phase, macrophages that are activated by

lipopolysaccharide (LPS) or inflammatory cytokines like interferon gamma (IFN-γ) continue the

7

job of neutrophils by phagocytizing bacteria and damaged tissue. Additionally, the M1

macrophages release pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α)

and interleukin-6 (IL-6) [3]. Later during inflammation, initiated by the phagocytosis of

apoptotic cells, M1 macrophages phenotypically convert to M2 macrophages [27]. M2

macrophages, activated by interleukin-4 (IL-4) and interleukin-13 (IL-13), play a critical role in

the resolution of inflammation. Additionally, they are a predominant component of the transition

to the proliferative phase by promoting angiogenesis, tissue remodeling, and repair [3, 28-35].

Lucas et al. demonstrated that a depletion of macrophages during the inflammatory phase

significantly delayed wound repair in a mouse model; thus, they demonstrated the critical need

for macrophages for wound healing completion [36].

1.4.3. Proliferation

Until now, the wound bed has been prepping for the active repair phase by preventing

exsanguination, accomplishing wound debridement, and controlling the bacterial load.

Proliferation or the reparative phase is subdivided into different stages including granulation

tissue formation, collagen deposition, re-epithelialization, and angiogenesis [7, 15, 16]. The

initiation of the proliferative phase is evident by the infiltration of dermal fibroblasts into the

wound bed. Fibroblasts in the non-wounded dermis are typically dormant and sparsely

populated; therefore, fibroblasts are recruited from neighboring uninjured cutaneous tissues and

migrate to the wound bed [16]. A concentration gradient provided by chemotactic growth

factors, cytokines, and chemokines in conjunction with the alignment of collagen fibrils in the

provisional ECM govern the direction of fibroblast migration [16, 37]. Once the fibroblasts have

8

reached the wound bed, they anchor in the scaffold of the interim ECM and begin to proliferate

and synthesize granulation tissue components such as collagen, elastin, and proteoglycans. The

creation of granulation tissue, named for its irregular grainy appearance, slowly replaces the

temporary fibrin clot. Initially collagen, predominantly Type III collagen, is laid down in

irregular bundles providing moderate stability and tensile strength. In the later part of

proliferation, fibroblasts assume the myofibroblast phenotype, expressing α-smooth muscle actin

(α-SMA) [2, 38]. The myofibroblast phenotype enables the wound to contract but is dependent

upon growth factors PDGF and TGF-β [39].

Simultaneously, the epidermis must replace the initial wound seal, the fibrin clot, with a

new epithelial barrier to protect the newly forming ECM. Growth factors released during the

healing process stimulate the migration and proliferation of keratinocytes. Actively migrating

cells are incapable of proliferating; therefore, the neo-epithelium is created using new cells from

the proliferating basal layer of the epithelium near the wound margin. The normal cuboidal basal

epithelial cells flatten in shape and begin to migrate as a monolayer over the newly deposited

granulation tissue. The epithelial cells move in a tumbling fashion (also known as the epithelial

tongue) until the edges establish contact and form a confluent sheet. Once the newly formed

monolayer of keratinocytes covers the wound’s surface, migration stops, and the sheet enters a

proliferative state to re-establish the stratified layers of the epidermis and restore barrier function.

This process has to happen succinctly with ECM foundation or else the wound can easily re-

open.

As dermal and epidermal cells migrate and proliferate within the wound bed, there is a

substantial need for adequate blood supply for nutrient delivery and gas and metabolite

exchange. Due to vascular disruption, ischemia, and high oxygen consumption by metabolically

9

active cells, the microenvironment of the early wound is hypoxic, oxygen depleted. Compared to

normal tissue PO2 levels of 45–50 mm Hg, wound oxygen tension is 6 –7 mm Hg after five days

[40]. An inadequately replenished oxygen supply suppresses wound-healing processes; as an

example, fibroblasts can survive in a low-oxygen state but cannot replicate or synthesize [16,

41]. Angiogenesis restores tissue nutrient exchange, reestablishes microcirculation, and increases

oxygen tension to 30 – 40 mm Hg. Ischemia and hypoxia are critical inducers of angiogenic

factors to the wound site [40]. Binding of angiogenic factors such as VEGF, FGF, and TGF-β

cause microvascular endothelial cells of the blood vessels next to the wound site to migrate in the

same fashion as the fibroblasts. Once in the wound bed, the microvascular endothelial cells

proliferate to form buds or sprouts. As sprouts grow and encounter other sprouts, they develop a

cleft that subsequently becomes the lumen of the new vessel and complete a new vascular loop.

This process continues until the capillary system is sufficiently repaired for nutrient delivery and

oxygenation [16]. VEGF is noted to be the most potent angiogenic factor for its effects on

multiple components of the angiogenesis cascade: VEGF is an endothelial cell mitogen,

chemotactic agent, and inducer of vascular permeability [3, 42]. However, the angiogenic effect

of VEGF appears to be dependent on nitric oxide (NO). It has been shown that VEGF increases

NO production by up-regulating endothelial NOS (eNOS). Conversely, it has been shown that

when blocking eNOS, VEGF angiogenic properties such as endothelial migration and mitogen

activities are prevented [43, 44].

10

1.4.4. Maturation

The maturation or remodeling phase is different from the previous phases in that it does

not have a time sensitive pressure and can take from months to years to complete. During this

phase cell proliferation slows, protein synthesis decreases, and most endothelial cells,

macrophages, and fibroblasts undergo apoptosis or exit the wound. The goal of the remodeling

phase is to convert the new ECM, with a tensile strength of only 25-30% compared to normal

skin, to the a final scar with maximum tensile strength. Though tensile strength increases, the

maximum a wound can achieve is 80% of unwounded skin’s tensile strength. Collagen is

remodeled, and Type III collagen that is predominant in the proliferation stage is replaced by

stronger Type I collagen. The collagen is further strengthened by cross-links between collagen

fibrils. The final result is a scar that is more brittle and less elastic than normal skin. It should be

noted that scar tissue lacks the appendages (sweat glands) seen in the undamaged skin. Thus,

scar represents wound repair rather than regeneration.

1.5. Cellular Energy Metabolism

Cellular energy metabolism extracts a utilizable form of energy, adenosine triphosphate

(ATP) from the nutrition we eat via an elaborate set of biochemical reactions. There are two

categories of metabolic pathways: catabolism and anabolism. Catabolism includes the digestion

of ingested macromolecules (carbohydrates and fats) into smaller components (sugars and fatty

acids), which are either fully metabolized for energy or stored for later use. As a result of these

pathways, the energy released (7.3 kCal/mole) from the hydrolysis of the tertiary (ATP to ADP)

and secondary (ADP to AMP) phosphate bonds is used to execute all biologically necessary

11

reactions in the body. In contrast, anabolism uses the newly created ATP to synthesize essential

biomolecules such as proteins, lipids, and nucleic acids (DNA and RNA). The ratio between

anabolism and catabolism is an important factor in determining wound-healing progression,

which will be discussed later in this section.

1.5.1. Catabolism

In a standard American diet (SAD), ingested carbohydrates are metabolized into

monosaccharides, predominantly glucose, as the main fuel source for ATP. Glucose is

transferred into the cellular cytosol by low affinity, high capacity membrane transporters

(GLUTs). While in the cytosol, the process of glycolysis, ten enzymatic steps involving a

number of intermediates and specific enzymes, converts the 6-carbon glucose into two molecules

of 3-carbon pyruvate. Glycolysis results in the net gain of 2 ATP (2 ATP are invested in

reactions 1-5 and 4 ATP are produced in reactions 6-10) and the formation of 2 molecules of

NADH, an electron carrier that will eventually play an important role in the production of

additional ATP. Glycolysis is the only pathway to produce ATP without the presence of oxygen.

The flux through glycolysis is enough to sustain both ATP generation and fueling of the

biosynthetic pathways. Additionally, glycolytic intermediates can funnel into multiple

biosynthetic pathways. As an example glucose-6-phospahte (G6P) can enter the pentose

phosphate pathway, which generates NADPH to maintain antioxidant capacity and nucleic acid

synthesis.

The fate of pyruvate is dependent on the cellular oxygen concentration. In the absence of

oxygen, lactate dehydrogenase (LDH) converts pyruvate to lactate in a process known as

12

anaerobic respiration or fermentation. Coupled to this reaction are the formation of another ATP

and the oxidation of NADH to NAD+, which is cycled back to glycolysis. In the presence of

oxygen, the cells will use aerobic respiration. The two molecules of pyruvate are shuttled into the

mitochondrial matrix where they are oxidized into acetyl-CoA by the pyruvate dehydrogenase

complex (PDH). Two additional NADH molecules are created during this step. Acetyl-CoA is

joined to oxaloacetate, beginning the Krebs cycle. The Krebs cycle or citric acid cycle

completely oxidizes the remaining carbons from glucose, generates additional electron carriers (6

NADH and 2 FADH2) to feed the electron transport chain (ETC), and generates 2 ATP. As of

now, these metabolic pathways have synthesized a net gain of only 4 ATP, 10 NADH, and 2

FADH2. The electron carriers are shuttled to ETC, in the inner mitochondrial membrane for

oxidative phosphorylation. NADH and FADH2 are oxidized by the components of the ETC and

their electrons are passed between the ETC complexes in a series of oxidation/reduction

reactions. The final electron acceptor of the ETC is molecular oxygen, which reduces to H2O.

During the transfer of electrons through the ETC, H+ protons are pumped into the inner

mitochondrial membrane space creating an electrochemical gradient. This electrochemical

gradient drives the proton motive force used to synthesize ATP. Thus, the ETC generates 32-34

ATP per glucose molecule (3 ATP per NADH and 2 ATP per FADH2).

Dietary fats and stored fats are both metabolized via lipolysis to release their glycerol

backbone and their fatty acid side chains. The free fatty acids undergo β-oxidation to produce

acetyl-CoA. The metabolic fate of acetyl-CoA is the same as the previously described steps of

aerobic respiration from glucose. Each round of the β-oxidation cycle reduces the length of the

free fatty acid’s aliphatic tail by two carbon atoms and produces one NADH and one FADH2.

This process continues until the entire chain is cleaved into acetyl-CoA units. The ATP yield for

13

every β-oxidation cycle is 17 ATP (3 per NADH, 2 per FADH2, and 12 for the acetyl-CoA/Krebs

cycle). Thus, the total ATP yield will depend on chain length. Two ATP are required to activate

the fatty acid; hence a net ATP yield for palmitic acid (C16:0) is 113 ATP.

1.5.2. Anabolism

Anabolic or biosynthetic pathways utilize the newly created energy to fuel the creation of

necessary biomolecules. These include glycogenesis (glycogen synthesis), gluconeogenesis

(glucose synthesis), protein synthesis, and the pentose phosphate pathway. The main

biosynthetic pathways to focus on for this dissertation are the pentose phosphate pathway (PPP)

and gluconeogenesis. As mentioned previously, the first step of glycolysis produces glucose 6-

phosphate (G6P), which can be shuttled to the PPP, an anabolic pathway parallel to glycolysis

that generates the reducing agent NADPH and synthesizes 5-carbon sugars. The PPP accounts

for approximately 60% of NADPH production in the cells. It is important to note that NADPH is

the reducing agent consumed during biosynthetic reactions, whereas NADH is a reducing agent

generated in energy-yielding reactions. NADPH is used for fatty acid synthesis and helps fight

oxidative stress by reducing the antioxidant glutathione via glutathione reductase. The ribose

sugars generated from the PPP are utilized in the synthesis of nucleotides and nucleic acids.

Gluconeogenesis (GNG) is a series of eleven enzyme-catalyzed reactions to synthesize

glucose from non-carbohydrate sources including pyruvate, amino acids (alanine and glutamine),

lactate, and glycerol. The system is a reciprocal of glycolysis, which together creates a feedback

mechanism to normalize and maintain blood glucose levels. While most steps in gluconeogenesis

14

are the exact reverse of glycolysis, there are three reactions that require different catalytic

enzymes to make them more kinetically favorable.

In general, normal metabolism is hormone-mediated to alter energy production to meet

energy need and to restore daily macronutrient balance, especially proteins, through the catabolic

and anabolic pathways [45-50]. This balanced metabolic profile significantly changes in the

presence of a wound.

1.6. Wound Healing Metabolism

The presence of any substantial wound increases metabolic energy demands from 30-

50% above normal requirements to produce the necessary biomolecules for increased cellular

proliferation and migration of the various cell types during wound healing [17, 51]. Cuthbertson

famously described the metabolic response to injury as having an “ebb” phase, the period of

traumatic shock or hypometabolism during the first few hours or days after injury and a “flow”

or hypermetabolic phase (proliferation) that may last weeks to months depending on the

complexity of the wound [17, 52, 53].

It has been well established that non-wounded epidermal cells, as well as wounded skin,

are mainly dependent upon carbohydrate metabolism via glycolysis and the PPP rather than the

Krebs cycle for their energy requirement [54-59]. In a hallmark study, Im et al. showed a 4-fold

increase of glycolytic enzymes of wounded skin compared to normal skin [56]. The contribution

of the Krebs cycle to glucose metabolism was reduced in wounded skin compared to normal skin

(8% to 4%) demonstrating a metabolic shift to the PPP and other biosynthetic pathways, needed

15

for repairing the wound. This metabolic shift towards glycolysis was confirmed by the

observations that the rate of nucleotide formation was higher in wounded skin compared to

normal skin and a 7-fold increase in the PPP enzymatic activities. Furthermore, glucose-6-

phosphate dehydrogenase (G6PDH) was increased 7-fold, corresponding to the increased

contribution to the PPP [56]. Additionally, there was evidence that lactate production was tripled

in day three post-wounding, further confirming the higher flux through glycolysis and

subsequently anaerobic fermentation. Even though glycolysis is less efficient at creating ATP

compared to flux through the TCA cycle, the rate of ATP formation was doubled after three days

of wound healing. The ATP production was seen as a gradient with higher concentrations of

ATP in the migrating epithelium compared to the marginal proliferating epithelium. The study

showed a decrease in energy production per unit of glucose in wounded skin compared to normal

skin, as would be expected in the switch to glycolytic metabolism compared to aerobic

respirations. The inefficiency of glycolysis forming ATP compared to the ETC is overcome by

the increased metabolic rate of wounded skin. It has been shown that elevated activities of

glycolytic enzymes may contribute to increased cellular demand for energy during healing of

acute wounds [51, 59, 60].

1.7. Nutrition and Wound Healing

Nutritional status is extremely critical for effective wound healing [61-81]. As mentioned

earlier in this chapter, maintenance of a balanced anabolism: catabolism ratio is fundamental to

optimizing the wound healing process. Since healing a wound is crucial to surviving, a wound

requires high priority of the circulating nutrients in the blood stream. Increased macronutrient

16

intake is required to keep up with the catabolic losses and to allow wound-healing processes. The

American Society for Parenteral and Enteral Nutrition and Wound Healing recommends

approximately 30 to 35 kcal/kg/d for optimal wound healing, which is roughly 1.2-1.5 times

more than a non-wounded patient would ingest [82]. These macronutrients are predominantly

used as fuel; however, they also have direct effects essential for healing [64]. It should be noted

that individual energy needs depend on age, gender, nutritional status, basal metabolic rate, body

mass index (BMI), comorbid conditions, activity level, the stress of illness, severity and number

of wounds, and wound size of that patient, which supports the heterogeneous classification of

wounds [17, 83].

Macronutrients (fats, proteins, and carbohydrates) provide the energy for all body

functions and are major building blocks for the reparative process. In a healing wound, the influx

of inflammatory cells, as well as fibroblasts, use carbohydrates as an energy source. Additionally

carbohydrates have been shown to have a variety of non-energy related roles (structural,

lubricant, immunologic, hormonal, and enzymatic) [17]. Fats and their derived lipid components

provide the building blocks for the cell membrane and certain inflammatory mediators, behave as

signaling molecules, control tissue growth, wound remodeling, and collagen and extracellular

matrix production [84-91]. Proteins are predominantly metabolized into amino acids and

peptides for the purpose of protein synthesis; only 5% of protein metabolism is used for energy

[83]. Protein derived amino acids provide the major structural building blocks of all proteins in

the body, notably collagen. Amino acids are necessary for the cell membrane, enzymes, and

cytokine production in the healing wound [64].

When dietary nutrients are not adequate, or if the catabolic state outweighs the anabolic

state, the wound can proficiently use substrates available from the rest of the body for energy

17

production, resulting in what is called protein-energy malnutrition (PEM). The body catabolizes

the muscle, skin, and bone to support the synthesis of proteins, inflammatory cells, and collagen

needed to fight infection and repair the wound, causing a loss of lean body mass (LBM). As an

individual loses more LBM, wound healing is more likely to be delayed. A loss of more than

15% LBM impairs wound healing, a 30% LBM loss stops wound healing, and a loss of 40%

LBM typically results in death [64, 92-95]. As will be important later, a body in starvation

(ketosis) has been shown to be muscle sparing, attenuating the catabolic effects [83]. LBM loss

becomes vital in older adults as many suffer from pre-existing malnutrition and PEM.

1.8. Chronic Wounds

Chronic wounds represent an under-acknowledged socioeconomic epidemic, affecting

1.8 million new patients per year in the US. [96]. The care of these chronic wounds costs the US

health care system at least $25 billion annually. This substantial cost is rapidly growing due to

increased healthcare costs, an aging population, an increase in diabetes and obesity, and the

expanding need for wound care for veterans [96-98]. As more of our population ages (>65

years), projected to be 20% of the population by 2030, there is a critical need to develop

effective wound healing treatments to alleviate the considerable medical, social, and economic

burden facing the country [99].

18

1.8.1. Classification of Chronic Wounds

Chronic wounds are defined as wounds that have failed to proceed through and orderly

and timely reparative process to produce anatomic and functional integrity. All wound types

have the potential to become chronic, and as such, chronic wounds are traditionally divided by

etiology. A prolonged inflammatory stage, persistent infections, drug-resistant microbial

biofilms, and the inability of cells to respond to reparative signals are pathophysiological features

of all chronic wounds [8, 100, 101]. The Wound Healing Society’s Chronic Wound Care

Guidelines divide chronic wound into four different phenotypes: vascular ulcers (venous and

arterial), pressure ulcers, and diabetic ulcers.

The venous ulcer accounts for more than half of ulcer cases and 70-90% of ulcers found

on the lower limbs [102]. Venous ulcers affect approximately 1.69% of older adults or 600,000

patients total in the US annually [96, 103]. They are associated with deep vein thrombosis,

varicose veins, and venous hypertension. Physically, venous ulcers have skin that is shiny,

smooth, with minimal to no hair, are superficial, shallow, irregularly shaped, and are

accompanied by pain and edema. Less common than venous ulcers, arterial ulcers affect

approximately 100,000 Americans annually [104]. Arterial ulcers occur from hypertension,

atherosclerosis, and thrombosis, where the reduced blood supply leads to an ischemic state.

Commonly, they are full thickness wounds with a punched out appearance and smooth edges. As

compared to the venous ulcers that have prevalence on the lower limbs (between the knee and

ankle), arterial ulcers usually occur more distal such as the tip of the toe and over distal bony

prominences.

19

Pressure ulcers, also known as bedsores, are a result of prolonged pressure, friction and

shear from body weight and usually occur at a bony prominence such as the sacrum or heel.

Clinically, they present with redness, itching, blistering, warmth, swelling, and discoloration of

the overlying skin. They are prevalent in any patient that is wheelchair bound, bedridden, or

suffering from immobility or sensory neuropathies [105]. 2.5 million pressure ulcers are treated

in the US per year [106]. The cost of treating one pressure ulcer is estimated to be as much as

$70,000, totaling to an estimated $11 billion a year for pressure ulcer care [107, 108]. About

15% of patients in an acute care facility and up to 29% of patients in long-term care will

experience a pressure ulcer [96]. According to the Centers for Medicare & Medicaid Services

(CMS), there were a staggering 257, 412 preventable pressure ulcers in 2007 [109]. Additionally,

pressure ulcers can be a major source of infection, leading to complications and even death.

Development of a pressure ulcer increases mortality rate by 7.23% [110]. Over the last ten years

nearly 60,000 deaths occurred annually from hospital-acquired pressure ulcers [96]. Even though

many resources have been devoted to rectify these overwhelming statistics, it is still extremely

challenging and costly to prevent pressure ulcers [111-114].

As of October 2014, 29.1 million people in the United States have diabetes mellitus of

which 8.1 million people are undiagnosed [115]. Diabetic ulcers are a common complication in

uncontrolled diabetes, resulting from impaired immune function, ischemia (due to poor blood

circulation from high glucose content as well as vascular insufficiencies), and neuropathy. It is

estimated that up to 25% of all diabetics will develop an ulcer in their lifetime [116]. In 2007, a

single foot ulcer cost between $7,439 and $20,622, totaling to a $9 billion expense per year

[117].

20

1.8.2. Current Standard of Care, Alternative Therapies, and Their Disadvantages

Identifying and treating the underlying etiology of chronic wounds such as venous

insufficiency, arterial perfusion, diabetes, or continuous pressure as well as systemic factors such

as age, nutritional status, immunosuppression, and infection is key to successful wound healing

therapies. However, the establishment of effective wound healing therapies has been

unsuccessful due to the magnitude of these underlying pathogenic factors and the heterogeneity

across and within wound types [118]. Furthermore, it is important to emphasize that no two

wounds are exactly the same; thus, a treatment that could work for one patient may not be

effective for another.

Most current wound therapies being studied target a specific molecular or cellular

mechanism where there are often multiple mitigating factors that prevent normal wound closure.

This is likely one reason most wound therapies are minimally effective. Many growth factor

supplements tested in animal models appeared to be promising therapeutic agents promoting

wound healing [119]; however, human clinical trials have not been very successful [120, 121].

Additionally, there is a multitude of topical therapies (antimicrobials, dressings, silver-containing

products) that are commercially available, with minimal data to support their effectiveness in

promoting wound healing [122].

In vitro models have aided in the understanding of basic biology of wound healing; however,

more complex models to simulate the wound environment (multiple cell types) in conjunction

with comorbid conditions have not been developed. Many studies in wound repair have relied on

rodent models; however, skin morphology and the mechanisms of repair are markedly different

between rodents and humans [118, 123]. Additionally, some pre-clinical studies have used acute

21

wounds in young animals and not appropriately aged animals, demonstrating that there are no

animal models that comprehensively mimic human chronic wounds [124].

1.8.2.1. TIME and Amputation

Standard clinical therapies for non-healing wounds use the wound bed preparation

concept, which focuses on optimizing the condition of the wound bed to encourage normal

healing. In 2002, the acronym TIME was developed as a practical guideline for wound

management. TIME encompasses four main components of wound bed preparation: Tissue

management (debridement), control of Infection and inflammation, manage Moisture, and the

Edge of wound advancement [125, 126].

Debridement is the surgical removal of necrotic, damaged, or infected tissue, biofilms,

exudate, and debris to improve the healing of the remaining healthy tissue [127-132]. The goal of

debridement is to transition an excessively inflamed chronic wound to more of an acute profile,

promoting resolution of inflammation [122, 133, 134]. Chronic wounds are well characterized as

being stuck in the inflammatory phase with a marked increase in degrading proteases such as

matrix metalloproteinases (MMPs) and elastases and persistence of inflammatory cells [126,

129, 135-137]. Resolution of prolonged inflammation renews tissue healing, lessens exudate, and

reduces bioburden. It is important to distinguish between normal inflammation associated with

physiological healing compared to excessive inflammation caused by underlying adverse

etiologies and infection. Inflammation and infection are controlled using antimicrobials

(disinfectants, antiseptics, and antibiotics) in a variety of applications including silver dressings,

iodine dressings, PHMB dressings, honey, and surfactants [126]. Balanced moisture is essential

22

for the action of growth factors, cytokines, and cell migration. Excessive moisture damages

surrounding skin and insufficient moisture inhibits cellular activities and leads to eschar (scab)

formation [137]. Wound edge advancement assessment determines if wound contraction or

epithelialization is progressing and evaluates the effectiveness of the wound treatment. An

increasing range of treatment modalities has been proposed to improve wound edge advancement

including electromagnetic therapy (EMT), laser therapy, ultrasound therapy, systemic oxygen

therapy, and negative pressure wound therapy (NPWT) [126].

Amputation is the final and last course of action against chronic wounds (when they

occur on an extremity). This is done to prevent the spread of infection to healthy tissue.

Amputations are classified as either major (an amputation of the leg above or below the knee) or

minor (amputation of toes or forefoot) [138]. Many diabetic patients eventually must undergo

lower extremity amputations as a result of infection brought on by untreated foot ulcers. Diabetic

foot ulcers are responsible for 20% of diabetes-related hospitalization and underlie up to 80% of

non-traumatic amputations per year. It is estimated that 12% of individuals with a foot ulcer will

require amputation. Mortality following amputation is 13-40% at one year, 35-65% at three

years, and 39-80% at five years, which is worse than most malignancies. Once amputation

occurs, 50% of patients will develop an ulcer in the contralateral limb within five years [117].

1.8.2.2. Adjunctive Therapies

Even with the standard care practices of wound healing, there is a multitude of treatment

options and modalities. As previously mentioned, most of these treatments have limited research

data to prove effectiveness and many have controversial clinical results. Adjunctive treatments

23

for wound healing include negative pressure therapy, hyperbaric oxygen therapy (HBOT),

dermal substitutes, growth factor supplementation therapy, and stem cell therapy [139, 140].

Negative Pressure Wound Therapy (NPWT), vacuum-assisted wound therapy or

commonly known as a wound VAC®, has become a popular adjunctive therapy[141-143].

Negative pressure is applied to the wound via foam or gauze dressing moistened with saline,

which is then covered with a transparent adhesive sheet attached to a vacuum pump. NPWT has

been shown to manage wound drainage, reduce edema, reduce the bio-burden of

microorganisms, increase wound perfusion, and loosen the exudate [126]. This therapy in

combination with debridement has been shown to promote granulation tissue formation,

contraction and epithelialization [135]. However, a systematic review and meta-analysis of 21

studies found no clear evidence that wounds heal either better or worse with NPWT compared to

standard of care [144]. Additionally, the mechanism of action for NPWT is poorly understood,

and few studies have focused on the efficacy of this therapy in older adults [118].

Hyperbaric oxygen therapy (HBOT), originally utilized for decompression illness, has

been an adjunctive therapy for wound healing for more than two decades. It involves placing a

patient in a sealed chamber where 100% oxygen is pressurized to 1.5-3 atmospheres absolute

(ATA) for 60-120 minutes over a course of multiple treatments [139]. There isn’t an optimized

protocol for treatment and the mechanism of action for HBOT to improve wound healing is

poorly understood. However, it is speculated that HBOT helps reduce edema via

vasoconstriction and fluid absorption, increases oxygen perfusion aiding collagen elongation and

deposition, and stimulates epithelial progenitor cells and stem cell release for vasculogenesis

[139]. Published systematic reviews have reported mix results regarding the efficacy of HBOT,

as observational studies have shown a significant advantage of HBOT for wound healing;

24

however, six randomized trials did not show a benefit [145]. There are some promising animal

data suggesting that HBOT is effective for all ages; however, there are no studies specifically

focused on the efficacy of HBOT in older adults [146, 147].

Both biosynthetic skin substitutes (synthetic) and cultured autologous-engineered skin

(natural) are available to provide the protective barrier of skin when placed over a wound [148-

154]. The synthetic substitutes are made of an acellular material, and the natural substitutes

consist of cultures allogeneic or autologous cell suspensions or sheets used alone or with a

dermal matrix. The purpose of engineered skin products is to expedite wound healing by serving

as a scaffold for cellular ingrowth, stimulating the release of growth factors from the wound bed,

and restoring the functional properties of the skin [154, 155]. The skin substitutes (SS) can be

either permanent or temporary, depending on design and composition. It’s important to note that

although each of skin substitutes has their advantages and have applications, none of them can

fully simulate native skin. Both types of SS have been shown to be quite effective in increasing

the speed of wound healing, decreasing recurrence, reducing the number of required surgical

procedures, and reducing patient hospitalization time [148, 153, 155, 156]. Nevertheless, the

commercially available skin substitutes have several limitations such as reduced vascularization,

scarring, failure to integrate, poor mechanical integrity, and immune rejection. Furthermore, the

cost associated with the use of the current skin substitutes is very high. For example, it is

estimated that the cost for each 1% body surface areas covered by the commercially available

Epicel ™ is more than $13,000 [157]. As it relates to this project, the effect of the patient age on

skin substitutes is poorly characterized [118].

It is well characterized that growth factors play a substantial role in both healing and non-

healing wounds. Wound healing products have been designed to accelerate repair by augmenting

25

or modulating these growth factors in hopes of mediating inflammation and promoting

angiogenesis. Among all the possible growth factors, platelet-derived growth factor (PDGF) has

been approved by the Food and Drug Administration (FDA) for the treatment of diabetic foot

ulcers [4]. PDGF has been shown to stimulate granulation, increased odds of wound healing and

decrease rates of amputations in diabetic patients [158-165]. Growth factors EGF and FGF have

also been studied as topical wound healing therapies; however, the heterogeneity of wounds,

applications, patient management protocols, and clinical trial outcomes has limited their use to

outside of the USA [122].

Stem cells have the unique capability to differentiate into the variety of tissues in the

body. Stem cells derived from various sources including bone marrow, adipose, peripheral and

umbilical blood, in addition to vascular progenitor cells have been shown to migrate towards the

wound site and contribute to the wound healing process [166-168]. These cells have been to

shown to increase angiogenesis, increase granular tissue formation, enhance wound healing, and

reduce scarring [166, 168-172]. Even though stem cell research seems promising, the use of stem

cells is hampered by immunogenicity, potential for malignancy, pluripotency maintenance,

ethical consideration, and limited supply [118]. Additionally, harvesting bone marrow derived

stem cells is painful and leads to donor site morbidity [173].

The field of wound care is ever expanding with advances in technology. However, there

is still no superior substitute to allowing the body to heal systemically on its own accord in

conjunction with reconstruction using patients’ own tissues and carefully thought-out surgical

procedures. The therapy proposed in the next chapter of this dissertation focuses on exploiting

the body’s systemic physiology to enhance wound healing.

26

1.9. Age Influence on Chronic Wounds

Chronic wounds disproportionally affect older adults. 72% of hospitalized patients with

pressure ulcers were 65 and older. Most chronic wounds are associated with co-morbid

conditions prevalent in older adults, including cardiovascular disease, unrelieved pressure,

obesity, or diabetes mellitus [96]. However, Wicke and associates in 2009 demonstrated that age

was an independent risk factor for delayed wound closure. By the age of 70, the frequency of

wound closure was significantly lower than the younger counterparts [174]. It should be noted

that the effect of aging on wound repair is primarily a temporal delay; a change in the quality of

wound closure has not been reported [175]. The exact mechanisms responsible for this increased

risk remain unknown. In this dissertation we focus on four physiological attributes that we feel

lead to the prevalence of chronic wounds in older adults: unresolved inflammation, unmitigated

ROS production, decreased angiogenesis/blood flow, and decreased metabolism.

1.9.1. Age-Related Changes in Skin Morphology

Though the skin is incredibly durable, like all other systems, it eventually succumbs to

the inevitable effects of aging. The skin is also the most visible indicator of age. Clinically,

drying, roughness, sagging, wrinkling, and alterations in pigmentation characterize aged skin.

Physiological changes in the skin include both structural and biochemical changes.

Morphologically, the skin is characterized by atrophy due to the flattening of the

epidermis/dermis junction and a decrease of epidermal and dermal cellular content, collagen

27

disorganization, a decrease in microcirculation, and an increase in time for keratinocyte

maturation [4, 176-179]. The thickness of the skin decreases about 6.4% per decade on average.

Keratinocytes change shape as the skin ages, becoming shorter and fatter while corneocytes

become bigger as a result of decreased epidermal turnover. The cellular content of the dermis

(fibroblasts, mast cells, and macrophages) and protein content (collagen) are reduced with age.

The collagen and elastin that remain in the dermis are less organized, resulting in a decreased

elasticity of the skin [180]. Enzymatically active melanocytes decrease at a rate of 8-20% per

decade, resulting in the irregular pigmentation of elderly skin. Alterations in the aging skin not

only have an impact on wound healing but also make the skin more susceptible to injury. The

flattening of the epidermis/dermis junction results in less resistance to shearing forces and

increased vulnerability to insult as well as increased separation.

1.9.2. Age-Related Changes in the Wound Healing Cascade

Aging affects every phase of wound healing [176]. It has been demonstrated that aged

wounds have decreased levels and response to growth factors, reduced microcirculation,

diminished cell proliferation and migration, and diminished ECM secretion [181-183]. Aging is

also associated with delays in macrophage and T cell infiltration, angiogenesis, and re-

epithelialization [118]. It has been hypothesized that age-associated disadvantages in healing

may arise from overexpression of matrix metalloproteinases, which normally aid in

phagocytosis, angiogenesis, cell migration during epidermal restoration, and tissue remodeling.

Protease expression and activity are elevated in older human adults, which leads to the

perpetuation of the pro-inflammatory state and inhibits progression to the reparative phase [184].

28

Inflammation is a major contributing factor in wound chronicity and the aging

phenotype. As previously mentioned, chronic wounds are characterized as being stuck in the

inflammatory phase. Without resolution, the wound cannot advance to the reparative phase. This

inability to resolve inflammation is believed to be the most significant delaying factor in the

healing of chronic wounds [185]. It has been established that aged individuals have a consistent

low-grade inflammation marked by elevated cytokines like TNF-α, IL-6, and CRP, even when

the individual is healthy, or infection is absent [186]. This is in direct contrast to young

individuals, who only produce elevated levels of cytokines in response to injury or infection.

This low-grade inflammation has been implicated in the higher prevalence of co-morbidities

such as cardiovascular disease and diabetes in older adults. It has been suggested that

inflammation may be a common underlying cause of several age-related diseases or could be a

common pathway by which multiple diseases lead to disability and adverse outcomes in older

adults [186]. This already heightened inflammatory response is worsened in the presence of a

wound. It has been shown that there are increased numbers of neutrophils with prolongation of

the pro-inflammatory state as well as a delay in macrophage recruitment [185]. Additionally, a

study by Mahbub and colleagues demonstrated that M1 macrophages fail to transition to the M2

phenotype, perpetuating the inflamed state in an aged population [187].

ROS are produced in the healing wound as a bactericidal tool; however, when the levels

are not quenched by downstream antioxidants elevated ROS levels transcend the beneficial effect

and cause mitochondrial dysfunction, DNA damage, lipid peroxidation, and even cell death [188,

189]. Additionally, ROS are produced as a normal by-product of oxidative phosphorylation;

thus in a wound environment cells are hit with ROS from neutrophilic respiratory bursts as well

as from enhanced energy metabolism necessary to heal the wound [23, 190, 191]. The energy

29

metabolism shift to anaerobic respiration during wound healing likely protects the mitochondria

from over usage and damage related to ROS production [55]. Skin mitochondria in the human

aged population demonstrated an increased incidence of mitochondrial DNA (mtDNA)

mutations leading to mitochondrial dysfunction [192, 193]. Electron microscope images of aged

mitochondria show disorganization and degeneration via absent cristae, vacuolation, and

swelling [194]. Additionally, it has been determined that both superoxide anion and H2O2 are

increased in aged dermal fibroblasts [195]. Several studies have shown that the aged phenotype

has decreased levels of antioxidants to quench the increased ROS [196-199]. Treiber et al.

demonstrated that the skin of manganese superoxide dismutase (MnSOD, SOD2) deficient mice

was phenotypically similar to aged animals with characteristics such as atrophy of dermal

connective tissue, muscle fibers, and subcutaneous fat, loosely packaged collagen fibrils,

damaged mitochondria, and decreased cellular proliferation [200].

It has been demonstrated that angiogenesis is decreased with age because of reduced

levels of the angiogenic factors: FGF, VEGF, and TGF-β, impaired vasodilation, proliferation

and migration of angiogenic, microvascular endothelial cells. It is important to note that the

coexistence of hypertension, diabetes, smoking, and hypercholesterolemia exacerbates the

inherent effects of aging that are detrimental to angiogenesis [176, 201].

The metabolic challenge of a wound is exacerbated in elderly patients, who have a higher

prevalence of malnutrition combined with reduced total energy expenditure, basal metabolic rate,

and protein synthesis [17]. Without appropriate energy (ATP) levels, wound healing is

significantly impaired. The ATP deficiency creates an imbalance of energy production versus

utilization. To compensate, anaerobic respiration increases in the wound bed, which only

produces 2 ATP per glucose molecule as compared to the potential 38 ATP made from normal

30

aerobic respiration [41]. Gupta et al. showed that key metabolic enzymes were decreased in aged

rats [51]. It has been illustrated that mitochondria become larger and less numerous with age,

mitochondrial respiratory chain (MRC) enzyme activities decrease as well as mitochondrial

membrane potential [202]. Furthermore, there is a significant reduction in mitochondrial

bioenergetic capacity with advancing age, which has been shown in numerous animal models

and recently in a study with human volunteers [202, 203].

1.10. Closing Remarks

In summary, there is come controversy regarding whether chronological age alone affects

wound healing. However, it is well documented that there is increased ROS production with age,

leading to cellular damage, reduced blood flow leading to the lessening of nutrient exchange,

decreased metabolic activity and ATP production at the wound bed, and unresolved chronic

inflammation. It is easily deduced how these factors may lead to increased prevalence of chronic

wounds in older adults. Most approaches to accelerate wound healing focus on delivery of

growth factors by topical application. The discouraging results of focusing on single molecular

targets are not surprising since wound healing is the product of a complex set of interactions

between numerous factors. It is here that we aim to switch the approach to optimizing the

body’s physiology to reach the wound bed systemically as well as targeting multiple facets of

impaired wound healing with one therapy- exogenous ketone supplementation, which will be

discussed further in the next chapter.

31

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203. 4-105-1-PB.pdf.

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CHAPTER 2: ORAL KETONE SUPPLEMENTATION AS A NOVEL THERAPEUTIC

FOR DELAYED WOUND HEALING

2.1. Chapter Synopsis

In the previous chapter, I provided a thorough overview of chronic wounds and the non-

efficacious therapies that are currently available, especially for the aged population. Here I

provide a rationalization for the use of an oral exogenous ketone supplement as a novel therapy

for wound healing. The ketogenic diet has been used since the 1920s for refractory epilepsy in

children. Though it has been proven to be effective, it is hard to maintain and is limited in a

clinical setting. Recently, it has been shown that ketone bodies themselves may confer

therapeutic benefits, paving the way for an exogenous ketone supplement that does not require

dietary restriction. Ketones have been shown to decrease inflammation, decrease ROS levels,

increase blood flow, and increase ATP hydrolysis and increase metabolism. As previously

described, these are the four underlying age-dependent features we will utilize to determine the

efficacy of our treatment. For these reasons, we hypothesize that exogenous ketone

supplementation will augment wound healing and we propose to test their effects in vivo using

an ischemic wound model in young and aged Fischer 344 rats and in vitro using patient-derived

primary human dermal fibroblasts isolated from discarded skin.

Portions of this chapter have previously been published in “Kesl SL, Poff AM, Ward NP,

47

Fiorelli TN, Ari C, Van Putten AJ, Sherwood JW, Arnold P, D’Agostino DP. Effects of

Exogenous Ketone Supplementation on Blood Ketone, Glucose, Triglyceride, and Lipoprotein

Levels in Sprague-Dawley Rats. Nutrition and Metabolism (2016)”, Poff AM, Kesl SL,

D’Agostino DP “Ketone Supplementation for Health and Disease” Book Chapter, Oxford Press

(Submitted), and “ Trujilio AN*, Kesl SL*, Sherwood J, Wu M, Gould LJ. (2014)

Demonstration of the Rat Ischemic Wound Model. Journal of Visualized Experiments: JoVE ”. *

Indicates that both authors contributed equally to the work. Copies of these articles may be found

in Appendix C. See Appendix B for copyright permissions. Materials and methods presented in

this chapter can be found in Appendix A.

2.2. KetoneBodySynthesisandMetabolism

Ketone bodies are naturally elevated to serve as alternative metabolic substrates for extra-

hepatic tissues during the prolonged reduction of glucose availability, suppression of insulin,

and depletion of liver glycogen, such as occurs during starvation, fasting, vigorous exercise,

calorie restriction, or the ketogenic diet (KD). As blood glucose levels drop, the body goes

through a metabolic shift from glucose-based metabolism towards fatty acid oxidation and

hepatic ketogenesis to generate ATP. As a result, hepatic cells synthesize necessary glucose

through gluconeogenesis (GNG) and β-oxidation metabolizes fats (dietary and stored) to form

Acetyl CoA. During GNG, the oxaloacetate stores are depleted, causing the Acetyl CoA from

fatty acid oxidation to accumulate and not enter the Krebs cycle. The excess Acetyl CoA

produces the ketone bodies beta-hydroxybutyrate (βHB) and acetoacetate (AcAc) via a 3-step

enzymatic process known as ketogenesis. From the liver, these substrates are transported

48

systemically to extra-hepatic tissues (i.e. muscle, brain, and skin) where Acetyl CoA is

reconstructed and used to generate ATP via the Krebs cycle and the ETC [1, 2]. Additionally,

AcAc can be decarboxylated to produce the third ketone body, acetone. Although acetone has

been considered mainly to be a metabolic by-product that is eliminated via the lungs and urine, it

has recently been shown to play an important role in the anticonvulsant properties of the

ketogenic diet [3-7].

2.3. Review of Ketogenic Diet and Therapeutic Uses

The classical ketogenic diet (KD) consists of a 4:1 ratio of fat to protein and carbohydrate

combined, with 80-90% of total calories derived from fat [8]. For most patients this equates to

eating less than 50 grams of carbohydrates and around 100 grams of protein a day [9]. Actual

carbohydrate and protein intake must be individually optimized to maintain therapeutic ketone

production. The macronutrient ratio of the KD induces the metabolic shift towards hepatic

ketogenesis, elevating AcAc and βHB in the blood. Compared to other low carb diets like the

Atkins diet, the ketogenic diet requires only adequate protein. Excess protein intake can fuel

GNG, resulting in glucose synthesis that disables ketogenesis. It is important to emphasize that

not all low carbohydrate diets are ketogenic. The ketogenic diet is really a high-fat diet, and not a

high-protein diet.

Emerging evidence supports the therapeutic potential of the ketogenic diet (KD) for a

variety of disease states, leading investigators to research methods of harnessing the benefits of

nutritional ketosis without the dietary restrictions. The KD has been used as an effective non-

pharmacological therapy for pediatric intractable seizures since the 1920s [10-12]. In a study of

49

150 epileptic children who averaged 400 seizures per month and were on multiple anti-epileptic

medications, 60% reported a significant decrease in seizure frequency [12]. In addition to

epilepsy, the ketogenic diet has elicited significant therapeutic effects for weight loss and type-2

diabetes (T2D) [13]. Several studies have shown significant weight loss on a high fat, low

carbohydrate diet without significant elevations of serum cholesterol [14-21]. Another study

demonstrated the safety and benefits of long-term application of the KD in T2D patients. Patients

exhibited significant weight loss, reduction of blood glucose, and improvement of lipid markers

after eating a well-formulated KD for 56 weeks [22]. Recently, researchers have begun to

investigate the use of the KD as a treatment for acne, polycystic ovary syndrome (PCOS),

cancer, amyotrophic lateral sclerosis (ALS), traumatic brain injury (TBI) and Alzheimer’s

disease (AD) with promising preliminary results [23-35].

2.3.1. Potential Concerns and Side Effects

Although the KD has clear therapeutic potential, several factors limit the efficacy and

clinical utility of the KD for metabolic therapy. Patient compliance to the KD can be low due to

the severe dietary restriction, the diet being perceived as unpalatable, and intolerance to high-fat

ingestion. Maintaining ketosis can be difficult as consumption of even a small quantity of

carbohydrates or excess protein can rapidly inhibit ketogenesis [36, 37]. Furthermore, enhanced

ketone production and tissue utilization can take several weeks (keto-adaptation), and patients

may experience mild hypoglycemic symptoms during this transitional period [38]. One common

concern regarding the KD is its purported potential to increase the risk of atherosclerosis by

elevating blood cholesterol and triglyceride levels [39, 40]. This topic remains controversial as

50

some, but not all studies have demonstrated that the KD elevates blood levels of cholesterol and

triglycerides [41-46]. Kwitervich and colleagues demonstrated an increase in low-density

lipoprotein (LDL) and a decrease in high-density lipoprotein (HDL) in epileptic children fed the

classical KD for two years [8]. In this study, total cholesterol increased by ~130%, but then

stabilized at the elevated level over the 2-year period. A similar study demonstrated that the lipid

profile returned to baseline in children who remained on the KD for six years [47]. Children

usually stay on the diet for approximately two years then return to a diet of normal fat and

carbohydrate ingestion [48]. In contrast, the majority of recent studies have suggested that the

KD can lead to significant benefits in biomarkers of metabolic health, including blood lipid

profiles [49-56]. In these studies, the KD positively altered blood lipids, decreasing total

triglycerides and cholesterol while increasing the ratio of HDL to LDL [52-61]. Although, the

KD is well established in children, the diet has only recently been accepted as a strategy to

control seizures in adults. In 2014, Schoeler et al reported on the feasibility of the KD for adults,

concluding that 39% of individuals achieved > 50% reduction in seizure frequency, similar to

results seen in pediatric studies. Patients experienced similar gastrointestinal adverse events that

have been previously described in pediatric patients, but they did not lead to discontinuation of

the diet in any patient [62].

2.4. Exogenous Ketone Supplementation

Considering both the broad therapeutic potential and limitations of the KD, an oral ketone

supplement that could rapidly induce and sustain therapeutic ketosis without the need for dietary

restriction would be an attractive and practical alternative. Recent studies suggest that many of

51

the benefits of the KD are due to the effects of ketone body metabolism. Veech et al. have

summarized the potential therapeutic uses for ketone bodies, which will be discussed later in this

chapter [3, 63]. Furthermore, there is potential for an additive therapeutic effect between a

standard KD and an oral ketone supplement for patients consuming a low carbohydrate or

ketogenic diet. Additionally, in some scenarios, a ketone supplemented ketogenic diet may be

more effective than KD or ketone supplementation alone. Several natural and synthetic ketone

supplements capable of inducing nutritional ketosis have been identified. There are numerous

sources of ketones and ketogenic precursors being developed and tested, including medium chain

triglycerides, diols, salts, and esters that have been shown to elevate blood ketone levels without

dietary restriction. The investigation of natural and synthetic ketogenic precursors to establish

nutritional ketosis without the need for dietary restriction has revealed that each formulation has

distinct properties in terms of extent and duration of ketosis as well as metabolic effects. Most of

the developed ketone supplements are currently under investigation for safety and efficacy in a

number of disease states. For this dissertation, we focus on five ketogenic agents including 1, 3-

butanediol (BD), medium chain triglyceride oil (MCT), sodium/potassium-βHB mineral salt

(BMS), combination of BMS+MCT, and 1,3-butanediol acetoacetate diester/ ketone ester (KE).

2.4.1. 1,3-Butanediol

1,3-Butanediol (BD) is a FDA approved organic alcohol used as a food flavoring solvent, an

intermediate in the manufacture of certain polyester plasticizers, and a humectant for cosmetics

[64, 65]. When ingested orally, BD is metabolized by the liver via alcohol dehydrogenase (ADH)

to β-hydroxybutyraldehyde, which is rapidly oxidized to βHB by aldehyde dehydrogenase [66].

52

BD contributes approximately 6 kcal/g of energy and can produce dose-dependent millimolar

concentrations of ketones in the blood [67-69]. Extensive toxicology studies have concluded that

BD is safe with very little adverse health effects in humans or animals [70-73]. BD has been

investigated as a therapy to reduce brain damage following cerebral ischemia. BD attenuated

hypoxic damage via a cerebral protective effect mediated by brain ketone metabolism [74].

Additionally, ketosis induced via exogenous BD supplementation has been shown to increase

survival time of mice exposed to hypoxia in a stroke model [75]. BD supplementation has been

shown to significantly prolong the survival time in mice with metastatic cancer by 51% [26].

Recently, BD has been investigated therapeutically as a backbone of ketone mono- and di-esters,

which are discussed later in this chapter.

2.4.2. MCT Oil

Medium chain triglycerides (MCTs) contain a glycerol backbone esterified to medium-

chain fatty acids (MCFAs), which are fatty acids with hydrocarbon side chains 6 to 12 carbons in

length. MCFAs include caproic acid (C6:0, hexanoic acid), caprylic acid (C8:0, octanoic acid),

capric acid (C10:0, decanoic acid), and lauric acid (C12:0, dodecanoic acid). MCTs are naturally

found in coconut oil (15%), palm kernel oil (7.9%), cheese (7.3%), milk (6.9%), butter (6.8%),

and yogurt (6.6%) [76, 77]. Compared to long-chain fatty acids (LCFAs), MCFAs have a lower

melting point, smaller molecule size, and are less calorically dense (8.3 calories per gram versus

9.2). These distinct physiochemical properties allow MCTs to be absorbed directly into the

bloodstream through the portal vein without the need for bile or pancreatic enzymes for

degradation. Additionally, MCTs do not require carnitine to enter the mitochondria, but rather

53

quickly cross the double mitochondrial matrix where they are metabolized to acetyl co-A, and

subsequently, to ketone bodies. Thus, they are easily and rapidly digested, transferred to the

liver, and used for energy rather than stored as fat. In comparison, LCFA metabolism is much

slower requiring re-esterification in the small intestine, transport by chylomicrons via the

lymphatic and vascular systems, and oxidation in the liver for energy or storage. MCTs are

metabolized as rapidly as glucose but have roughly twice the energy density [78]. In the early

1980s, Dr. Vigen K. Babayan of the Nutrition Laboratory at Harvard University developed a

process to produce MCTs in large quantities [79]. These commercialized MCTs are acquired

through lipid fractionation from natural fats such as coconut oil and milk and are predominantly

comprised of C:8 and C:10 MCTs [80, 81].

Since the 1970s, MCTs have been used in a modification of the classical KD as an

alternative fat source [82]. The ketogenic properties of MCTs allow patients to eat less total fat

in their diet and include more carbohydrate and protein without sacrificing their nutritional

ketosis. Although MCTs have the potential to be an efficient and beneficial ketogenic fat, they

are currently limited in clinical use due to gastrointestinal (GI) side effects stimulated by the

large dose needed to induce ketonemia (greater than 40 g/day) [78]. The original modified KD

with MCT allowed 60% of its energy to be derived from MCTs; however, this amount caused GI

distress in some children [83-86]. For this reason, an additional modified KD with MCT was

developed using only 30% of its energy from MCTs; however, it induced much lower levels of

ketosis [87, 88].

54

2.4.3. βHB Salt

Originally, researchers attempted to administer βHB or AcAc in their free acid forms;

however, this was shown to be too expensive and ineffective at producing sustained ketosis.

Subsequently, it was suggested to buffer the free acid from of βHB with sodium salts, but this

causes potentially harmful sodium overload and mineral imbalance at therapeutic levels of

ketosis, and is largely ineffective at preventing seizures in animal models [89]. A study showed

that oral administration of Na+/βHB in doses from 80-900mg/kg/day elevated blood ketone

levels to 0.19-0.36 mM in children with acyl CoA dehydrogenase deficiency [90]. However, to

achieve the same level of ketosis a 70 kg man would need to ingest between 5.6 to 63 g/day,

costing up to $200 a day. Considering the potential effects of such a large sodium load, the costs

of the administration of Na+ /βHB salts to achieve ketosis made this approach unrealistic [63].

Recently, a βHB mineral salt was developed in collaboration with Savind Inc. These

ketone salts are balanced with minerals to prevent sodium overload and can include arginine,

potassium, calcium, magnesium, lithium, lysine, histidine, ornithine, creatine, agmatine

(guanidine), or citrulline. Maintaining an optimal sodium-mineral ratio should help offset any

potential adverse effects of sodium on blood pressure. It is speculated that this formulation will

be especially beneficial for elderly patients most susceptible to sodium-induced hypertension

[91]. The mineral salt has to be delivered as a concentrated liquid because potassium βHB is too

hygroscopic to isolate as a powder (it picks up water from the atmosphere and turns into a paste

at an astonishing rate, whether by itself or mixed with sodium βHB). Many people experience

the “low carb flu” during keto-adaptation (2-3 week phase characterized by enhancement of

ketone production and tissue utilization from being on a ketogenic diet) caused by glucose

withdrawal in the brain and a depletion of minerals, especially sodium and potassium, in the

55

plasma [38]. These symptoms can be attenuated or reversed with sufficient supplementation of

sodium, potassium, calcium, and magnesium.

Several mineral combinations are currently being tested for safety and efficacy. In a recent

case study, a 100 kg male subject was administered a 4% Na+/K+ βHB salt solution (containing

11 grams of sodium and 7.1 grams βHB) and his plasma levels of βHB were significantly

elevated 30-60 minutes after administration. After receiving the same dose for three days, the

patient had sustained elevated blood ketone levels from 15-120 minutes after administration.

Similar results were seen in a 70kg male who fasted three days prior to administration of the

ketone salt supplement [92]. In a 15-week study, Sprague-Dawley rats were administered

Na+/Ca+2-β-hydroxybutyrate mineral salt (S-BHB), 20% by weight (~25 g/kg/day) in their food

fed ad libitum. The S-BHB supplemented rats exhibited sustained elevated blood ketone levels

(1mM βHB) at 1, 4, 8, 10, and 13 weeks of chronic feeding which did not affect blood glucose

levels compared to controls (unpublished data).

2.4.4. Combination BMS+MCT

Considering the variety of ketogenic precursors available, researchers are investigating

unique combinations of the individual supplements in hopes of optimizing their benefits. A

combination of βHB salts and MCT oil has been administered in 1:1 to 1:2 mixtures. This

mixture allows for a lower dosing of the components compared to administering the individual

compounds, thus reducing potential for side effects (sodium-induced hypertension, GI side

effects, etc) and resulting in distinct synergistic blood ketone profile [92].

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In a case study, a 100 kg male was administered a combination of a 4% Na+/K+ βHB salt

solution (containing 11 grams of sodium and 7.1 grams βHB) + 20 mL MCT oil. This

combination demonstrated higher elevation of blood ketone levels than either βHB salts or MCT

oil alone, starting at 15 minutes post consumption and lasting for 4 hours. Similar results were

observed in a 70-kg male who fasted 3 days prior to administration; however elevated blood

ketone levels were observed sooner after supplementation and were sustained for a considerable

longer time after administration (8 hours). This effect was accompanied by a reduction in blood

glucose, and a lower starting blood glucose concentration on each subsequent day of

supplementation [92]. In a 15-week study, Sprague-Dawley rats were administered a 1:1 mixture

of Na+/Ca+2-β-hydroxybutyrate mineral salt + Medium Chain Triglyceride Oil (S/MCT) (20%

by weight (~25 g/kg/day) in their food fed ad libitum. The combination-supplemented rats had

significantly sustained and elevated blood ketone levels as weeks 3, 4, 8, 10, and 13 without

significantly affecting blood glucose levels during the study (unpublished data).

2.4.5. Ketone Esters

Researchers have developed and investigated several synthetic ketone mono- and di-

esters to produce nutritional ketosis without dietary restriction. In these compounds, gastric

esterases rapidly liberate ketones as a free acid from a backbone molecule, which varies

depending upon the specific formulation, but is often R,S-1,3-butanediol. As previously

discussed, 1,3-BD is subsequently metabolized by the liver to produce βHB [4]. Thus, the ketone

esters currently available are unique amongst the aforementioned ketone supplements in that

many contain, and thus directly elevate, AcAc rather than only βHB.

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In the late 1970s, Birkhahn et al. were the first to synthesize a monoester of glycerol and

AcAc (monoacetoacetin) for parenteral nutrition. These studies demonstrated that

monoacetoacetin induced hyperketonemia comparable to fasted rats at a dose of 50 g/kg per day

[93-95]. In attempts to increase the caloric density of monoacetoacetin, they synthesized both a

monoester and triester of glycerol and βHB. These esters are hydrolyzed to release free βHB,

thus elevating blood ketones. Later, Desrochers and colleagues synthesized mono- and di-esters

of 1,3-BD with either AcAc or βHB [96]. These and other esters developed by or in collaboration

with Henri Brunengraber and Richard Veech have been shown to induce various degrees of

hyperketonemia (2-7mM) in rats, mice, dogs, pigs, and humans [97-102]. Clarke and colleagues

demonstrated the safety of these ketone esters in rats and humans [103, 104]. Recently, a R,S-

1,3-butanediol acetoacetate diester (KE) was developed in collaboration with Savind Inc. A

single dose of the KE administered via intragastric gavage elevated both AcAc and βHB blood

levels to >3mM in rats [4]. In a 15-week study, the KE was administered to Sprague-Dawley rats

in a low dose (5%, 10 g/kg/day) (LKE) and a high dose (20%, 25 g/kg/day) (HKE) in food fed

ad libitum. Both doses significantly elevated blood ketone levels without affecting blood glucose

levels. Serum clinical chemistry of both LKE and HKE did not reveal any changes in any major

markers for kidney and liver function compared to standard diet fed rats (unpublished data).

2.4.6. Potential Concerns and Side Effects: Ketoacidosis

Ketoacidosis is one major concern for the use of oral ketone supplements without dietary

restriction. Ketoacidosis is normally only seen in patients with diabetes mellitus. In this

circumstance, the lack of insulin prevents glucose delivery to the tissues to be used as energy.

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High glucose levels spill over into the urine, taking water and solutes (sodium and potassium)

with it in a process known as osmotic diuresis. The body senses that it is in starvation mode and

enhances b-oxidation of fatty acids for ketogenesis. The resulting ketone bodies are used as fuel.

Additionally, insulin acts as a negative feedback loop to ketogenesis. Any rise in insulin and/or

glucose normally inhibits ketogenesis immediately or excess ketones are expelled through

ketonuria. Without insulin, the body is overwhelmed by ketone bodies. Since the ketone bodies

are acidic, the uncontrolled elevation of ketone bodies (20-25 mM) drops the blood pH and can

lead to organ failure, cerebral edema, and even death. If not properly administered, oral ketone

supplementation does have the potential to induce ketoacidosis; however, optimal dosing

circumvents this danger. Oral ketone supplementation will be optimized to elevate blood ketone

levels to the 2-7 mM range, which is far below the elevation seen in ketoacidosis.

2.5. Potential Physiology Factors Affected to Enhance Wound Healing

It has been well established that optimized nutritional support aids wound healing. [105,

106]. Demling et al. stated that in order to optimize healing, a substrate that is more dependent

on intake rather than on the bodily breakdown of protein needs to be available [107]. In the

traditional classification, ketone bodies are known as a carrier of energy from the liver to extra-

hepatic tissue. However they can also be considered as a nutritional supplement and an anabolic

agent [108]. Indeed, Nair et al. concluded that βHB promotes protein synthesis in human beings

[109]. Recently, it has been shown that βHB possess a variety of signaling functions that may

regulate a broad range of cellular functions. [110-113]. It has been shown that βHB can regulate

cellular processes via histone deacetylase (HDAC) inhibition, binding to cell surface receptors,

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and altering the levels of other regulatory metabolites including acetyl-CoA, succinyl-CoA, and

NAD+ [110]. These unique cellular and physiological properties support the assertion that

ketone bodies themselves confer many of the benefits associated with the KD. We hypothesize

that ketone bodies may augment wound healing via the suppression of inflammation, inhibition

of oxidative stress, enhancement of blood flow, and enhancement of metabolic efficiency.

Additionally, ketones may aid diabetic foot ulcer healing via modulation of blood glucose levels

and enhancement of insulin sensitivity.

2.5.1. Ketones and Inflammation

As mentioned in the previous chapter, chronic wounds are stuck in the inflammatory

phase. A therapy that promotes resolution of inflammation may push the chronic wound to a

more acute wound phenotype, therefore progressing to closure. Several studies have determined

that the KD reduces circulating inflammatory markers in animals and in humans [114-117].

Additionally, Ketone bodies themselves have been shown to decrease inflammatory biomarkers

including TNF-a, IL-6, IL-8, MCP-1, E-selectin, I-CAM, and PAI-1 [17, 118]. In an unpublished

study from the D’Agostino laboratory, rats were fed one of three exogenous ketone supplements

LKE, HKE, S-BHB, or S/MCT mixed into standard rodent chow at 5- 20% by weight for 15

weeks. Inflammatory profiling was performed on serum collected from the animals at the end of

the chronic feeding study and revealed decreases in several pro-inflammatory cytokines

including IL-1β, IL-6, IFN-γ, MCP-1, and RANTES.

Recently, Dixit and colleagues demonstrated in both in vitro and in vivo that βHB inhibits

the NLRP3 inflammasome, which controls activation of caspase-1 and release of the pro-

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inflammatory cytokines IL-1β and IL-18 from M1 macrophages. The authors concluded that

elevating blood ketones could be a therapeutic option for patients with NLRP3-mediated chronic

inflammatory diseases [119]. It has been established that elevated levels of IL-1β are present in

wounds of diabetic humans and mice, consistent with increased inflammasome activity. Since

M1 macrophages are activated by IL-1β, the NLRP3 is thought to perpetuate this pro-

inflammatory phenotype, leading to impaired wound healing [120]. Two studies have shown that

blocking the activation of the inflammasome decreased pro-inflammatory cytokine levels and

accelerated wound healing in diabetic mice [120, 121]. This evidence supports the hypothesis

that oral ketone supplementation could produce similar effects on the inflammasome.

2.5.2. Ketones and ROS

Veech and others have determined some of the molecular mechanisms by which ketone

bodies suppress oxidative stress [122-124]. In non-wounded skin, the major source of free

radicals is the half-reduced semiquinone of co-enzyme Q in the electron transport chain of the

mitochondria. At physiological levels, ketone bodies increase the oxidation of co-enzyme Q,

decreasing mitochondrial oxygen radical generation, thereby reducing the amount of ROS

produced [3, 63, 124]. Additionally, ketone metabolism induces reduction of the mitochondrial

NAD+ and cytoplasmic NADP electron carriers. As previously mentioned in Chapter 1, NADH

is important for energy producing pathways and NADPH is necessary for the regeneration of the

endogenous antioxidant reduced glutathione (GSH) [125].

Eric Verdin and colleagues have recently demonstrated βHB is an endogenous and

specific inhibitor of class 1 histone deacetylases (HDACs) [126]. Elevated βHB levels by fasting,

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calorie restriction, and exogenous administration increased global histone acetylation in mouse

tissues and induced the transcription of genes encoding oxidative resistance factors FOXO3A

and MT2 via selective depletion of HDAC 1 and HDAC2. Furthermore, the authors

demonstrated in vivo that exogenous ketone supplementation could prevent oxidative stress.

Mice were pretreated with βHB via a subcutaneous pump for 24 hours prior to receiving an

injection of paraquat, which induces the production and accumulation of ROS. Protein

carbonylation was suppressed by 54%, lipid peroxidation was completely suppressed, and

endogenous antioxidants mitochondrial superoxide dismutase (MnSOD, SOD2) and catalase

(CAT) were elevated in the renal tissue of βHB pre-treated mice.

Regulated ROS levels are critical for optimal wound healing. Normal oxidative

phosphorylation, transient hypoxia, and increased neutrophil and macrophage respiratory bursts

release ROS during wound healing. The increased level of ROS transcends the beneficial effect

and causes additional tissue damage and healing delay. This unquenched ROS production is

prevalent in the aged population. Recently, Moor et al demonstrated age-dependent deficiencies

in the glutathione and SOD2 antioxidant pathways. In an ischemic wound model, wounds from

aged rats had lower SOD2 protein and activity, decreased ratio of reduced/oxidized glutathione,

and decreased glutathione peroxidase activity [127]. These age-exaggerated insufficiencies lead

to excessive inflammation and impaired wound healing.

One of the hallmarks of aging is mitochondrial dysfunction [128]. Electron microscope

images of aged mitochondria show disorganization and degeneration via absent cristae,

vacuolation, and swelling. Treatment with lipoic acid and carnitine stimulates repair of

mitochondrial membranes, returns functionality to the mitochondria, and demonstrates a young

phenotype [129]. Bough et al showed increased mitochondrial biogenesis in rats maintained on a

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KD for 4-6 weeks [130, 131]. Interestingly, exogenous ketone supplementation with a ketone

ester has been shown to induce mitochondrial biogenesis [132]. Mice in this study were fed a

diet from which approximately 30% of calories were derived from the D-β-hydroxybutyrate-R-

1,3-butanediol monoester for one month. The mitochondrial content and expression of electron

transport chain proteins were significantly increased in the intrascapular brown adipose tissue as

compared to control mice, although calorie intake was matched between the two groups. These

experiments support the hypothesis that ketone supplementation could have therapeutic benefits

for an aged population.

2.5.3. Ketones and Blood Flow and Angiogenesis

Restoration of blood flow via angiogenesis is critical for healing a wound, as well as the

integration of skin substitutes. Venous, arterial and diabetic blood flow insufficiencies are major

underlying contributors to chronic wound development. Additionally, most older patients have

hypertension or other comorbidities that affect blood flow. In a study by Hasselbalch and

colleagues, 8 volunteers (4 male, 4 female, 24±4 years of age, normal weight) were given an

infusion of Na+-βHB via antecubital vein at a rate of 4-5 mg/kg/min corresponding to an infusion

of ~350mL/hr for 3-3.5 hours. Global cerebral blood flow was measured via the Kety-Schmidt

technique; ketone supplementation showed a 39% increase in cerebral blood flow [133].

As noted in Chapter 1 of this dissertation, VEGF is the most potent angiogenic factor for

its effects on multiple components of the angiogenesis cascade: endothelial cell mitogenesis,

chemotaxis, and induction of vascular permeability [134, 135]. In addition to VEGF, endothelin-

1 (ET-1) is another cytokine produced in the response to hypoxia and is a crucial promoter of

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cell proliferation, migration and chemotaxis, and angiogenesis in wound healing [136-138]. A

study by Isales and colleagues demonstrated that supplementation of mouse brain microvascular

endothelial cells with βHB and AcAc both independently increased ET-1 and had an added affect

when applied together. βHB but not AcAc increased VEGF levels [139]. Though this area needs

further exploration, this may be one mechanism that oral ketone supplementation may augment

wound healing.

2.5.4. Ketones and Metabolism

In the 1940s Henry Lardy demonstrated that βHB and AcAc had distinct energetic

efficiencies compared to 16 major carbohydrate, lipid, and intermediary metabolites in their

ability to increase bull sperm mobility while simultaneously decreasing oxygen consumption.

[140, 141]. Nearly 50 years later, Richard Veech and colleagues confirmed that ketone bodies

increased metabolic efficiency and elucidated the molecular mechanisms in the working perfused

rat heart [124, 142]. They demonstrated that supplementation of glucose-containing perfusate (10

mM glucose) with 5 mM ketones (4 mM βHB, 1 mM AcAc) increased cardiac hydraulic work

by approximately 25% while simultaneously reducing oxygen consumption [124]. Their study

demonstrated the ketone-mediated increase in metabolic efficiency was facilitated by a reduction

of the mitochondrial NAD couple and an oxidation of the coenzyme Q couple increasing the

energy span between the sites (mentioned previously to decrease ROS production). This results

in an increase in energy released by electrons in the ETC, causing more protons to be pumped

into the inner mitochondrial space. The electrochemical gradient is enhanced, hyperpolarizing

the cell, and increasing the ΔG0 (free enthalpy) of ATP hydrolysis; thus, increasing metabolic

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efficiency. Additionally, ketone bodies were shown to cause a 16-fold elevation in acetyl-CoA

content and increased Krebs cycle intermediates. Furthermore, thermodynamic tables for heat of

combustion, calculated with bomb calorimeter experiments, show that βHB produces more

energy than glucose per carbon molecule [143].

Decreased availability of ATP negatively impacts nearly every aspect of the healing

process [144]. Decreased nutrient and oxygen exchange during wound healing limits ATP

production to fuel wound healing processes. Additionally, the skin predominantly uses anaerobic

respiration, which decreases the potential ATP production by 90% compared to aerobic

respiration [145-147]. Chiang and colleagues developed a new intracellular ATP delivery

technique in which highly fusogenic lipid vesicles (ATP-vesicles) are used to encapsulate

magnesium-ATP (Mg-ATP). When the vesicles come into contact with the cell membrane, they

fuse together and deliver the contents into the cytosol. Using eleven controlled-pairs of adult

nude mice, they demonstrated accelerated wound healing via enhanced development of

granulation tissue, more rapid re-epithelialization, and elevated VEGF levels [31, 63].

2.5.5. Ketones Suppress Blood Glucose via Increasing Insulin Sensitivity

Failure to heal in diabetic lower limbs is associated with hyperglycemia, insulin

resistance, tissue hypoxia, chronic inflammation, oxidative stress, impairment of the immune

system, and metabolic dysfunction [148, 149]. A study by Rubinstein et al. showed that once

diabetes was a controlled, diabetic foot ulcers in 11 of 15 patients healed in 4 to 13 weeks [150].

In addition, diabetic patients with glucose under 200 mg/dl showed a decrease in surgical site

infections after foot and ankle surgery [151]. Recent studies suggest that many of the benefits of

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the KD are due to the effects of ketone body metabolism. Interestingly, in studies on T2D

patients, improved glycemic control, improved lipid markers, and retraction of insulin and other

medications occurred before weight loss became significant, suggesting physiological effects of

ketone metabolism [3, 63]. Exogenous ketone supplements may provide therapeutic benefits for

the underlying hyperglycemia and insulin resistance as reports have demonstrated that oral

ketone administration lowers blood glucose by increasing insulin sensitivity. This could be

critical for older adults as all have demonstrable insulin resistance, even if not diabetic. Richard

Veech has suggested this hypoglycemic effect is the result of ketones activating pyruvate

dehydrogenase (PDH), which enhances insulin-mediated glucose uptake and the production of

acetyl-CoA. In addition, the body regulates ketone production via ketonuria and ketone-induced

insulin release, which shuts off hepatic ketogenesis. The insulin from this process may increase

glucose disposal which, when coupled with PDH activation, could drive down blood glucose

levels. The administration of 5 mM ketone has been shown to increase acetyl-CoA production

16-fold in the glucose-perfused isolated rat heart. Additionally, in this model, ketones and insulin

increased cardiac hydraulic efficiency to a similar degree, approximately 25-35% [124].

Male rats were fed a standard diet with 30% of calories replaced with the R-3-

hydroxybutyrate-R-1,3-butanediol monoester for 14 days. The ketone ester-supplemented diet

induced nutritional ketosis (3.5mM βHB), and both plasma glucose and insulin were decreased

by approximately 50% [132]. Glucose was decreased from 5 mM to 2.8 mM, and insulin was

decreased from 0.54 ng/mL to 0.26 ng/mL. In a similar study by the same group, mice receiving

a KE diet exhibited a 73% increase in the Quantitative Insulin-Sensitivity Check Index

(QUICKI), a surrogate marker of insulin sensitivity, compared to control, calorie-matched mice

[132]. Fasting plasma glucose levels were not altered in these mice, but fasting plasma insulin

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levels were reduced by approximately 85% in the KE-fed mice compared to controls,

demonstrating that exogenous ketones enhance insulin sensitivity [152].

The histone deacetylase inhibitor (HDACI) activity of βHB could also be beneficial in

T2D by altering the direct regulation of HDAC-dependent glucose metabolism and by inducing

resistance to oxidative stress. HDACs regulate the expression of genes encoding many metabolic

enzymes, and HDAC3 knockout animals exhibit reduced glucose and insulin. SAHA, a class I

HDAC inhibitor, has been shown to improve insulin sensitivity and increase oxidative

metabolism and metabolic rate in a mouse model of diabetes [153]. Butyrate, a short-chain fatty

acid that is structurally similar to β-hydroxybutyrate and also acts as a HDACI lowers blood

glucose and insulin levels and improves glucose tolerance and respiratory efficiency [154]. The

vascular dysfunction in T2D is thought to be caused by oxidative stress [155]. HDAC inhibition

prevents renal damage in mouse models of diabetic nephropathy through modulation of redox

mechanisms [156]. Therefore, βHB suppression of oxidative stress through HDAC inhibition

may help restore insulin sensitivity and manage complications of diabetes.

2.6. Ischemic Wound Healing Model

As mentioned previously, a chronic wound is by definition one that entirely fails to heal.

In this respect, there is no standardized animal model that reflects the human chronic wound

environment. However, animal models have been developed that attempt to mimic these

conditions for the purpose of furthering our understanding of the complexity of chronic wounds.

The rat species, often employed due to its wide availability, size and docile nature is used for

wound healing studies as it is large enough to provide a suitable skin area for incisional and

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excisional wounding, imaging and tissue collection [157]. Yet, it should be noted that the skin of

a rat and a human are different morphologically, as rats are loose-skinned animals. This distinct

anatomical characteristic enhances wound contraction over re-epithelialization in rat integument

[157]. Additionally, the presence of a subcutaneous panniculus carnosus muscle in rats,

contributes to healing by both contraction and collagen formation [158, 159]. These very

important morphological distinctions were considered in the optimization of the following rat

ischemic skin wound model used for this dissertation. Additionally, specific modifications were

implemented to decrease wound contraction and reduce the influence of the panniculus carnosus

muscle [160].

In the rat ischemic wound model, a dorsal bi-pedicle 10.5 x 3 cm flap is surgically

created on day 0. A silicone sheet is placed under the panniculus carnosus fascia and above the

paraspinous muscles, limiting revascularization from the underlying tissue, which increases the

duration of flap ischemia. Two 6mm punches are created within the flap, extending just above

the fascia, creating ischemic wounds. Additionally, two 6 mm punches are created lateral to the

flap, where there isn’t silicone sheet, providing control non-ischemic wounds within the same

animal.

One of the main factors in the development of a chronic wound is localized tissue

ischemia (reduced blood flow) contributing to the inability to clear inflammation [160]. In 2005,

Dr. Lisa Gould developed and validated this model as a modification of the ischemic wound

model originally described by Schwartz et al. and subsequently used in modified form by Chen

et al. [161, 162]. The wound model was modified to test the induction of angiogenesis in the

wound bed.

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Wound healing in rats has often been the subject of debate due to their ability to heal

infected wounds and high rate of inter-animal variability [160]. One of the original goals of the

model during its development was to decrease this variation. Modifications to the width of the

flap, reducing the number of wounds with specific placement (centered on the flap with

consistent cranio-caudal location) and introduction of a silicone sheet has accomplished this

goal. Wound healing by contraction has also been reduced and healing by epithelialization, as in

humans, is the measured outcome. Adaptation of the model to a different strain of rat, ie the

F344, has also proven successful and reproduces the degree of ischemia observed using Sprague

Dawley rats.

To achieve consistency with this model while performing multiple surgeries, it was found

that it is important to create the ischemic wounds prior to elevation of the flap for silicone sheet

placement [160]. Additionally, not punching through the panniculus carnosus fascia is critical to

provide a viable wound bed to remain over the silicone. The silicone acts not only to prevent

vascular regrowth but also as a “splint” that reduces wound contraction. The application of the

adhesive and dressings to prevent infection and maintain a moist environment for wound healing

is also important. Product choice can be what is preferred or used in the researcher’s animal

facility. However, it is not uncommon for some of the animals to be able to remove their

dressings, no matter what type of adhesive/dressing combination is used.

The bi-pedicled flap should remain viable throughout a time course of healing which is

approximately 28 days, depending on rat strain and other co-morbidities present. Rarely,

abscesses can form in the flap (particularly near sutures) and seromas may form under the flap.

Fluid can be drained and antibiotics administered if necessary. However, if the flap loses

viability and becomes necrotic it is recommended that that animal no longer be used. Wound

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excision for biochemical analysis does introduce variability due to (1) some normal tissue must

be retained for support (2) the choice of tissue homogenization and preparation for isolation of

RNA, DNA or protein and (3) inherent inter-animal variability [127, 160, 163]. One could

consider this last point a limitation to the model. It was found that reducing the size of the flap

(<2.0 cm) or flap trauma can cause necrosis, indicating that minor variations in technical or

environmental factors such as temperature or stress levels, may also lead to biochemically

detectable variation between wound samples from one rat to another [160].

In summary, this model, with a longitudinal, bi-pedicle flap ranging from 2.0-3.0 cm in

width and a strategically placed silicone sheet, is a reliable model of prolonged tissue ischemia.

Once the user is adept at using the techniques to create a consistent ischemic wound, they should

be able to adapt it to additional ages and species of rodents (mice included). The excisional

wounds can be treated topically, or systemic treatments utilized to further explore the

mechanism(s) involved in chronic wound formation, exacerbated inflammatory responses,

aberrant angiogenesis and delayed wound closure.

2.7. Use of Primary Human Dermal Fibroblasts in vitro

Though immortal cells lines are more abundant and more cost efficient to maintain, in this

dissertation, we utilized primary human dermal fibroblasts isolated from discarded skin.

Compared to immortal cell lines, primary cell lines maintain a physiology that is more closely

identified to in vivo physiology. The caveat is that they must be used at early passage, generally

passage 2-8. Additionally, due to the heterogeneity of patients, the in vitro work was repeated in

at least two different patient-derived cell lines thereby reducing individual confounding. Also,

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fibroblasts behave differently depending on the location from which they are isolated; therefore,

skin for these studies was collected from similar anatomic locations. The complex extracellular

environment, paracrine, and autocrine interactions and range of cell types involved in wound

repair limits in vitro models to confirm what we see in in vivo models. Additionally, since there

is such a gamut of players and interactions, by focusing on just one cell type we may miss some

of the potential mechanism for our therapy.

2.8. Central Hypothesis and Project Goals

There is a dire need to discover novel and effective treatments for chronic wounds. There

are often multiple mitigating factors that prevent normal wound closure leading to minimally

effective wound therapies as few multifactorial treatments are available.

Exogenous ketone supplements are being developed as an alternative or adjuvant method

of inducing therapeutic ketosis aside from the classic ketogenic diet. It seems clear that these

novel compounds have the potential to offer benefits for both healthy and diseased individuals

alike. It is likely that most, if not all, of the conditions which are known to benefit from the KD

would receive some benefit from exogenous ketone supplementation. Importantly, ketone

supplementation provides a tool for achieving therapeutic ketosis in patients who are unable,

unwilling, or uninterested in consuming a low carbohydrate or ketogenic diet. It may also help

circumvent some of the difficulties associated with KD therapy, as it allows for rapid induction

of ketosis in a dose-dependent fashion, which can be sustained with prolonged consumption.

Simultaneously, it could provide patients with the opportunity to reap the benefits of ketosis

without the practical and social difficulties of a highly restrictive diet.

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Therefore, we hypothesize that exogenous ketone supplementation will improve metabolic

and physiological attributes to promote accelerated healing in the impaired wound environment

associated with aging.. To date, the majority of ketone-based metabolic enhancement strategies

have focused on enhancing brain and heart metabolism; thus, this dissertation proposes a novel

therapy, which will be evaluated in a cutaneous wound-healing model [164, 165]. Although this

dissertation focuses on the underlying features of the aging phenotype, similar molecular deficits

have been described in diabetes and other disorders [166], suggesting that administration of

ketones as an alternative fuel may have broader applications. The studies completed in this

dissertation provide an important step in the potential discovery of effective and novel treatments

for chronic wounds.

2.9. Closing Remarks

In this chapter I have provided scientific rationale for the potential utility of oral ketone

supplementation against age impaired wound healing. Approaching the healing wound from a

metabolic perspective provides an innovative opportunity to stimulate beneficial physiological

and cellular mechanisms that may be lacking or attenuated in the aged population. Chapters 3-5

will consist of the results and a detailed discussion of this dissertation work. A summary of the

findings, closing remarks, and major implications of this data will be discussed in Chapter 6.

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22. Dashti HM, Al-Zaid NS, Mathew TC, Al-Mousawi M, Talib H, Asfar SK, Behbahani AI: Long term effects of ketogenic diet in obese subjects with high cholesterol level. Molecular and cellular biochemistry 2006, 286(1-2):1-9.

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CHAPTER 3: ESTABLISHING THERAPEUTIC KETOSIS (METABOLIC THERAPY)

WITH EXOGENOUS KETONE SUPPLEMENTATION

3.1. Chapter Synopsis

In this chapter, we present data demonstrating the effects of five oral ketone supplements

on blood ketones, glucose, triglyceride, and lipoprotein levels in Sprague-Dawley rats. Though

each ketogenic precursor had a distinctive effect on each of the parameters, overall oral ketone

supplementation rapidly elevated blood ketone levels and suppressed blood glucose levels

without significantly affecting heart health biomarkers. Additionally we sent serum and

hippocampal samples from KE (5 g/kg) and BMS+MCT (10 g/kg) supplemented rats to

Metabolon Inc. to determine the effects that oral ketone supplementation would have on the

metabolome. Ketone supplements increased Kreb’s cycle intermediates, antioxidants, and

adenosine, which supports our hypothesis that oral ketone supplementation, will enhance wound

healing.

Portions of this chapter have previously been published in “Kesl SL, Poff AM, Ward NP,

Fiorelli TN, Ari C, Van Putten AJ, Sherwood JW, Arnold P, D’Agostino DP. Effects of

Exogenous Ketone Supplementation on Blood Ketone, Glucose, Triglyceride, and Lipoprotein

Levels in Sprague-Dawley Rats. Nutrition and Metabolism (2016)”. A copy of this article may

be found in Appendix C. See Appendix B for copyright permissions. Materials and methods

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presented in this chapter can be found in Appendix A.

3.2. Effects of Exogenous Ketone Supplementation on Blood Ketones, Glucose,

Triglyceride, and Lipoprotein Levels in Sprague-Dawley Rats

Nutritional ketosis induced with the KD has proven effective for the metabolic

management of seizures and potentially other disorders [1-26]. We hypothesized that oral

administration of ketone supplements could safely produce sustained nutritional ketosis (>0.5

mM) without carbohydrate restriction. Thus, we tested the effects of 28-day administration of

five ketone supplements on blood glucose, ketones, and lipids in male Sprague-Dawley rats. The

supplements included: 1,3-butanediol (BD), a sodium/potassium β-hydroxybutyrate (βHB)

mineral salt (BMS), medium chain triglyceride oil (MCT), BMS+MCT 1:1 mixture, and 1,3

butanediol acetoacetate diester (KE). Rats received a daily 5-10g/kg dose of their respective

ketone supplement via intragastric gavage. Weekly whole blood samples were taken for analysis

of glucose and βHB at baseline and 0.5, 1, 4, 8, and 12 hrs post-gavage, or until βHB returned to

baseline. In this section, we present evidence that chronic administration of ketone supplements

can induce a state of nutritional ketosis without the need for dietary carbohydrate restriction with

little or no effect on lipid biomarkers. The notion that we can produce the therapeutic effects of

the KD with exogenous ketone supplementation is supported by our previous study which

demonstrated that acutely administered KE supplementation delays central nervous system

(CNS) oxygen toxicity seizures without the need for dietary restriction [27]. We propose that

exogenous ketone supplementation could provide an alternative method of attaining the

therapeutic benefits of nutritional ketosis, and as a means to further augment the therapeutic

potential of the KD.

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3.2.1 Ketone supplementation causes little to no change in triglycerides and lipoproteins

One common concern regarding the KD is its purported potential to increase the risk of

atherosclerosis by elevating blood cholesterol and triglyceride levels [28, 29]. This topic

remains controversial as some, but not all studies have demonstrated that the KD elevates blood

levels of cholesterol and triglycerides [30-35]. Kwitervich and colleagues demonstrated an

increase in low-density lipoprotein (LDL) and a decrease in high-density lipoprotein (HDL) in

epileptic children fed the classical KD for two years [36]. In that study, total cholesterol

increased by ~130%, and stabilized at the elevated level over the 2-year period. A similar study

demonstrated that the lipid profile returned to baseline in children who remained on the KD for

six years [37]. Children typically remain on the diet for approximately two years then return to a

diet of common fat and carbohydrate ingestion [38]. The implications of these findings are

unclear, since the influence of cholesterol on cardiovascular health is controversial and

macronutrient sources of the diet vary per study. In contrast, more recent studies suggest that the

KD can actually lead to significant benefits in biomarkers of metabolic health, including blood

lipid profiles [39-46]. In these studies, the KD positively altered blood lipids, decreasing total

triglycerides and cholesterol while increasing the ratio of HDL to LDL [42-51]. Although, the

KD is well established in children, it has only recently been utilized as a strategy to control

seizures in adults. In 2014, Schoeler and colleagues reported on the feasibility of the KD for

adults, concluding that 39% of individuals achieved > 50% reduction in seizure frequency,

similar to the results reported in pediatric studies. Patients experienced similar gastrointestinal

adverse events to those previously described in pediatric patients, but they did not lead to

discontinuation of the diet in any patient [52].

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With oral ketone supplementation, we observed a significant elevation in blood βHB

without dietary restriction and with little change in lipid biomarkers (Figure 3.1). Over the 4

week study, MCT-supplemented rats demonstrated decreased HDL compared to controls. No

significant changes were observed in any of the triglycerides or lipoproteins (HDL, LDL) with

any of the remaining exogenously applied ketone supplements. It should be noted that the rats

used for this study had not yet reached full adult body size [53]. Their normal growth rate and

maturation was likely responsible for the changes in triglyceride and lipoprotein levels observed

in the control animals over the 4 week study (baseline data not shown, no significant differences)

[54, 55]. Future studies are needed to investigate the effect of ketone supplementation on fully

mature and aged animals. Overall, our study suggests that oral ketone supplementation has little

effect on the triglyceride or lipoprotein profile after 4 weeks. However, it is currently unknown if

ketone supplementation would affect lipid biomarkers after a longer duration of consumption.

Further studies are needed to determine the effects of ketone supplements on blood triglyceride

and lipoproteins after chronic administration and as a means to further enhance the

hyperketonemia and improve the lipid profile of the clinically implemented (4:1) KD.

LDL is the lipoprotein particle that is most often associated with atherosclerosis. LDL

particles exist in different sizes: large molecules (Pattern A) or small molecules (Pattern

B). Recent studies have investigated the importance of LDL-particle type and size rather than

total concentration as being the source for cardiovascular risk [29]. Patients whose LDL particles

are predominantly small and dense (Pattern B) have a greater risk of cardiovascular disease

(CVD). It is thought that small, dense LDL particles are more able to penetrate the endothelium

and cause in damage and inflammation [56-59]. Volek et al. reported that the KD increased the

pattern and volume of LDL particles, which is considered to reduce cardiovascular risk [47].

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Though we did not show a significant effect on LDL levels for ketone supplements, future

chronic feeding studies will investigate the effects of ketone supplementation on lipidomic

profile and LDL particle type and size.

3.2.2. Therapeutic levels of hyperketonemia suppress blood glucose levels

We demonstrated that therapeutic ketosis could be induced without dietary (calorie or

carbohydrate) restriction and that this acute elevation in blood ketones was significantly

correlated with a reduction in blood glucose (Figure 3.2-3.4). The BMS ketone supplement did

not significantly induce blood hyperketonemia or reduce glucose in the rats. The KE

supplemented rats trended towards reduced glucose levels; however, the lower dose of this agent

did not lower glucose significantly, as reported previously in acute response of mice (60). MCTs

have previously been shown to elicit a slight hypoglycemic effect by enhancing glucose

utilization in both diabetic and non-diabetic patients [60-62]. Kashiwaya et al. demonstrated that

both blood glucose and blood insulin decreased by approximately 50% in rats fed a diet where

30% of calories from starch were replaced with ketone esters for 14 days, suggesting that ketone

supplementation increases insulin sensitivity or reduces hepatic glucose output [63]. This

ketone-induced hypoglycemic effect has been previously reported in humans with IV infusions

of ketone bodies [64, 65]. Recently, Mikkelsen et al. showed that a small increase in βHB

concentration decreases glucose production by 14% in post-absorptive healthy males [66].

However, this has not been previously reported with any of the oral exogenous ketone

supplements we studied. Ketones are an efficient and sufficient energy substrate for the brain,

and will therefore prevent side effects of hypoglycemia when blood levels are elevated and the

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patient is keto-adapted. Owen et al. most famously demonstrated this in 1967 wherein keto-

adapted patients (starvation induced therapeutic ketosis) were given 20 IU of insulin. The blood

glucose of fasted patients dropped to 1-2mM, but they exhibited no hypoglycemic symptoms due

to brain utilization of ketones for energy [67]. Therefore, ketones maintain brain metabolism and

are neuroprotective during severe hypoglycemia. The rats in the MCT group had a correlation of

blood ketone and glucose levels at week 4, whereas the combination of BMS+MCT produced a

significant hypoglycemic correlation both at baseline and at week 4. No hypoglycemic

symptoms were observed in the rats during this study. Insulin levels were not measured in this

study; however, future ketone supplementation studies should measure the effects of exogenous

ketones on insulin sensitivity with a glucose tolerance test. An increase in insulin sensitivity in

combination with our observed hypoglycemic effect has potential therapy implications for

glycemic control in T2D [68]. Furthermore, it should be noted that the KE metabolizes to both

AcAc and βHB in 1:1 ratio [27]. The ketone monitor used in this study only measures βHB as

levels of AcAc are more difficult to measure due to spontaneous decarboxylation to acetone;

therefore, the total ketone levels (βHB +AcAc) measured were likely higher, specifically for the

KE (14). Interestingly, the 10 g/kg dose produced a delayed blood βHB peak for ketone

supplements MCT and BMS+MCT. The higher dose of the ketogenic supplements elevated

blood levels more substantially, and thus reached their maximum blood concentration later due

to prolonged metabolic clearance. It must be noted that the dosage used in this study does not

translate to human patients, since the metabolic rate of rats is considerably higher. Future studies

will be needed to determine optimal dosing for human patients.

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3.2.3. Effects of ketone supplementation on organ weight and body weight percentage

Ketone supplementation did not affect the size of the brain, lungs, kidneys or heart of

rats. As previously mentioned, the rats were still growing during the experimental time frame;

therefore, organ weights were normalized to body weight to determine if organ weight changed

independently to growth. There could be several reasons why ketones influenced liver and spleen

weight. The ratio of liver to body weight was significantly higher in the MCT supplemented

animals (Figure 3.5). MCTs are readily absorbed in the intestinal lumen and transported directly

to the liver via hepatic portal circulation. When given a large bolus, such as in this study, the

amount of MCTs in the liver will likely exceed the β-oxidation rate, causing the MCTs to be

deposited in the liver as fat droplets [69]. The accumulated MCT droplets in the liver could

explain the higher liver weight to body weight percentage observed with MCT supplemented

rats. Future toxicology and histological studies will be needed to determine the cause of the

observed hepatomegaly. It should be emphasized that the dose in this study is not optimized in

humans. We speculate that an optimized human dose would be lower due to the higher metabolic

rate of the rats and may not cause hepatomegaly or potential fat accumulation. Nutritional ketosis

achieved with the KD has been shown to decrease inflammatory markers such as TNF-α, IL-6,

IL-8, MCP-1, E-selectin, I-CAM, and PAI-1 [8, 70], which may account for the observed

decrease in spleen weight. As previously mentioned, Veech and colleagues demonstrated that

exogenous supplementation of 5mM βHB resulted in a 28% increase in hydraulic work in the

working perfused rat heart and a significant decrease in oxygen consumption [71-73]. Ketone

bodies have been shown to increase cerebral blood flow and perfusion [74]. Also, ketone bodies

have been shown to increase ATP synthesis and enhance the efficiency of ATP production [14,

68, 73]. It is possible that sustained ketosis results in enhanced cardiac efficiency and O2

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consumption. Even though the size of the heart did not change for any of the ketone

supplements, further analysis of tissues harvested from the ketone-supplemented rats will be

needed to determine any morphological changes and to understand changes in organ size. It

should be noted that the Harlan standard rodent chow 2018 is nutritionally complete and

formulated with high-quality ingredients to optimize gestation, lactation, growth, and overall

health of the animals. The same cannot be said for the standard American diet (SAD). Therefore,

we plan to investigate the effects of ketone supplements administered with the SAD to determine

if similar effects will be seen when the micronutrient deficiencies and macronutrient profile

mimics what most Americans consume.

MCT oil has recently been used to induce nutritional ketosis although it produces dose-

dependent gastrointestinal (GI) side effects in humans that limit the potential for its use to

significantly elevate ketones (>0.5 mM). Despite these limitations, Azzam and colleagues

published a case report in which a 43-year-old-man had a significant decrease in seizure

frequency after supplementing his diet with 4 tablespoons of MCT oil twice daily [75]. An

attempt to increase his dosage to 5 tablespoons twice daily was halted by severe GI intolerance.

Henderson et al. observed that 20% of patients reported GI side effects with a 20g dose of

ketogenic agent AC-1202 in a double blind trial in mild to moderate Alzheimer’s patients [76].

We visually observed similar gastrointestinal side effects (loose stools) in the rats treated with

MCT oil in our study. Rats were closely monitored to avoid dehydration, and gastric motility

returned to normal between 12-24 hrs. Interestingly, the BMS+MCT supplement elevated βHB

similarly to MCT oil alone, without causing the adverse gastrointestinal effects seen in MCT-

supplemented rats. However, this could be due to the fact in a 10 g/kg dose of BMS+MCT, only

5 g/kg is MCT alone, which is less than the 10 g/kg dose that elicits the GI side effects. This

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suggests that this novel combination may provide a more useful therapeutic option than MCT oil

alone, which is limited in its ability to elevate ketones in humans.

Exogenously delivered ketone supplements significantly altered rat weight gain for the

duration of the study (Figure 3.6). However, rats did not lose weight and maintained a healthy

range for their age. Rats have been shown to effectively balance their caloric intake to prevent weight

loss/gain [77-79]. Due to the caloric density of the exogenous ketone supplements (Table 3.1) it is

possible for the rats to eat less of the standard rodent chow and therefore fewer carbohydrates while

maintaining their caloric intake. Food intake was not measured for this study. However, if there

was a significant carbohydrate restriction there would be a significant change in basal blood

ketone and blood glucose levels. As the hallmark to the KD, carbohydrate restriction increases

blood ketone levels and reduces blood glucose levels. Neither an increase in basal blood ketone

levels nor a decrease in basal blood glucose levels was observed in this study (Figure 3.7).

Additionally, if there were an overall blood glucose decrease due to a change in food intake, this

would not explain the rapid reduction (within 30 minutes) in blood glucose correlated with an

elevation of blood ketone levels after an intragastric bolus of ketone supplement (Figures 3.2-

3.4).

Several studies have investigated the safety and efficacy of ketone supplements for disease

states such as AD and Parkinson’s disease, and well as for parenteral nutrition [68, 80-86]. Our

research demonstrates that several forms of dietary ketone supplementation can effectively

elevate blood ketone levels and achieve therapeutic nutritional ketosis without the need for

dietary carbohydrate restriction. We also demonstrated that ketosis achieved with exogenous

ketone supplementation can reduce blood glucose, and this is inversely associated with the blood

ketone levels. Although preliminary results are encouraging, further studies are needed to

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determine if oral ketone supplementation can produce the same therapeutic benefits as the classic

KD in the broad-spectrum of disease states that are currently under investigation. Ketone

supplementation could be used as an alternative method for inducing ketosis in patients who

have previously had difficulty implementing the KD because of palatability issues, gall bladder

removal, liver abnormalities, or intolerance to fat. Additional experiments should be conducted

to see if ketone supplementation could be used in conjunction with the KD to assist and ease the

transition to nutritional ketosis and enhance the speed of keto-adaptation. In this study we have

demonstrated the ability of several ketone supplements to elevate blood ketone levels, providing

multiple options to induce therapeutic ketosis based on patient need. Though additional studies

are needed to determine the therapeutic potential of ketone supplementation, many patients that

previously were unable to benefit from the KD may now have an alternate method of achieving

therapeutic ketosis. Ketone supplementation may also represent a means to further augment

ketonemia in those responsive to therapeutic ketosis, especially in those individuals where

maintaining low glucose is important.

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Figure 3.1 Effects of ketone supplementation on triglycerides and lipoproteins: Ketone supplementation causes little change in triglycerides and lipoproteins over a 4-week study. Graphs show concentrations at 4-weeks of total cholesterol (A), Triglycerides (B), HDL (C), and LDL (D). MCT supplemented rats had significantly reduced concentration of HDL blood levels compared to control (p<0.001)(B). One-Way ANOVA with Tukey’s post hoc test, results considered significant if p<0.05. Error bars represent mean (SD).

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Figure 3.2 Effects of ketone supplementation on blood βHB. (A, B) Blood βHB levels at times 0, 0.5, 1, 4, 8, and 12 hours post intragastric gavage for ketone supplements tested. (A) BMS+MCT and MCT supplementation rapidly elevated and sustained significant βHB elevation compared to controls for the duration of the 4-week dose escalation study. BMS did not significantly elevate βHB at any time point tested compared to controls. (B) BD and KE supplements, maintained at 5 g/kg, significantly elevated βHB levels for the duration of the 4-week study. Two-Way ANOVA with Tukey's post hoc test, results considered significant if p<0.05. Error bars represent mean (SD).

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Figure 3.3 Effects of ketone supplementation on blood glucose. (A, B) Blood glucose levels at times 0, 0.5, 1, 4, 8, and 12 hours (for 10 dose) post intragastric gavage for ketone supplements tested. (A) Ketone supplements BMS+MCT and MCT significantly reduced blood glucose levels compared to controls for the duration of the 4-week study. BMS significantly lowered blood glucose only at 8 hrs/week 1 and 12hrs/week (B) KE, maintained at 5 g/kg, significantly reduced blood glucose compared to controls from week 1-4. BD did not significantly affect blood glucose levels at any time point during the 4-week study. Two-Way ANOVA with Tukey's post hoc test, results considered significant if p<0.05. Error bars represent mean (SD)

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Figure 3.4 Relationship between blood ketone and glucose levels: (A) BMS+MCT (5 g/kg) supplemented rats demonstrated a significant inverse relationship between elevated blood ketone levels and decreased blood ketone levels (r2=0.4314, p=0.0203). (B) At week 4, BMS+MCT (10 g/kg) and MCT (10 g/kg) showed a significant correlation between blood ketone levels and blood glucose levels (r2=0.8619, p<0.0001; r2=0.6365, p=0.0057). Linear regression analysis, results considered significant if p<0.05.

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Figure 3.5 Effects of ketone supplementation on organ weight Data is represented as a percentage of organ weight to body weight. (A, B, D, F) Ketone supplements did not significantly affect the weight of the brain, lungs, kidneys or heart. (C) Liver weight was significantly increased as compared to body weight in response to administered MCT ketone supplement compared to control at the end of the study (day 29) (p<0.001). (E) Rats supplemented with BMS+MCT, MC and BD had significantly smaller spleen percentage as compared to controls (p<0.05, p<0.001, p<0.05). Two-Way ANOVA with Tukey's post-hoc test; results considered significant if p<0.05. Error bars represent mean (SD).

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Figure 3.6 Effects of ketone supplementation on body weight: Rats administered ketone supplements gained less weight over the 4-week period; however, did not lose weight and maintained healthy range for age. KE supplemented rats gained significantly less weight during the entire 4-week study compared to controls. BMS+MCT, BMS, and BD supplemented rats gained significantly less weight than controls over weeks 2-4.MCT supplemented rats gained significantly less weight than controls over weeks 3-4. Two-Way ANOVA with Tukey's post hoc test, results considered significant if p<0.05. Error bars represent mean (SD).

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Figure 3.7 Effects of ketone supplementation on basal blood ketone and basal blood glucose levels: Rats administered ketone supplements did not have a significant change in basal blood ketone levels (A) or basal blood glucose levels (B) for the four week study. Two-Way ANOVA with Tukey’s post-hoc test, results considered significant if p<0.05. Error bars represent mean (SD)

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3.3. Effect of Sustaining Dietary Ketosis Through Exogenous Ketone Supplementation on

the Serum and Hippocampal Metabolome of Sprague-Dawley rats

In the previous section, we reported that oral administration of ketone supplements

produced nutritional ketosis (>0.5 mM) without carbohydrate restriction and the effects of a 28-

day administration of five ketone supplements on blood glucose, ketones, and lipids in male

Sprague-Dawley rats. We hypothesized that the 28-day administration would affect metabolomic

markers. Serum (~300 µL) and hippocampal tissues for ketone supplements KE and BMS+MCT

were collected on day 28 at 4 hours post-intragastric gavage (peak ketone elevation). Metabolon

analyzed samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas

chromatography-mass spectrometry (GC-MS). Both ketone supplements significantly increased

serum Krebs cycle intermediates: citrate, fumarate and malate. KE supplement significantly

increased alpha-ketoglutarate and BMS+MCT supplement significantly increased succinate in

the rat serum. Additionally, both supplements affected medium chain fatty acids, antioxidants,

and adenosine in both serum and hippocampal tissue. Although both forms of ketone

supplementation increased brain and blood ketone levels, the global metabolic profiles were

different.

3.3.1 Oral administration of ketone supplements elevates blood ketone levels, increases

Krebs cycle intermediates, MCFAs, antioxidants, and adenosine in serum and hippocampal

tissue and implications for wound healing

Global metabolomic profiling of serum from KE and BMS+MCT supplemented rats

exhibited a total of 142 out of 388 metabolites that were significantly changed from control for

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KE-treated rats and 119 out of 388 metabolites were significantly altered compared to control for

BMS+MCT treated rats. Additionally, 12 of 290 metabolites were significantly changed in the

hippocampal tissue for KE-treated rats and 36 of 290 metabolites were significantly altered for

BMS+MCT treated rats (Table 3.1). These metabolic changes provide preliminary insight to

possible mechanisms by which ketones could enhance wound healing as well as potential

mechanisms to target other disease states.

Metabolomics analysis confirmed elevated ketone bodies βHB as well as AcAc in the

serum and elevated βHB in the hippocampus (Figure 3.8). βHB is a more stable molecule since

it cannot be decarboxylated to acetone; therefore, it is the predominant ketone body within the

blood stream and likely a reason only βHB was elevated in the hippocampus. It should be noted

that KE metabolizes to both βHB and AcAc as compared to BMS+MCT that only metabolizes to

βHB. However, both supplements elevated AcAc, KE showed a 9-fold increase and

interestingly, BMS+MCT demonstrated a 15-fold increase. It should be noted that KE was at a 5

g/kg dose and BMS+MCT was given at a 10 g/kg dose. Both ketone supplements demonstrated a

2:1 ratio of βHB: AcAc (KE 15.78:8.84, BMS+MCT 35.33:15.39) in the serum. Thus, total

blood ketones seen in our previous study were likely higher in the KE-treated and BMS+MCT-

treated animals. The commercially available blood ketone meters used in previous study offer the

most cost feasible method of measuring blood ketones, but are only able to measure βHB, not

AcAc. Additionally, BMS+MCT treated animals had significantly lower serum glucose (0.87,

p<0.05). This observation supports the hypoglycemic effect demonstrated in the previous study

as well as previous studies by Veech and colleagues [87, 88]. As discussed, this hypoglycemic

effect has been attributed to the enhancement insulin sensitivity, which may help diabetic foot

ulcers specifically. As discussed, MCFAs are one of the main components of MCT oil namely

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caprylic acid (C8:0) and Capric acid (10:0); these medium chain fatty acids were significantly

elevated in the serum and hippocampal tissue of BMS+MCT supplemented rats (Figure 3.9).

This demonstrates that the MCT oil is being metabolized into its MCFAs, which are available in

the blood stream and tissues to be used for energy. Interestingly, KE supplemented rats also had

significantly elevated capric acid in the serum and hippocampal tissue.

Krebs cycle intermediates citrate, fumarate and malate were significantly elevated in the

serum of both KE-treated and BMS+MCT-treated animals (Figure 3.10). Additionally, KE

significantly elevated the intermediate α-ketoglutarate and BMS+MCT significantly elevated

succinate. These elevations were not seen in the hippocampal tissue. As discussed, non-wounded

skin predominantly uses anaerobic respiration with only about 30% going to the Krebs cycle; this

is exaggerated during wound healing when hypoxia due to tissue damage shifts the metabolic

profile to only using 4% Krebs cycle [89-92]. However, ketones have been shown to increase

blood flow; thus, increased blood flow would lead to potential increased oxygenation and the

restoration of the non-wounded skin metabolic profile. Even with a 30% Krebs cycle flux, an

increase in Acetyl CoA via ketolysis would produce a net increase in ATP production. Indeed,

Veech and colleagues demonstrated a 16-fold increase in acetyl CoA with a ketone ester

supplementation [88]. Acetyl co-A was significantly elevated via BMS+MCT supplementations

but not KE in the hippocampal tissue, supporting this finding. Levels of pyruvate and lactate

were unchanged in both ketone supplements in both the serum and hippocampal tissue

demonstrating that there wasn’t an increased flux of glycolysis and anaerobic respiration. Even

though there initially wouldn’t be sufficient oxygen for the wound bed to use the intermediates in

the ETC, Krebs cycle intermediates have other anabolic fates that would be important to wound

healing. Citrate is a precursor to fatty acid synthesis; α-ketoglutarate has been shown to scavenge

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H2O2 in cell cultures and is an amino acid synthesis precursor [93]. Krebs cycle intermediates

were shown to be decreased in aged rats; this supplementation may restore these levels to those

of a young person or at least elevate them [94]. Significant amino acid synthesis is supported by

significant serum elevations in the amino acids glycine and serine. Additionally, KE-treated rats

had a significant increase in PPP intermediates ribose-5-phosphate and ribulose/xylulose-5-

phosphate demonstrating an increase flux through the pathway and potentially nucleic acid

synthesis needed for wound healing. From this data, we can speculate that if the ketones were

being used as energy a higher portion of the glucose can be shunted to the PPP instead of being

metabolized for energy.

Surprisingly, oxidative stress (oxidized glutathione, GSSG (serum)) and antioxidant

capacity (reduced glutathione (hippo), carnosine (serum), and anserine (serum)) were elevated,

suggesting a heightened state of oxidative stress and antioxidant defense with ketone

supplementation. As discussed by Moor and colleagues, age alone affects the levels of SOD2 and

glutathione antioxidant profile [95]. Without appropriate oxidative stress, neutrophil respiratory

bursts cannot effectively clear bacteria from the wound bed; however, without appropriate

antioxidant capacity, the reactive oxygen species become damaging to neighboring tissue. This

ketogenic elevation of both oxidative stress and antioxidant defense may help clear bacteria as

well as quench ROS to promote wound healing in an aged phenotype. Additionally, carnosine (β-

alanyl-L-histidine) and anserine (β-alanyl-N-methylhistidine) were elevated by KE and

BMS+MCT supplementation (Figure 3.11). Carnosine is a natural dipeptide widely and

abundantly distributed in excitable tissue. Although its physiologic role is not completely

understood, many beneficial actions have been attributed to carnosine, such as being an

antioxidant, antiglycating and ion-chelating agent, and a free-radical scavenger. Several studies

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have shown that supplementation with carnosine accelerates wound healing [96-100]. Anserine

is a dipeptide containing beta alanine and histidine and has been shown to exhibit equal

antioxidant activity to carnosine by reducing the primary molecular products of lipid

peroxidation [101-103]. A study by Altavilla et al. demonstrated that reduction of lipid

peroxidation restores impaired VEGF expression leading to accelerated wound healing and

angiogenesis in a diabetic wound model [104]. This supports a possible mechanism of action for

ketone supplementation to augment wound healing. However, further experiments need to be

performed to optimize dose as too high of an oxidative state, which can be seen in the KE

supplementation can potentially be damaging, especially in an aged patient.

Commonly known for its role in energy transfer via phosphorylation, adenosine also

plays a role in the regulation of blood flow as a potent vasodilator. Adenosine was significantly

elevated in the serum via KE supplementation and trended towards significance for BMS+MCT;

however, BMS+MCT supplemented rats showed a significant elevation of adenosine in their

hippocampal tissue (KE not significant). Even though BMS+MCT did not show a significant

elevation in the blood stream, this suggests that the BMS+MCT supplemented rat tissues were

more efficient at using it; therefore a larger portion had already been sequestered in the

hippocampal tissue. High levels of AMP could reflect a metabolic insufficiency for the KE

supplement, which may be due to the high dose; further studies optimizing the dose of ketone

supplements are needed. Masino and colleagues have demonstrated that increased adenosine of

the A1 subtype plays a key role in the anticonvulsant success of ketogenic strategies [105-108].

Additionally, several studies have shown that topical adenosine application accelerates wound

healing via adenosine A2A receptors and promoting angiogenesis [109-117].

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A recent study by Sood et al, was the first to document a global metabolomic profile of

diabetic and non-diabetic wounds, 7 days post injury. In non-diabetic mice, 88 of the 129

detected metabolites had a significant response to injury, 85 up regulated and 3 down-regulated.

In diabetic wounds, 81 metabolites had a significant response to injury with 76 up regulated and

5 down regulated. Interestingly, they found 62 unique metabolites that differed between the non-

diabetic and diabetic wound phenotype [118]. From their study, glycine was dysregulated in

diabetic wounds and both ketone supplements significantly increased glycine in the serum (1.3,

1.27 fold change). A recent metabolic profiling of cancer cells has correlated glycine with

increased cell proliferation; however, it remains to be determined whether glycine is essential for

cell proliferation during wound healing [119]. Two recent studies suggest that Kynurenine, a

kynurenate precursor, may play a role in both anti-inflammatory activity and fibroblast

proliferation during wound repair; additionally, it has been shown to be dysregulated in diabetic

wounds [120, 121]. KE supplemented rats showed an increase in kynurenate but didn’t reach

significance (2.61 fold) (0.05<p<0.10). Additionally, metabolite OH-phenylpyruvate was shown

to be dysregulated and KE supplementation significantly enhanced the levels 3.59 fold in the

serum. Little is known about the role during wound healing. Further experiments need to be

conducted to determine the meaning of this observation.

The metabolomics profiling data presented here help us to understand the metabolic

consequences of KE and BMS+MCT administration and offers insights into potential

mechanisms of action by which ketone supplementation may augment wound healing as well as

affect other disease states for future studies.

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Table 3.1 Global metabolism profile of serum and hippocampal tissue following chronic administration of KE and BMS+MCT: Rats received a daily 5-g/kg dose of water (control) (n=11), KE (n=11), or BMS+MCT (n=12) via intragastric gavage for 29 days. On day 29, blood serum (~300 µL) and hippocampal tissues were collected 4-hours post-intragastric gavage (peak ketone elevation) and global metabolomics profiling was performed at Metabolon Inc. using gas and liquid chromatography and tandem mass spectrometry. Tx1=KE and Tx2=BMS+MCT. Results were considered significant when p<0.05 and direction is indicated by red or green arrows. 388 known metabolites were identified in the serum and 290 were identified in the hippocampus. KE significantly increased 106 metabolites and significantly decreased 36 in the serum compared to control; increased 10 and decreased 2 compared to control in the hippocampus (p<0.05, Welch’s two sample t-test). BMS+MCT significantly increased 57 metabolites and significantly decreased 62 metabolites compared to control in the serum; increased 28 and decreased 8 compared to control in the hippocampus (p<0.05). An additional 24 metabolites trended towards significance compared to control for KE and 58 for BMS+MCT (0.05<p<0.10).

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Figure 3.8 Ketone supplementation elevates blood ketone levels in serum and hippocampal tissues: KE and BMS supplemented rats had elevated blood ketone levels in their serum. KE elevated βHB 15.76 fold and AcAc 8.84 fold. BMS+MCT elevated βHB 35.33 fold and AcAc 15.39 fold. KE elevated βHB 2.7 fold in hippocampus and BMS+MCT elevated βHB 3.39 fold.

Figure 3.9 Ketone supplementation increases medium chain fatty acids in serum and hippocampal tissue: KE significantly increased medium chain fatty acid (MFCA) Caprate in the serum (3.15 fold) and in the hippocampus (1.92 fold) (p<0.05). BMS+MCT significantly increased Caprate in the serum (3.98 fold) and in the hippocampus (3.16 fold) (p<0.05). BMS+MCT significantly increased Caprylate in the serum (13.19 fold) and in the hippocampus (3.24) fold (p<0.05).

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Figure 3.10 Ketone supplementation increases Krebs cycle intermediates: KE and BMS+MCT supplemented rats had significantly elevated Krebs cycle intermediates in their serum. Both, KE and BMS+MCT increased citrate (1.99 fold, 2.34 fold) (A), fumarate (1.92, 2.45 fold) (B), and malate (2.03, 2.68 fold) (C) Additionally KE increased alpha-ketoglutarate (1.98 fold) (D) and BMS+MCT increased succinate (1.66 fold) (E). Krebs cycle precursor Acetyl CoA was significantly elevated by BMS+MCT in the hippocampus (1.37 fold) (F). Krebs cycle intermediates were unchanged in hippocampal tissues.

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Figure 3.11 Ketone supplementation increases antioxidants: KE and BMS+MCT significantly increased antioxidants carnosine (3.23, 1.79 fold) and anserine (5.66, 3.70 fold) in the serum (p<0.05). Carnosine and anserine were unchanged in the hippocampus.

Figure 3.12 Ketone supplementation increases Adenosine: KE significantly increased adenosine levels in the serum (9.18 fold) (p<0.05). BMS+MCT significantly increased adenosine levels in the hippocampus (10.95 fold).

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CHAPTER 4: ENHANCING WOUND HEALING WITH EXOGENOUS KETONE

SUPPLEMENTATION

4.1. Chapter Synopsis

In the previous chapter, we determined that exogenous ketone supplementation

significantly elevated blood ketone levels and reduced blood glucose levels in juvenile Sprague-

Dawley rats. In this chapter, we present data demonstrating that both BD and BMS+MCT

ketone supplements elicit age-dependent rapid elevation of blood ketone levels and reduction of

blood glucose levels in young (8 month) and aged (20 month) Fischer 344 rats similar to those

determined in Chapter 3. Additionally, we present data demonstrating the effects of food-

integrated oral ketone supplementation on an ischemic wound-healing model in young and aged

Fischer 344 rats. In the ischemic wound model, the flap placement on the dorsum prevents the

use of oral gavage; therefore, the ketone supplements were mixed into the food and fed ad

libitum. Nonetheless, this allowed continuous delivery of the ketone supplements in smaller

doses as the rats fed throughout the day. Compared to a bolus administration, multiple daily

doses or inclusion in the food may provide more sustained benefits. Additionally, we chose one

synthetic (BD) and one natural (BMS+MCT) ketone supplement for the following studies.

Though KE would seem to be a logical choice to continue with, the Fischer 344 rats refused to

eat the food mixed with this supplement; therefore, BD was used.

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Portions of this chapter have been taken from a manuscript Poff AM, Kesl SL,

D’Agostino DP “Ketone Supplementation for Health and Disease” Book Chapter, Oxford Press

(Submitted). A copy of this article may be found in Appendix C. Materials and Methods

presented in this chapter may be found in Appendix A.

4.2. Dietary Ketone Supplementation Increases Blood Flow and Wound Closure in an

Ischemic Wound Model in Young and Aged Fischer Rats

It has been well established that the ketogenic diet (KD) induces “keto-adaptation”, a

physiologic state characterized by a shift away from glucose metabolism and towards fat and

ketone body metabolism. [1-3]. As discussed in previous chapters, there are many

physiological changes associated with sustained ketosis that may contribute to its multifaceted

therapeutic potential for many disease states including chronic wounds. Thus, we previously

measured blood glucose, ketones, lipids, and other biochemical metabolites in response to

chronic oral ketone administration to show physiological equivalence to KD [4]. Increasing

evidence shows that limited energy and nutrient exchange is associated with age-related

impairment of wound healing. We hypothesized that oral ketone supplementation without

dietary restriction would enhance wound closure in young and aged Fischer rats by improving

blood flow and supplying an alternative energy substrate. In our preliminary studies, we

measured the magnitude and duration of ketosis following administration of a single 6.5g/kg

dose of ketone precursors: 1,3-Butanediol (BD), Na+/K+ βHB salt and medium chain

triglyceride (MCT) oil 1:1 mixture (BMS+MCT), or water in young and aged Fischer 344 rats

(n=6). Substances were administered through an intragastric gavage, and whole blood samples

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(10 µl) were acquired for analysis of glucose and βHB at 0, 0.5, 1, 1.5, 2, 4, 8, 12, and 24 hours

following administration. Following the creation of ischemic wounds, the ketogenic

supplements were added to a standard diet fed ad libitum for 28 days. Laser Doppler imaging of

the ischemic peri-wound tissue every seven days demonstrated significantly increased blood

flow in young rats (n=10) fed BD at day 14 and 28 (p<0.001) and BMS+MCT at day 7, 14, and

28 (p<0.01). In aged rats, blood flow was significantly increased in BD-fed at day 14 and

BMS+MCT-fed at days 7 and 14 (p<0.05). Wound size was significantly smaller in young rats

fed BD and BMS+MCT compared to control at 11 and 14 days following wound creation

(p<0.05). In aged rats, BD-fed wounds were significantly smaller at days 11 and 14 (p<0.05)

and in BMS+MCT-fed at days 11, 14, and 28 (p<0.05). Wound healing improved by three days

in aged BD-fed, seven days in young BMS+MCT-fed, and ten days in aged BMS+MCT-fed

compared to the healing time line of the control animals.

4.2.1 Aged rats metabolize exogenous ketone supplements differently than young rats

In Chapter 3, we studied if oral ketone administration could elicit similar physiological

effects as the KD by determining how blood glucose, ketones, and lipids and other biochemical

metabolites are affected by chronic ketone administration [4]. Here we present evidence that

chronic administration of ketone supplements can induce a state of nutritional ketosis without

the need for dietary carbohydrate restriction, enhance blood flow, and augment wound closure

in young and aged Fischer 344 rats.

Both young and aged rats exhibited elevated ketones within 30 minutes of the bolus

administration for both BD and BMS+MCT supplements and were sustained for 12 hours

(p<0.05). However, BD supplemented aged rats demonstrated a peak elevation of blood βHB

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levels at 8 hours, which was delayed compared to their younger counterparts who had peaked

blood βHB levels at 4 hours post intragastric gavage. Additionally, aged animals supplemented

with either of the ketone supplements demonstrated a ~1.5x higher elevation of blood ΒHB

levels for the same dose compared to the young animals (Figures 4.1A, B). An additional set of

young and aged animals were tested with a 10 g/kg dose of their respective ketone supplements

and a similar pattern emerged, young and aged animals’ blood βHB levels both peaked at 12

hours post gavage (data not shown). Aged animals administered BD appeared sedated, had

increased blood BHB up to 8 mM, and hypoglycemia (<50 mg/dL). We speculated that these

sedative effects might be because BD is metabolized as an alcohol; thus, we decided that an

intragastric gavage at 10 g/kg was not optimal for aged animals. Young animals were able to

tolerate the dose.

The metabolic challenge of a wound is exacerbated in elderly patients [5]. Without

sufficient energy (ATP) levels, wound healing is significantly impaired. As discussed, the ATP

deficiency creates an imbalance of energy production versus utilization (the catabolism to

anabolism ratio) leading to loss of lean body mass (LBM) [6, 7]. Additionally, Gupta and

colleagues demonstrated that metabolic enzymes hexokinase, citrate synthase,

phosphofructokinase, and lactate dehydrogenase were decreased in aged rats. Additionally, the

metabolic enzyme glucose-6-phosphate dehydrogenase exhibited elevated activity and the

beginnings stages followed by a significant decreased activity in a later phase compared to

controls [8]. These age-dependent metabolic changes could explain the difference between the

young and aged rats’ response to acute ketone supplementation. However, ketones have been

shown to increase Kreb’s cycle intermediates, mitochondrial biogenesis, and enhance overall

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metabolic efficiency; thus, this is one potential mechanism of how the prolonged ketone

administration could have enhanced wound healing.

4.2.2. Hypoglycemic effect of hyperketonemia attenuated with age

In Chapter 3, we demonstrated that at baseline and 4 weeks, 4 hours after intragastric

gavage with BMS+MCT (5 g/kg), the elevation of blood ketones was inversely correlated with

the reduction in blood glucose (r2=0.4314, p=0.0203, r2=0.8619, p<0.0001). This correlation

was not observed at any time point for BD supplemented rats [4]. Veech and colleagues have

demonstrated that administration of similar ketone supplement simultaneously decreased blood

glucose and blood insulin by approximately 50%. Our data show the same correlation between

elevated blood ketone levels and reduced blood glucose levels with exogenous BMS+MCT

supplementation in 8-month-old Fischer rats, supporting the work of Veech and confirming our

previous work (Figure 4.2). However, this hypoglycemic effect is not apparent in the 20-month

animals (Figure 4.2). As discussed, metabolic changes that occur during aging may play a role

in diminishing this relationship. Furthermore, insulin resistance has been shown to increase with

age [9]. Veech and colleagues have determined that ketone-induced hypoglycemia occurs via

increasing insulin sensitivity [10-12]. In the older adult, with pre-existing has insulin resistance,

the same ketone-induced effect on insulin sensitivity may not occur or it may not be enough to

cause hypoglycemia. It should be noted that even though there wasn’t a significant correlation

between blood ketone levels and blood glucose levels in aged ketone supplemented rats, there

was suppression of glucose based on the linear regression analysis (data not shown). This

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observation supports the speculation that an age-dependent metabolic change diminishes the

significant correlation but doesn’t completely neutralize the relationship.

Additionally, hyperketonemia was observed throughout the 28-day wound healing study

in the BD supplemented young and aged rats, but was not noted in the BMS+MCT

supplemented rats (Figure 4.5A). In Chapter 3, we demonstrated that ketone supplementation

caused a sustained reduction in glucose over the time course of the intragastric gavage study;

however, this sustained reduction of glucose was not observed over the course of the wound

healing study (Figure 4.5B). In a follow-up chronic feeding study (15 weeks), food-integrated

ketone supplementation resulted in elevated blood ketone levels without affecting the blood

glucose levels throughout the study, which supports our findings in this study (data not shown,

unpublished data).

4.2.3. Ketone supplementation increases blood flow in both young and aged rats

Restoration of blood flow via angiogenesis is critical for healing a wound and to support

revascularization of grafts including tissue engineered skin substitutes. Venous, arterial, and

diabetic blood flow insufficiencies are major underlying contributors to chronic wound

development. Additionally, older patients have a greater prevalence of hypertension, diabetes,

and smoking, which exacerbate the age-dependent angiogenic insufficiencies [13, 14]. In this

study, oral ketone supplementation significantly increased blood flow in young and aged rats

supplemented with BD or BMS+MCT (Figure 5.3B). In the raw Laser Doppler data, darker

colors are indicative of low blood flow/ischemia and lighter colors demonstrate increased blood

flow. The increased blood flow in the ketone-supplemented rats is clearly visible in the raw data

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(Figure 5.3A). This wound-healing model was created to induce ischemia and thus delay

wound healing [15, 16]. Further studies need to be conducted to measure oxygen concentrations

during wound healing to establish that increased blood flow reverses flap ischemia leading to

accelerated wound healing. In a study by Hasselbalch and colleagues, exogenous ketone

supplementation exhibited a 39% increase in cerebral blood flow [17]. Additionally, VEGF, a

potent angiogenic factor, has shown to be elevated with exogenous ketone supplementation [18-

20]. As discussed in Chapter 3, ketone supplementation has been determined to increase the

potent vasodilator, adenosine. These two mechanisms may explain how ketone supplementation

increased blood flow in the ischemic wound model.

4.2.4 Ketone supplementation accelerates wound closure in young and aged rats

As the population continues to age, there is a critical need to develop effective wound

healing therapies. In this study, we have demonstrated that exogenous ketone supplementation

enhanced wound closure by ten days (36% faster) in aged rats supplemented with BMS+MCT

and three days (10% faster) in aged rats supplemented with BD compared to control aged rats

(Figure 4.4). Additionally, young rats supplemented with BMS+MCT healed three days earlier

(14% faster) compared to standard diet fed young rats (Figure 4.4). Future studies are designed

to determine the wound healing effect of topical ketone administration alone and in combination

with the oral exogenous ketone therapy. As is, this therapy could allow patients to benefit from

nutritional ketosis without dietary restriction.

Studies by Nevin and colleagues investigated the influence of a topical application of

virgin coconut oil (VCO) on the healing of dermal wounds in young rats [21-23]. Their results

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demonstrated a significant beneficial effect of VCO administration on intracellular and

extracellular matrix components compared to controls including increased cross-linking

collagen molecules indicating greater wound tensile strength and increased total DNA of the

granulation tissue. Additionally, VCO therapy induced greater antioxidant capacity (superoxide

dismutase 2, glutathione reductase, glutathione peroxidase) during wound healing, which led to

a decrease in lipid peroxides (MDA). They concluded that the wound healing property of VCO

might be due to its minor biologically active components and antimicrobial fatty acids. Previous

studies have demonstrated that food integrated coconut oil was able to eliminate bacterial

infection and stimulate the immune response [24]. Coconut oil is a natural source of medium

chain triglycerides (MCTs), which have been shown to modulate cellular proliferation, cell

signaling, and growth factor activities as well as elicit ketogenesis as seen in this study and our

previous study [4, 25-27].

The BMS contains potassium and other minerals to prevent sodium overload.

Maintaining an optimal sodium-mineral ratio should help offset any potential adverse effects of

sodium on blood pressure. For example, multiple studies have shown that potassium provides

an antihypertensive effect and protects cardiovascular damage in salt-sensitive hypertension

[28, 29]. It is speculated that this formulation will be especially beneficial for elderly patients

most susceptible to sodium-induced hypertension [11]. The dose of the salt solution is easily

adjusted to benefit the patients’ needs. In this study a 10% ketone salt solution is mixed in a 1:1

ratio with MCT oil, which allows for reduced dosing of each component compared to

administering the compounds individually. This reduces the potential for side effects (sodium-

induced hypertension, gastric side effects, etc.) and results in distinct synergistic blood ketone

profile [30]. A noteworthy observation from our previous study revealed that in rats, MCT alone

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was more effective than BMS+MCT or BMS alone at inducing ketosis. However, preliminary

human data suggests that the BMS+MCT mixture is most effective at inducing ketosis and

MCT the least. This suggests that there is inter-species variability in the metabolic response to

ketone supplements, which will need to be further characterized to fully understand effects in

humans. In the rats, the BMS+MCT supplement elevated blood ketones similar to that of MCT

alone; however, the gastric side effects were not observed suggesting a potential method for

avoiding this unwanted adverse effect [31].

4.2.5. Food- integrated ketone supplementation did not elicit weight loss in young and

aged rats

The KD is hypothesized to induce weight loss by reducing appetite through the satiety

effect of ketone bodies, reducing lipogenesis and increasing lipolysis, and enhancing metabolic

efficiency with fat and ketone metabolism [32]. Recently, preliminary studies have shown that

exogenous ketone supplementation can also induce weight loss. The administrations of both

βHB and BD have been shown to decrease food intake in rats and pigmy goats [33-37].

Similarly, it is suggested that MCTs increase satiety, resulting in reduced food intake and

weight loss as a consequence of their rapid oxidation into ketone bodies [38-40]. MCTs may

further counteract fat deposition in adipocytes by increasing thermogenesis [41]. Several studies

in animals and humans have revealed increased energy expenditure and lipid oxidation with

MCTs compared to LCTs [42-51]. Ketone esters have also been shown to affect weight in

mice, rats, and humans [20, 52-55]. In Chapter 3, all five ketogenic supplements tested via

intragastric oral gavage (BD, KE, MCT, BMS, BMS+MCT) inhibited weight gain compared to

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control animals [4]. Similarly, a 15-week chronic feeding study in which KE, BMS, or

BMS+MCT replaced approximately 20% of the diet by weight, fed ad libitum, led to reduced

weight gain compared to control animals (data not shown, unpublished data).

Even though weight loss has been noted in a variety of studies, significant weight loss

was not observed in either young or aged rats of this study (Figure 4.6A). To improve post-

anesthetic appetite, thereby limiting the initial weight loss that is standard with an invasive

surgery, the rats were fasted for 18 hours before surgery. . Weight loss was noted during the

first week after surgery, but did not reach significance in any group, and was similar across all

groups demonstrating that it wasn’t a ketone-mediated effect. Weight was maintained in all

groups for the remainder of the study. This initial fasting may be a reason that the initial weight

loss during the first week post-surgery did not reach significance. In the previously discussed

studies, rats were Sprague Dawley and were either juvenile or still in a growth phase, whereas

in this study the rats were mature or elderly Fischer 344s. Age and strain variability may

account for the lack of weight loss in this study compared to previously discussed experiments.

Particularly in elderly patients where malnutrition may already be present, it is critical to

prevent the wound from parasitizing substrates from the rest of the body for energy production,

resulting in what is called protein-energy malnutrition (PEM). The body catabolizes the

muscle, skin, and bone to support the synthesis of proteins, inflammatory cells, and collagen

needed to fight infection and repair the wound, causing a loss of lean body mass (LBM). As an

individual loses more LBM, wound healing is more likely to be delayed. A loss of more than

15% LBM impairs wound healing, a 30% LBM loss stops wound healing, and a loss of 40%

LBM typically results in death [56-60]. A body in ketosis has been shown to be muscle sparing

as it is adapted to using the readily available fat stores, attenuating the catabolic effects [7]. The

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weight maintenance seen in this wound healing study may reflect this protein-sparing effect.

Moreover, if ketosis enhances insulin sensitivity, glucose uptake by the insulin-sensitive

skeletal muscle should be increased. Together, these effects would help support muscle tissue

health and function, suggesting another possible mechanism of ketone supplementation in

diminishing LBM and PEM.

4.3 Closing Remarks

It has been debated if chronological age alone affects wound healing. However, with the

aged population showing decreased blood flow leading to the decline of nutrient exchange,

decreased metabolic activity and ATP production at the wound bed, it is clear how these factors

may result in an exacerbation of chronic wounds in older adults. The discouraging results from

studies focusing on single molecular targets are not surprising since wound healing is the

outcome of a complex set of interactions between numerous factors. This is likely one reason

most wound therapies are minimally effective. Our approach utilizes endogenous physiology to

reach the wound bed systemically with one therapy, exogenous ketone supplementation, that

has multiple downstream effects.. Even though there is much work still to be done, this

approach shows promising results as a potential wound therapy.

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Figure 4.1 Effects of ketone supplementation on blood ketone and blood glucose levels: (A, B) Blood βHB and blood glucose levels at times 0, 0.5, 1, 4, 8, 12, and 24 hours post (6.5 g/kg) intragastric gavage for ketone supplements tested. BMS+MCT and BD supplementation rapidly elevated and sustained significant βHB elevation (p< 0.05) (A) and significantly reduced glucose (p < 0.05) (B) compared to controls in both young (8M) and aged (20M) rats. Two-Way ANOVA with Tukey’s post-hoc test, results considered significant if p<0.05. Error bars represent mean (SD).

Figure 4.2 Relationship between blood ketone and blood glucose levels attenuated with age: At four hours post intragastric gavage, BMS+MCT (6.5 g/kg) supplemented rats demonstrated a significant correlations between elevated blood ketone levels and decreased blood glucose levels in young (8M) rats (r2=0.4775, p=0.0393); however, correlation was not present in aged (20M) rats (r2=0.0088, p=0.8431). Linear regression analysis, results considered significant if p<0.05. Error bars represent mean (SD).

8M Fisher Rats-BMS+MCT

60 80 100 1200

1

2

3

4

5

Glucose (mg/dL)

BH

B (m

M)

r2=0.4775p=0.0393 *

20M Fisher Rats- BMS+MCT

60 80 100 1200

1

2

3

4

5

Glucose (mg/dL)

BH

B (m

M)

r2=0.0088p=0.8431

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Figure 4.3 Ketone Supplementation increases blood flow in both young and aged rats: Laser Doppler was used to measure blood flow weekly in the ischemic flap. Raw data from aged rats fed SD, BD and BMS ketone supplements at day 14 demonstrates the difference in blood flow seen by the laser doppler (A). Laser Doppler imaging of the ischemic peri-wound tissue every 7 days for 28 days demonstrated significantly increased blood flow in young rats (n=10 per group) with the BD at day 14 (p<0.0001) and 28 (p=0.0002) and the BMS+MCT at day 7 (p=0.0007), 14(p<0.0001), and 28 (p=0.0036) compared to control. There was an increase in blood flow in the aged rat flaps (n=10 per group) treated with the BD at day 14 (p=0.0039) and BMS+MCT at days 7 (p=0.0305) and 14 (p=0.0008) compared to control. Two-Way ANOVA with Tukey’s post-hoc test, results considered significant if p<0.05. Error bars represent mean (SD).

136

Figure 4.4 Ketone supplementation enhances wound closure time in young and aged rats: Visualized using Kaplan-Meier survival plot. Wound healing (closure) was determined to be significantly different in the young (n=10 per group) Fischer rats in BD at day 11 (p=0.0493) and in BMS+MCT at day 11 (p=0.0022) and 14 (p=0.0349). In the aged Fischer rats (n=10 per group), the BD was significantly different at day 11 (p=0.0230) and 14 (p=0.0233) and BMS+MCT was significantly different at day 11 (p=0.0115), 14 (p=0.0016) and 28 (p=00010). Two-Way ANOVA with Tukey’s post-hoc test, results considered significant if p<0.05.

137

Figure 4.5 Ketone supplementation elevates blood ketones levels but does not affect blood glucose levels: (A) Hyperketonemia was sustained for the duration of the wound healing progression in BD supplemented young and aged animals. (B) Ketone supplementation did not significantly reduce blood glucose levels at any point of the healing period. Two-Way ANOVA with Tukey’s post-hoc test, results considered significant if p<0.05. Error bars represent mean (SD).

138

Figure 4.6 Body and Spleen Weight: (A) Ketone supplementation did not result in significant weight loss through the study in young and aged animals. (B) Though aged animals’ spleen size trended towards splenomegaly, results did not reach significance. Two-Way ANOVA with Tukey’s post-hoc test, results considered significant if p<0.05. Error bars represent mean (SD).

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CHAPTER 5: POTENTIAL MECHANISMS FOR EXOGENOUS KETONE

SUPPLEMENTATION TO ENHANCE WOUND HEALING

5.1. Chapter Synopsis

In this chapter, we present data defining some potential mechanisms by which exogenous

ketone supplementation augments ischemic wound healing. We focus on four main age

dependent factors: inflammation, ROS production, angiogenesis, and metabolism. Since ketone

supplementation enhanced wound healing, we hypothesized that there would be measurable

physiological changes in wound healing as early three days post-wounding. Periwound tissue

was harvested at day three and day seven post-wounding for mechanistic analysis. Additionally

mechanistic studies were conducted using primary human dermal fibroblast (HDFs) to confirm

ex vivo observations. From these studies we conclude that exogenous ketone supplementation

decreases ROS production, increases migration and proliferation, and decreases lactate

production. Ketone supplementation did not affect inflammatory markers or antioxidants SOD2

and NQO1. Though there were similarities in effects of ketone supplements, BD and BMS+MCT

elicited distinctive mechanistic profiles. We propose further studies at later time points of wound

healing to determine if exogenous ketone supplementation will affect wound healing at a later

phase than we originally hypothesized.

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5.2. Potential Mechanisms of Action for Exogenous Ketone Enhancement of Ischemic

Wound Healing in Young and Aged Fisher Rats

In the previous chapter, we reported that oral ketone supplementation without dietary

restriction enhanced wound closure and increased blood flow in young and aged Fisher 344 rats.

We hypothesized that exogenous ketone supplementation promoted wound healing via

enhancement of physiological factors such as increasing proliferation, advancing migration,

reducing ROS production, and resolving inflammation. Experiments in vitro with young and

aged primary human dermal fibroblasts supplemented with 5mM βHB for 72 hours ahead of an

oxidative stimulus (100μM tert-butyl-hydrogen peroxide) resulted in significantly decreased

cellular ROS production, enhanced cell migration, and augmented cellular proliferation (p<0.05).

Comparing peri-wound tissue lysates isolated from rats fed 1,3 Butanediol (BD), βHB NA+/K+

salt mixed with MCT oil in a 1:1 ratio (BMS+MCT), or standard diet (SD) we show that ketone

administration during wound healing induced changes in cytokines, including a significant

elevation in epidermal growth factor (EGF) at day 7 of wound healing compared to control

(p<0.05). BMS+MCT supplementation in aged rats significantly decreased tumor necrosis

factor- alpha (TNF-α) levels on day 7 compared to control (p<0.05). Furthermore, ketone

supplementation significantly reduced leptin levels in ischemic wound tissue lysates in both

young and aged rats. Changes in pro-inflammatory (IRF-5 labeled, M1) to anti-inflammatory

(CD206 labeled, M2) macrophage ratio and blood vessel density were demonstrated by

immunohistochemistry. We conclude that ketone supplementation in vivo and in vitro modifies

systemic physiology to enhance wound closure.

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5.2.1 Effects of ketone supplementation on inflammation

Inflammation is a key regulatory step in the progression of wound healing. Chronic

wounds are stuck in the inflammatory phase characterized by a proteolytic environment in which

pro-inflammatory cells, cytokines, and chemokines inhibit the normal progression of wound

healing [1]. Pro-inflammatory cytokines including IL-6, IL-1β, and TNF-α are upregulated

during the inflammatory phase of wound healing [2]. IL-6 is produced by neutrophils and

monocytes and has been shown to be important in initiating the healing response, has mitogenic

and proliferative effects on keratinocytes, and is chemoattractive to neutrophils [3-8]. TNF-α

alone has been shown to enhance wound healing in a concentration-dependent manner. Low

levels of TNF-α can promote wound healing indirectly by stimulating inflammation and

macrophage-produced growth factors. However, at higher levels, TNF-α has a detrimental effect

on wound healing by suppressing ECM synthesis and TIMPs while increasing MMPs, inhibiting

re-epithelialization. Additionally, levels of TNF-α and IL-1β are elevated in chronic wounds and

have been shown to work synergistically and perpetuate each other’s expression to amplify the

pro-inflammatory environment [9-11].

Exogenous ketone supplementation did not affect pro-inflammatory markers IL-6 or IL-

1β in young or aged periwound tissue lysates on day three or day seven of wound healing

(Figure 5.2). BMS+MCT supplementation significantly reduced TNF-α levels in periwound

tissue lysates of aged rats after seven days of wound healing compared to aged controls.

Significant changes were not observed in selected anti-inflammatory cytokines including IL-4,

IL-10, or IL-13 (Figure 5.2). In an unpublished study from our laboratory, rats were fed one of

three exogenous ketone supplements (1, 3-butanediol diacetoacetate ester (KE), Na+/Ca+2-β-

hydroxybutyrate mineral salt (ΒHB-S), or a 1:1 mixture of Na+/Ca+2-β-hydroxybutyrate mineral

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salt: Medium Chain Triglyceride Oil (S/MCT) mixed into standard rodent chow at 5-20% by

weight for 15 weeks. Inflammatory profiling was performed on serum collected from the rats at

the end of the chronic feeding study and revealed decreases in several pro-inflammatory and

anti-inflammatory cytokines including IL-1β, IL-6, IFN-γ, MCP-1, RANTES, IL-4, IL-10, IL-13.

These findings suggest that ketone supplementation may suppress total inflammation and not

affect pro-inflammatory or anti-inflammatory markers independently; however, further

experiments need to be conducted to confirm this observation. Because ketone supplementation

hastened wound healing, we hypothesized that we would see early resolution of inflammation.

Further studies need to be conducted on subsequent days of wound healing to see if ketone

supplementation affects inflammatory markers after day 7. Epidermal growth factor (EGF) is

secreted by platelets, macrophages and fibroblasts to accelerate epithelialization and increase the

tensile strength of wounds [12-15]. Topical EGF administration has shown promising results for

venous ulcers and diabetic ulcers; however, the heterogeneity of wounds, modes of

administration, patient management protocols, and varied clinical trial outcomes has limited

clinical application to outside of the USA [16]. Exogenous ketone supplementation with BD

elevated EGF levels in day seven periwound tissue lysates of young rats (Figure 5.3B).

Leptin is a hormone that works in opposition of ghrelin to regulate energy homeostasis by

inhibiting hunger and establishing satiety. The KD has been demonstrated to slow weight gain by

altering leptin levels. Juvenile rats fed a KD for two weeks had slower weight gain, had higher

leptin levels, and lower insulin levels compared to those fed an SD [17]. We demonstrated a

similar slowing of weight gain in juvenile Sprague-Dawley rats in Chapter 3, consistent with this

observation. In the same study, they demonstrated that calorie restriction did not elicit the same

leptin elevation as the KD. However, Kolacynski and colleagues investigated the responses of

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leptin following a short term fasting, demonstrating that leptin levels declined following a 12

hours fast. Re-feeding prompted leptin levels to rise and return to normal values within 24 hours

[18]. They demonstrated a reverse relationship between levels of βHB and leptin. Small amounts

of glucose added to inhibit hepatic ketogenesis was enough to prevent the fall of leptin.

However, exogenous ketone administration did not reduce leptin, causing the team to conclude

that the relationship was due to a physiological response to ketogenesis and not ketone bodies

themselves.

An increasing number of studies have determined that leptin has a broad spectrum of

physiological effects in the wound-healing environment. Leptin has been shown to directly cause

T cells and monocytes to release pro-inflammatory cytokines, to produce angiogenesis in

endothelial cells, and to induce neo-vascularization in corneal cells. Leptin production has been

documented in a variety of non-adipose cells including placental trophoblasts, mammary

epithelial cells, gastric fundic mucosa, and ovarian follicle cells [19-29]. In a study by Murad and

colleagues, it was demonstrated that cells within an incisional dermal wound produced a burst of

leptin shortly after surgery [30]. The study concluded that leptin might have a multifunctional

role during wound healing and serves a critical functional role as an autocrine/paracrine regulator

of normal wound healing.

As discussed previously, leptin is produced locally in the wound bed. In our experiment

leptin was only measured in the wound bed tissue lysates, not in circulating blood serum. BD and

BMS+MCT significantly reduced leptin levels in ischemic wound tissue lysates in young

animals compared to controls on day three post-wounding but this observation was no longer

apparent after seven days of wound healing (Figure 5.3C). In aged animals, leptin levels were

decreased in both ketone-supplemented groups on both day three and day seven post-wounding

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(Figure 5.3C). This decrease in leptin levels in the ischemic wound bed may play a role to the

enhanced wound healing via exogenous ketone supplementation. The circulating blood serum

levels may give more insight to the data. If these measurements were in systemic blood serum, it

may indicate that the rats were not eating the food-integrated ketone supplements, as leptin levels

were elevated in the SD. Additionally, this would suggest that the elevations in blood ketone

levels would be due to caloric restriction and not ingestion of the supplement. Further

experiments monitoring food intake would be required to decipher this observation. Further

experiments need to be conducted to determine the full impact of decreasing leptin levels with

ketone supplementation.

As discussed in Chapter 1, macrophages in the wound bed can exhibit two distinct

functional phenotypes: M1 (classically activated, pro-inflammatory) and M2 (alternatively

activated, anti-inflammatory). During the early inflammatory phase, macrophages that are

activated by lipopolysaccharide (LPS) or inflammatory cytokines like interferon gamma (IFN-γ)

continue the job of neutrophils by phagocytizing bacteria and damaged tissue. Additionally, the

M1 macrophages release pro-inflammatory cytokines such as TNF-α and IL-6 [31]. During the

late inflammatory phase, initiated by the phagocytosis of apoptotic cells, M1 macrophages

phenotypically convert to M2 macrophages [32]. M2 macrophages, activated by interleukin-4

(IL-4) and interleukin-13 (IL-13), play a critical role in the resolution of inflammation.

Additionally, they facilitate transition to the proliferative phase by promoting angiogenesis,

tissue remodeling, and repair [31, 33-40]. In this study, we did not see an effect of exogenous

ketone supplementation on IFN-γ, IL-4, or IL-13 as previously hypothesized (Figure 5.2).

Additionally, exogenous ketone supplementation did not elicit a significant change in M1 to M2

phenotype in day seven aged ischemic wounds (Figure 5.4). This observation supports the

150

inflammatory array data and further supports that the aged ischemic wounds were still in a pro-

inflammatory environment seven days post-wounding. Heterogeneity seen in the wound healing

in this model is consistent with patient heterogeneity seen in the clinical setting and accounts for

the lack of statistical significance in the immunohistochemical analysis (Figure 5.1). In general,

ketone supplemented wounds demonstrated more epithelial advancement, granulation tissue

formation, and fewer labeled macrophages (Figure 5.1, Figure 5.4). Further studies need to be

conducted to confirm these histological observations.

5.2.2 Effects of ketone supplementation on ROS production

As discussed in previous chapters ROS production is an important bactericidal

component in acute wound healing. However, in a chronic wound environment, ROS production

isn’t properly mitigated, leading to mitochondrial dysfunction, DNA damage, lipid peroxidation,

and even cell death, which delays wound healing [41, 42]. Ketones have been shown to decrease

ROS production through a variety of physiological effects [43-46]. Additionally, ketones have

been shown to decrease oxidative stress by increasing the downstream antioxidant glutathione

via the Nrf2-ARE pathway [47]. Nrf2 is the primary transcription factor that responds to

oxidative stress. Under normal conditions, Nrf2 is targeted for proteasomal degradation by the

E3 ubiquitin ligase, Keap1. Oxidants and electrophiles disrupt the Nrf2/Keap1 binding allowing

Nrf2 to translocate to the nucleus, bind DNA at the antioxidant or electrophilic response element

(ARE), and transcribe downstream antioxidants including SOD2, NAD (P) H: quinone

oxidoreductase (NQO1) and enzymes for glutathione synthesis [48-50]. Additionally, Nrf2 has

been shown to play a role in wound healing as disruption of Nrf2 has shown to impair the

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angiogenic capacity of endothelial cells [51]. It has been shown that Nrf2 and antioxidant

capacity are decreased with age, leading to increased oxidative stress [52, 53]. Recently, Moor

and colleagues demonstrated age-dependent deficiencies in the glutathione and SOD2

antioxidant pathways. In an ischemic wound model, wounds from aged rats had lower SOD2

protein expression and activity, decreased ratio of reduced/oxidized glutathione, and decreased

glutathione peroxidase activity [54]. These age-exaggerated insufficiencies lead to excessive

inflammation and impaired wound healing. New data indicate that nutrient intake, energy

metabolism, and ROS are linked at the nuclear level via the Nrf2/ARE pathway [55]. The KD

has been shown to increase glutathione synthesis via the Nrf2/ARE pathway [56-59].

Additionally, the age-dependent deficit in Nrf2 has been reversed with lipoic acid

supplementation [60]. Lipoic acid is derived from the MCFA caprylic acid (a main component of

MCT oil); thus we hypothesized that exogenous ketone supplementation would result in similar

effects.

In this study, we were unable to measure Nrf2 levels via Western blot due to inefficient

antibodies; the downstream antioxidants SOD2 and NQO1, measured by Western blot analysis,

were not significantly altered by exogenous ketone supplementation in either primary human

dermal fibroblasts (HDFs) or ischemic wounds (Figure 5.6). Western blot sensitivity may not be

able to detect the subtle changes in these antioxidants during the first few days of wound healing;

thus, further experiments need to be conducted to determine antioxidant levels. However, DHE

and Mitosox Red labeled ROS production in primary human dermal fibroblasts was significantly

reduced with 5mM βHB supplementation (Figure 5.5). Metabolomic studies demonstrated that

ketone supplementation increased antioxidants carnosine and anserine. Further studies are

152

needed to investigate other antioxidant pathways and determine how ketone supplementation is

suppressing ROS production in a wound-healing environment.

5.2.3. Effects of ketone supplementation on angiogenesis

As a continuation of Chapter 4, we sought to delineate if the observed increase in blood

flow occurred via enhanced angiogenesis or vasodilatation. Immunohistochemical analysis of

CD31 blood vessel density demonstrated a trend of increased blood vessels in the ischemic

wounds of aged rats supplemented with BD and BMS+MCT seven days post-wounding;

however, this observation did not reach significance (Figure 5.7). As discussed, VEGF is a

potent angiogenic factor. Periwound tissue lysates were analyzed for VEGF levels, revealing a

trend of elevation of VEGF on day seven of wound compared to day three in both young and

aged animals though this did not reach significance. Ketone supplementation did not affect levels

of VEGF in this study, (Figure 5.2) but in the unpublished chronic feeding study, in which rats

were ketone supplemented via food-integration for 15-weeks, low dose KE significantly

increased serum (systemic) VEGF levels. As discussed in the metabolomics data in Chapter 3,

exogenous ketone supplementation leads to an increase in the potent vasodilator adenosine.

These observations suggest that the increased blood flow may be due to increased vasodilation

and not angiogenesis. Further studies need to be conducted to determine the impact of

vasodilation on ketone supplemented wound healing and to determine the exact mechanisms of

the observed increase in blood flow.

153

5.2.4. Effects of ketone supplementation on metabolism

Proliferation and migration represent crucial processes by which epidermal and dermal

layers restore anatomical integrity to the wound site. During the formation of granulation tissue

in the dermis, platelets, monocytes, and other cellular blood constituents release various peptide

growth factors, stimulating fibroblasts to migrate into the wound site where they proliferate to

reconstitute the various connective tissue components [61]. We investigated if ketone

supplementation enhanced proliferation and migration of primary human dermal fibroblasts;

indeed, 5mM HB supplementation elicited a significant increase in migration and proliferation

in both young and aged HDFs (Figure 5.8 and Figure 5.9). Further experiments need to be

conducted using keratinocytes of the epidermal layer to examine if ketone supplementation also

increases proliferation and migration in those cells. These further studies will help determine if

ketone supplementation affects all layers of the wound healing process or the dermal layer

independently. Additionally, the enhanced proliferation and migration may explain the enhanced

granulation tissue detected in the histological analysis (Figure 5.1, Figure 5.4).

Proliferating and migrating cells require more nutrients and energy production to perform

their functions; thus, an increase in these physiological factors may be a reflection of a higher

metabolic state. As discussed in Chapter 1, one of the fates of pyruvate is lactate production and

is indicative of anaerobic respiration. The hypoxic wound environment shifts to higher anaerobic

respiration and thus higher lactate production. Throughout the last decade, wound-healing

studies have revealed that high lactate levels (10-15 mM) are characteristic in healing, stimulate

collagen deposition, and enhance angiogenesis [62-66]. Subsequent studies demonstrated that

lactate has several sources in wounds, remains elevated even in the presence of oxygen, and

intense neutrophil respiratory bursts drive lactate and ROS accumulation through aerobic

154

glycolysis (Warburg effect) [64, 67-71]. These studies have concluded that lactate is produced in

both aerobic and anaerobic environments during tissue repair and the presence of both lactate

and oxygen is required for optimal wound healing. However, it is the quantity of lactate that is

critically significant [72]. Systemic lactate concentration rarely exceeds 10 mM due to tight

metabolic control; accumulation exceeding this level is associated with a poor wound-healing

prognosis [73]. When lactate levels reach ~20 mM it becomes deleterious to the wound bed as

high lactate levels reduce cell viability, diminish cell motility, and decrease polymerized

cytoskeletal actin, which impedes angiogenesis, fibroplasia, and wound contraction [74]. Aging

further complicates this environment as cells of older patients have shown decreased sensitivity

to growth factor and cytokine stimulation, diminished antioxidant capacity, and reduced cell

motility and proliferation, causing their tissues to be very susceptible to hypoxic and oxidant

stress and worsening patient survival outcome [75-78]. In this study, we demonstrated the 5 mM

βHB supplementation in young and aged HDFs significantly reduced lactate production (Figure

5.10). Further research needs to be conducted to investigate lactate levels in the wound bed with

exogenous ketone supplementation and explore lactate production in keratinocytes of the

epidermal layer to determine the full impact of ketone supplementation on lactate production

during wound healing.

5.3. Closing Remarks

In summary, we have just begun to elicit some of the mechanisms by which exogenous

ketone supplementation impacts ischemic wound healing. As ketone supplementation represents

a multifaceted tool, it is likely that multiple mechanisms are acting simultaneously to provide a

155

therapeutic effect and unlikely that a single, clearly delineated mechanism of action could be

elucidated. However, understanding these mechanistic attributes will help to optimize exogenous

ketone supplementation as a wound healing therapy.

Figure 5.1 Heterogeneity of Aged Ischemic Wound Healing: Histological hematoxylin and

eosin staining of aged ischemic wounds on day seven post-wounding demonstrated heterogeneity

in healing within rats supplemented with SD, BD, and BMS+MCT.

SD1 SD2

BD1 BD2

BMS+MCT1 BMS+MCT2

Non-Wounded

156

Figure 5.2 Effects of ketone supplementation on pro-inflammatory and anti-inflammatory cytokines: Multiplex immunoassay

on peri-wound tissue lysates isolated from SD, BD, and BMS+MCT fed rats on day three and day seven post-wounding. BMS+MCT

supplementation in aged rats significantly decreased tumor necrosis factor- alpha (TNF-α) levels on day 7 of wound healing compared

to controls (p<0.05) (A). No other pro-inflammatory (IL-1β, IL-6) or anti-inflammatory (IL-4, IL-10, IL-13) cytokines were

significantly changed (A, B). Two-way ANOVA with Tukey’s post-hoc analysis, results were considered significant when p<0.05.

Error Bars Represent mean (SD).

157

Figure 5.3 Effects of ketone supplementation on growth factors and the hormone leptin:

Peri-wound tissue lysates isolated from SD, BD, and BMS+MCT fed rats on day three and day

seven post-wounding. Eve Technologies analyzed samples with a 27-biomarker multiplex

immunoassay. Neither ketone supplement affected vascular endothelial growth factor (VEGF)

levels in young nor aged animals (A). BD ketone supplementation elicited a significant elevation

in epidermal growth factor (EGF) at day 7 of wound healing in young rats compared to control

(p<0.05)(B). Ketone supplementation with BMS+MCT and BD significantly reduced leptin

levels in both young and aged rats (C). Two-way ANOVA with Tukey’s post-hoc analysis,

results were considered significant when p<0.05. Error Bars Represent mean (SD).

VEGF

Day 3 Day 7 Day 3 Day 70

200

400

600

800

pg

/mL

Young Aged

EGF

Day 3 Day 7 Day 3 Day 70

2

4

6

pg

/mL

Young Aged

**

Leptin

Day 3 Day 7 Day 3 Day 70

200

400

600

pg

/mL

Young Aged

* **

* * *

A

B

C

SD

BD

BMS+MCT

SD

BD

BMS+MCT

158

Figure 5.4 Ketone supplementation does not affect M1 to M2 ratio of day 7 ischemic

wounds in aged rats: Paraffin embedded wound tissues harvested from SD, BD, and

BMS+MCT supplemented rats were stained with H&E for morphological analysis or labeled

with IRF-5 (M1, pro-inflammatory macrophage, red) or CD206 (M2, anti-inflammatory

macrophage, red) and CD68 (all macrophages, green) and DAPI (blue). Immunofluorescent

scans demonstrated that there was not a significant transition from M1 to M2 macrophage

phenotype with exogenous ketone supplementation, suggesting a pro-inflammatory environment

at day seven of wound healing.

H&E IRF-5/M1 CD206/M2

SD

BD

BMS+MCT

159

Figure 5.5 Ketone supplementation deceases ROS production: Primary human dermal

fibroblasts from young and aged donors (24 and 88 years) were incubated in the presence or

absence of 5mM βHB for 72 hours. The cells were stressed with 100uM tert-butyl-hydrogen

peroxide for 3 hrs. ROS production was measured via dihydroethidium (DHE) and Mitosox Red

fluorescence (A, B). DHE fluorescence: Aged HDFs produced 25% more ROS compared to their

young counterparts (p<0.05). βHB treatment decreased ROS production in young HDFs by 40%

and aged HDFs by 41% (p<0.05) (A). Mitosox Red fluorescence: Aged HDFs produced 27%

more ROS than young HDFs (p<0.05). βHB treatment decreased ROS production in young

HDFs by 29% and by 51% in aged HDFs compared to control (p<0.05) (B). One-way ANOVA

with Tukey’s post-hoc analysis, results were considered significant when p<0.05. Error Bars

Represent mean (SD).

160

Figure 5.6 Ketone supplementation does not affect antioxidants SOD2 and NQO1 in vitro and ex vivo: Primary human dermal

fibroblasts from young and aged donors (24 and 88 years) were incubated in the presence or absence of 5mM βHB for 72 hours. The

cells were stressed with 100uM tert-butyl-hydrogen peroxide for 3 hrs. Western blot protein expression analysis of antioxidants SOD2

and NQO1 demonstrated no change fibroblast expression of these antioxidants with ketone supplementation (A). Periwound tissue

lysates were isolated on day three and day seven post wounding. Western blot protein analysis showed no change in SOD2 or NQO1

expression with BD or BMS+MCT supplementation (B). Two-way ANOVA with Tukey’s post-hoc analysis, results were considered

significant when p<0.05. Error Bars Represent mean (SD).

161

Figure 5.7 Ketone supplementation does not significantly increase blood vessel density in

day 7 aged ischemic wounds: Quantification of histological staining with CD31 demonstrated

that exogenous ketone supplementation did not significantly increase blood vessel density at this

time point. One-way ANOVA, Tukey’s post-hoc analysis, results were considered significant

when p<0.05. Error Bars Represent mean (SD).

Vascularization of Aged Day 7 Ischemic Wounds

Aged SD Aged BD Aged BMS+MCT0

20

40

60

80

CD

31

Po

sitiv

e C

ells

SD BD BMS+MCT

162

Figure 5.8 Ketone supplementation increases migration in young and aged HDFs: Young

and aged HDFs were grown to confluence in 0.2% FBS 5mM glucose DMEM media, cells were

treated with or without the presence of 5mM βHB for 72 Hrs, cells were scratched with 200uL

yellow pipette tip. After 18 hours, migrated cells were counted using Dapi staining. Migration

was increased in both young and aged HDFs compared to the controls (p=0.0260 and p=0.0004)

(A, B). One-way ANOVA, Tukey’s post-hoc analysis, results were considered significant when

p<0.05. Error Bars Represent mean (SD).

Ketones Increase Migration in Young and Aged HDFs

0

10

20

30

40

50

Ce

ll N

um

be

r

Young

Aged*

*

+

+-

+ +

+-

+

5 mM BHB

100 uM TBOOH

A

B

163

Figure 5.9 Ketone supplementation increases proliferation in young and aged HDFs: Young

and aged HDFs were seeded with 75,000 cells in 10% FBS, 5mM Glucose DMEM media with or

without the presence of 5mM βHB. Total cell number was counted at 24, 48, 72, 96, and 120

hours post seeding. Young and aged HDFs treated with 5mM βHB had significantly more cells

at 72 and 96 hours (p<0.05). Two-way ANOVA with Tukey’s post-hoc analysis, results were

considered significant when p<0.05. Error Bars Represent mean (SD).

Proliferation of Young HDFs

24 48 72 96 1200

200000

400000

600000

Hours

To

tal N

um

be

r o

f C

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Young +BHB

Young -BHB

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Proliferation of Aged HDFs

24 48 72 96 1200

200000

400000

600000

Hours

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tal N

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be

r o

f C

ells

Aged +BHB

Aged -BHB

**

*

A

B

164

Figure 5.10 Ketone supplementation decreases lactate production in young and aged HDFs:

Young and aged primary human dermal fibroblasts were seeded with 250,000 cells in 10% FBS,

5mM glucose DMEM media with or without the presence of 5mM βHB. Extracellular lactate

was measured in cellular media at 24, 48 and 72 hours using Nova Biomedical Lactate Plus

blood lactate measuring meter. Young and aged HDFs treated with 5mM βHB produced

significantly less extracellular lactate at 72 hours p<0.05). Two-way ANOVA with Tukey’s post

hoc analysis, results were considered significant when p<0.05. Error Bars Represent mean (SD).

Lactate Production in Young HDFs

24 48 720

2

4

6

8

Ex

tra

ce

llula

r L

ac

tate

Co

nc

en

tra

tio

n (m

M)

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Young +BHB

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*

*

*

Lactate Production in Aged HDFs

24 48 720

2

4

6

8

Hours

Ex

tra

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Aged +BHB

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*

A

B

165

5.4. References for Chapter 5:

1. Barrientos S, Stojadinovic O, Golinko MS, Brem H, Tomic‐Canic M: PERSPECTIVE

ARTICLE: Growth factors and cytokines in wound healing. Wound Repair and

Regeneration 2008, 16(5).

2. Singer AJ, Clark RA: Cutaneous wound healing. The New England journal of medicine

1999, 341(10):738-746.

3. Di Vita G, Patti R, D'Agostino P, Caruso G, Arcara M, Buscemi S, Bonventre S, Ferlazzo

V, Arcoleo F, Cillari E: Cytokines and growth factors in wound drainage fluid from

patients undergoing incisional hernia repair. Wound repair and regeneration : official

publication of the Wound Healing Society [and] the European Tissue Repair Society

2006, 14(3):259-264.

4. Finnerty CC, Herndon DN, Przkora R, Pereira CT, Oliveira HM, Queiroz DMM, Rocha

AM, Jeschke MG: Cytokine expression profile over time in severely burned pediatric

patients. Shock (Augusta, Ga) 2006, 26(1):13-19.

5. Gallucci RM, Sloan DK, Heck JM, Murray AR, O'Dell SJ: Interleukin 6 indirectly

induces keratinocyte migration. The Journal of investigative dermatology 2004,

122(3):764-772.

6. Grellner W, Georg T, Wilske J: Quantitative analysis of proinflammatory cytokines

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CHAPTER 6: IMPLICATIONS FOR WOUND HEALING

6.1. Chapter Synopsis

In this chapter, we describe the major findings of this work based on the detailed

discussion of the data in previous chapters. This chapter will discuss the major implications for

wound healing as well as future directions for the project.

6.2. Implications for Wound Healing

The major goals of this dissertation work were to establish a method of sustaining

nutritional ketosis via exogenous ketone administration, determine the healing capabilities of

exogenous ketone supplementation in an aged rat population, and to acquire data of potential

mechanisms of action. As mentioned earlier, there is a dire need for a therapy that can accelerate

wound healing and efficiently restore normal tissue functionality. Chronic wounds, as discussed

in Chapter 1, impact several million people annually and is a billion dollar growing market. The

therapy presented within this dissertation exhibits great potential to accelerate cutaneous wound

healing closure and to be used as a multifaceted tool to stimulate wound repair and regeneration

in poor healing tissues especially chronic wounds in the aged population.

The KD has proven effective for the metabolic management of seizures and potentially

numerous other disorders, but is limited clinically [1-26]. Exogenous ketone supplements are

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primarily being developed as an alternative or adjuvant method of inducing therapeutic ketosis to

free patients from the restrictiveness of the KD. In Chapter 3, we were able to clearly

demonstrate that nutritional ketosis can be established with exogenous ketone supplementation

without negatively affecting blood triglyceride and lipoprotein profiles. Importantly, exogenous

ketone supplementation provides a tool for achieving ketosis in patients that are unable,

unwilling, or uninterested in consuming a low carbohydrate or ketogenic diet. It may also help

circumvent some of the difficulties associated with KD therapy initiation and maintenance, as it

allows for rapid induction of ketosis in a dose-dependent fashion, eases the transition to

nutritional ketosis, enhances the speed of keto-adaptation, and easily sustains ketosis with

prolonged consumption. Simultaneously, it could provide patients with the opportunity to reap

the benefits of ketosis without the practical and social difficulties of a highly restrictive diet.

Additionally, we demonstrated the ability of several ketone supplements to elevate blood ketone

levels, providing multiple options to induce therapeutic ketosis based on patient need. Many

patients that previously were unable to benefit from the KD may now have an alternative method

of achieving therapeutic ketosis. Ketone supplementation may also represent a means to further

augment ketonemia in those that respond to the ketogenic diet, especially in those individuals

where maintaining low glucose and insulin through carbohydrate restriction is important.

In Chapter 3, we demonstrated the exogenous ketone supplementation produced a

hypoglycemic effect, which in Chapter 4, was no longer apparent in the aged rats. Further

experiments need to be performed to determine what role age has explicitly on the observed

hypoglycemic effect. Additionally, further experiments need to be conducted to determine the

role that insulin sensitivity has in the age-dependent diminished hypoglycemic effect. As Chapter

3’s hypoglycemic effect was the result of an intragastric bolus and Chapter 4 transitioned to

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food-integrated administration, future studies need to be conducted to determine the effects of

administration on the observed hypoglycemic effect.

It should be noted that in these studies, rats were administered high doses due to their

higher basal metabolic rate (5.6x faster than humans). Future studies are needed to optimize

doses for humans. Preliminary data from the D’Agostino laboratory demonstrates that a 100 kg

male can obtain elevated ketone levels at a 0.4 g/kg dose compared to the 5 g/kg and 10 g/kg

doses that were administered to the rats. Follow up studies are currently being conducted to

determine the safety and toxicity profile of the ketone supplements KE and BMS as BD is

already FDA approved and MCT is a naturally occurring food source. Clarke and colleagues

have already demonstrated the safety of a similar ketone ester administration in humans; thus, we

don’t anticipate difficulty with the feasibility of using ketone supplements in humans [27].

However, one obstacle to overcome in the synthetic ketone supplements tested in this project is

their palatability. Both BD and KE are not pleasing to taste and will need to be optimized for

flavor without sacrificing their functionality. Additionally, future studies are needed to determine

the best route of administration for ketone supplements in patients, it may be more suitable to

have the supplements available in a variety of applications including parenteral nutrition,

integrated into food that the patients could ingest, meal replacement drinks, and oral pills.

As described in Chapter 2, exogenous ketone supplementation has the potential to

simultaneously target several underlying etiologies associated with age-impaired wound healing,

including unrelieved inflammation, unquenched ROS production, insufficient blood flow, and

dysfunctional metabolism. This is very different from most current wound therapies being

studied, which target a specific molecular or cellular mechanism. Because of the complexity of

wound healing, and the fact that there are often multiple mitigating factors that prevent normal

175

wound closure, most wound therapies are minimally effective. We hypothesize that using

exogenous ketone supplementation, as a component of standard care would target the

multifaceted etiologies underling most chronic wounds in the aged population. In Chapter 4, we

demonstrated that both BD and BMS+MCT ketone supplement therapies were effective in

enhancing wound healing in aged animals and the BMS+MCT also enhanced wound healing in

young animals. Further studies are needed to confirm the optimal dosing protocol to maximize

wound-healing efficacy.

Additionally, we elucidated some potential mechanisms of actions by which this novel

proposed therapy was able to significantly enhance wound healing. We demonstrated that

exogenous ketone supplementation altered the serum and hippocampal metabolome including

increasing Kreb’s cycle intermediates, antioxidants carnosine and anserine, and the potent

vasodilator adenosine. Further metabolomic studies need to be conducted to determine the

effects of age on ketone supplementation changes to the metabolome. Furthermore, young and

aged metabolomic studies of the wound healing environment with and without ketone

supplementation would offer great insights to the wound healing process. Mechanistically, we

demonstrated that ketone supplementation increases fibroblast proliferation and migration,

reduces ROS production, reduces lactate production, and increases blood flow. However, we did

not demonstrate a signifcant effect of ketone supplementation on antioxidants SOD2 or NQO1,

further studies need to be conducted to determine if the administration of ketone supplements

elicit the same physiological changes in other antioxidants. Further investigation into exploring

potential mechanisms of action are needed to fully understand the impact exogenous ketone

supplementation has on wound healing, specifically in an aged population. However, the

complexity of wound healing and the multifaceted targets of ketone bodies may constrain

176

elucidating a single, clearly delineated mechanism of action.

Moreover, futures studies will explore a topical application of ketone bodies. Even

though there was a significant increase in blood flow, this may not efficient for some patients;

thus, a direct application of the ketones or a combination of both therapies may be more

beneficial. Base on the results included in this dissertation we propose that exogenous ketone

supplementation could offer a novel therapeutic option for chronic wounds, especially in the

aging population.

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APPENDIX A: MATERIALS AND METHODS

A.1 Ethics Statement

All animal studies were approved by and performed within strict adherence to the

University of South Florida and James A. Haley Veterans Hospital’s Institutional Animal Care

and Use Committee (IACUC) protocols R00006 and V4251.

A.2 Materials and Methods for Experiments included in Chapter 3: Establishing

Therapeutic Ketosis (Metabolic Therapy with Exogenous Ketone Supplementation)

A.2.1 Synthesis and Formulation of Ketone Supplements

KE was synthesized as previously described [1]. BMS is a novel agent

(sodium/potassium- βHB mineral salt) supplied as a 50% solution containing approximately

375mg/g of pure βHB and 125 mg/g of sodium/potassium. Both KE and BMS were developed

and synthesized in collaboration with Savind Inc. Pharmaceutical grade MCT oil (~65% caprylic

triglyceride; 45% capric triglyceride) was purchased from Now Foods (Bloomingdale, IL). BMS

was formulated in a 1:1 ratio with MCT at the University of South Florida (USF), yielding a final

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mixture of 25% water, 25% pure βHB mineral salt and 50% MCT. BD was purchased from

Sigma-Aldrich (Prod # B84785, Milwaukee, WI).

A.2.2. Daily Gavage to Induce Dietary Ketosis

Animal procedures were performed in accordance with the University of South Florida

Institutional Animal Care and Use Committee (IACUC) guidelines (Protocol #0006R). Juvenile

male Sprague-Dawley rats (275-325 g, Harlan Laboratories) were randomly assigned to one of

six study groups: control (water, n=11), BD (n=11), KE (n=11), MCT (n=10), BMS (n=11), or

BMS+MCT (n=12). Caloric density of standard rodent chow and dose of ketone supplements are

listed in Table 1. On days 1-14, rats received a 5 g/kg body weight dose of their respective

treatments via intragastric gavage. Dosage was increased to 10 g/kg body weight for the second

half of the study (days 15-28) for all groups except BD and KE to prevent excessive

hyperketonemia (ketoacidosis). Each daily dose of BMS would equal ~1000-1500 mg of βHB,

depending on the weight of the animal. Intragastric gavage was performed at the same time

daily, and animals had ad libitum access to standard rodent chow 2018 (Harlan Teklad) for the

duration of the study. The macronutrient ratio the standard rodent chow was 62.2%, 23.8% and

14% of carbohydrates, protein and fat respectively.

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Table A.1. Caloric density of standard rodent chow and dose of ketone supplements

A.2.3. Measurement and analysis of blood glucose, ketones, and lipids

Every 7 days, animals were briefly fasted (4 hrs, water available) prior to intragastric

gavage to standardize levels of blood metabolites prior to glucose and βHB measurements at

baseline. Baseline (time 0) was immediately prior to gavage. Whole blood samples (10 µL) were

taken from the saphenous vein for analysis of glucose and βHB levels with the commercially

available glucose and ketone monitoring system Precision Xtra™ (Abbott Laboratories, Abbott

Park, IL). Blood glucose and βHB were measured at 0, 0.5, 1, 4, 8, and 12 hrs after test

substance administration, or until βHB returned to baseline levels. Food was returned to animals

after blood analysis at time 0 and gavage. At baseline and week 4, whole blood samples (10 µL)

were taken from the saphenous vein immediately prior to gavage (time 0) for analysis of total

cholesterol, high-density lipoprotein (HDL), and triglycerides with the commercially available

CardioChek™ blood lipid analyzer (Polymer Technology Systems, Inc., Indianapolis, IN). Low-

density lipoprotein (LDL) cholesterol was calculated from the three measured lipid levels using

the Friedewald equation: (LDL Cholesterol = Total Cholesterol - HDL - (Triglycerides / 5))[2,

Macronutrient Information

Standard Diet Water BMS+MCT BMS MCT KE BD

% Cal from Fat 18.0 0.0 N/A 50.0 100.0 N/A N/A % Cal from Protein 24.0 0.0 N/A N/A 0.0 N/A N/A

% Cal from Carbohydrates 58.0 0.0 N/A N/A 0.0 N/A N/A

Total Caloric Density (Kcal/g) 3.1 0.0 5.1 1.9 8.3 5.6 6.0

Dose 0-14 Days (g/kg) ad libitum N/A 5.0 5.0 5.0 5.0 5.0 Dose 15-28 Days (g/kg) ad libitum N/A 10.0 10.0 10.0 5.0 5.0

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3]. Animals were weighed once per week to track changes in body weight associated with

hyperketonemia.

A.2.4. Organ Weight and Collection

On day 29, rats were sacrificed 4-8 hrs after intragastric gavage which correlated to the

time range where the most significantly elevated blood βHB levels were observed. Brain, lungs,

liver, kidneys, spleen and heart were harvested, weighed (AWS-1000 1kg portable digital scale

(AWS, Charleston, SC)), and flash-frozen in liquid nitrogen or preserved in 4%

paraformaldehyde for future analysis.

A.2.5. Statistics

All data are presented as the mean ± standard deviation (SD). Data analysis was

performed using GraphPad PRISM™ version 6.0a and IBM SPSS Statistics 22.0. Results were

considered significant when p<0.05. Triglyceride and lipoprotein profile data were analyzed

using One-Way ANOVA. Blood ketone and blood glucose were compared to control at the

applicable time points using a Two-Way ANOVA. Correlation between blood βHB and glucose

levels in ketone supplemented rats was compared to controls using ANCOVA analysis. Organ

and body weights were analyzed using One-Way ANOVA. Basal blood ketone and blood

glucose levels were analyzed using Two-Way ANOVA. All mean comparisons were carried out

using Tukey’s multiple comparisons post-hoc test.

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A.2.6. Metabolomics Profiling of KE and BMS+MCT Treated Rats

On day 29, approximately 500 uL of whole blood was collected from the saphenous vein

of control, KE, and BMS+MCT-treated animals 4 hours post-intragastric gavage. Serum was

separated by centrifugation using Microtainer© Tubes with Serum Separator (Becton

Dickinson). Serum was transferred to screw-to cryovials and flash frozen in liquid nitrogen.

Frozen serum samples were shipped to Metabolon, Inc. for global metabolomics profiling using

liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography-mass

spectrometry (GC-MS). The concentration of 388 named biochemicals of known identity was

analyzed in the serum samples. Welch’s two-sample t-test was used to determine which

metabolites differed significantly in concentration between ketone supplemented animals and

controls. Results were considered significant when p<0.05.

A.3. Materials and Methods for Experiments included in Chapter 4: Enhancing Wound

Healing With Metabolic Therapy

A.3.1. Synthesis and Formulation of Ketone Precursors

BMS is a novel agent (sodium/potassium- βHB mineral salt) supplied as a 50% solution

containing approximately 375mg/g of pure βHB and 125 mg/g of sodium/potassium. Both KE

and BMS were developed and synthesized in collaboration with Savind Inc. Pharmaceutical

184

grade MCT oil (~65% caprylic triglyceride) was purchased from Now Foods (Bloomingdale,

IL). BMS was mixed in a 1:1 ratio with MCT at the University of South Florida (USF), yielding

25% water, 25% pure βHB mineral salt and 50% MCT. BD was purchased from Sigma-Aldrich

(Prod # B84785, Milwaukee, WI).

A.3.2 Animal Gavage to Induce Dietary Ketosis

Animal procedures were performed in accordance with the University of South Florida

Institutional Animal Care and Use Committee (IACUC) guidelines (Protocol #V4251) and

abided by all requirements of the Animal Welfare Act and the Guide for Care and Use of

Laboratory Animals. Healthy 8 month (young) and 20 month (aged) male Fisher 344 rats (Harlan

Laboratories and NIA) were randomly assigned to one of three groups: Control (water, n≥6), BD

(n≥6), or BMS+MCT (n≥6). Rats received one 6.5 g/kg dose of their respective treatments via

intragastric gavage. Animals had ad libitum access to standard rodent chow 2018 (Harlan

Teklad) for duration of the study. Whole blood samples (10 µL) were taken from the saphenous

vein for analysis of glucose and βHB levels with the commercially available glucose and ketone

monitoring system Precision Xtra™ (Abbott Laboratories, Abbott Park, IL). The Precision

Xtra™ ketone monitoring system only measures βHB blood levels; therefore, total blood ketone

levels would be higher than measured. Blood glucose and βHB were measured at 0, 0.5, 1, 4, 8,

12, and 24 hrs after test substance administration.

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A.3.3. Surgical Procedure

Healthy 8 month (young) and 20 month (aged) Fisher 344 rats (National Institutes of

Aging, Bethesda, MD) were utilized to create and ischemic wound model adapted from our

previous work [4-6]. In all Fisher 344 rats, a dorsal bi-pedicle 10.5x3 cm flap was surgically

created on day 0 (time of wounding), in which a silicone sheet was placed under the panniculus

carnosus fascia and above the paraspinous muscles, limiting revascularization and causing the

flap to become ischemic. Two 6mm punch biopsies created full thickness wounds extending just

above the fascia within the flap (ischemic wounds) and two 6mm punches were created laterally

to ischemic flap giving the control (non-ischemic) wounds in the same animal.

A.3.4. Diet Administration

The day before flap creation, animals are fasted overnight (~18hrs) to enable initiation of

the diet immediately upon waking from surgery. On the day of ischemic flap creation (day 0),

young and aged rats were randomly assigned to one of three study groups: Control standard diet

(SD), BD or BMS+MCT. Control rats were fed a combination of standard rodent chow (2018

Teklad Global 18% Protein Rodent Diet, Harlan Laboratories), water, and peanut butter mixed

into a pudding and fed ad libitum. Rats in BD group received a pudding diet of standard rodent

chow, peanut butter, water, 1% saccharine, and 20% 1,3-Butanediol by weight ad libitum. Rats

in the BMS+MCT group received a pudding diet of standard rodent chow, peanut butter, water,

1% saccharine, and 10% MCT oil by weight and 10% βHB mineral salt solution (10% βHB) by

weight ad libitum. Diets were continuously replaced to maintain freshness and to allow rat to

feed ad libitum.

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A.3.5. βHB, Glucose, and Weight Measurements

Every 7 days, whole blood samples (10 µL) were taken from the saphenous vein for

analysis of glucose and βHB levels with the commercially available glucose and ketone

monitoring system Precision Xtra™ (Abbott Laboratories, Abbott Park, IL) to confirm that the

dietary ingestion of BD and BMS+MCT induced continuous ketosis in the blood. Rats were

weighed 2x weekly with scales (Mittler Toledo SB16001) available in the Morsani College of

Medicine vivarium. Blood and weight measurements were taken at the same time of day each

week to limit variation based on normal metabolism fluctuations.

A.3.6. Analysis of Blood Flow and Wound Size

Every 7 days, while under anesthesia, Laser Doppler scanning was used to measure blood

flow over the course of the study. On days 3, 7, 10, 14, 21, 25, and 28 animals were anesthetized,

both ischemic and non-ischemic wounds were digitally photographed, and wound sizes were

determined (Bamboo Create v5, Wacom Co, Ltd, and Image J 64 Version 10.2).

A.3.7. Extraction of Tissue

On day 28 of the study, the rats were euthanized by carbon dioxide (CO2) asphyxiation

where periwound tissue (tissue surrounding the wound) including the wound, spleen and liver

were surgically harvested. Portions of the spleen, liver and wounds were flash frozen by liquid

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nitrogen, formalin-fixed, or preserved in optimal cutting temperature (OCT) freezing media for

future analysis.

A.3.8. Statistics

All data are presented as the mean ± standard deviation (SD). Data analysis was performed using

GraphPad PRISM™ version 6.0a. Results were considered significant when p<0.05. Blood

ketone and blood glucose were compared to controls at the applicable time points using a Two-

Way ANOVA. Correlation between blood βHB and glucose levels was determined by linear

regression analysis. Blood flow and wound healing progression were analyzed compared to

controls at the applicable time points using Two-Way ANOVA. Organ and body weights were

analyzed using One-Way ANOVA. All mean comparisons were carried out using Tukey’s

multiple comparisons post-hoc test.

A.4. Materials and Methods for Experiments included in Chapter 5: Potential Mechanisms

for Exogenous Ketone Supplementation to Enhance Wound Healing

A.4.1. Cytokine Array

After 3 days and 7 days post-wounding, young and aged Fischer rats were humanely

euthanized and per-wound tissue was harvested and preserved in liquid nitrogen. 500 mg of

periwound tissue was measured and razor blade minced on a petri dish in 400 µL of RIPA Lysis

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Buffer (Sigma Chemical Co, St. Louis, MO) with 10 µL / mL of Halt protease and phosphatase

inhibitor cocktail (Pierce, Rockford, IL). Minced tissue with liquid was placed into lysing

matrix-A tubes (MP Biomedical, Santa Ana, CA). An additional 400 µL of RIPA Lysis Buffer

was used to wash petri dish and added to the tube. Tissues were homogenized for 20 seconds

using FastPrep®-24 Instrument (MP Biomedical, Santa Ana, CA). Tubes were centrifuged at

16,000xg for 10 minutes, supernatants were transferred to a clean 1.5 mL conical tube, and

lysates were sonicated at low power setting and centrifuged at 16,000xg for 10 minutes. The

supernatant of the lysates was frozen at -80° for future use. Protein concentration of the lysate

supernatant was determined using Pierce BCA protein Assay Kit (Rockford, IL) reading

absorbance at 540 nanometers. 50µg of samples were sent to Eve Technologies Inc. (Calgary,

Alberta, Canada), where they were analyzed on Bio-Plex 200 Systems (Bio-Rad Laboratories,

Inc., Hercules, CA) via Rat Cytokine Array/ Chemokine Array 27-Plex/ Discovery Assay, a

multiplex assay that measures twenty-seven inflammatory biomarkers in a single microwell

(Millipore MILLIPLEX®, Billerica, MA).

A.4.2. Macrophage Profile Analysis with Immunofluorescence of CD68, IRF-5, CD206

After 3 days and 7 seven days post-wounding, young and aged Fischer 344 rats were

humanely euthanized and peri-wound tissue, livers, and spleen were harvested and preserved in

10% neutral, paraformaldehyde or phosphate buffered formalin. The peri-wound tissue was

paraffin-embedded, cut into 5-micron sections, and mounted onto microscope slides at the USF

Morsani College of Medicine Histology Core. One set of slides was stained with hematoxylin

and eosin and mounted with cover slips for morphological analysis. Another set of slides was co-

189

probed with either interferon regularity factor-5 (IRF-5) to label M1 pro-inflammatory

macrophages or CD206 to label M2 anti-inflammatory macrophages and CD68 to label all

macrophages. These slides were rehydrated through a series of xylene and ethanol washes, and

antigen was retrieved via standard heat-mediated citrate buffer method. Slides were incubated in

0.3% H2O2 in dH2O to inhibit endogenous peroxide activity. Immunofluorescent analysis of the

slides was performed using the VECTASTAIN Elite ABC Kit (Vector Laboratories Inc.,

Burlingame, CA). Slides were blocked for 60 minutes in normal anti-rabbit blocking serum, and

then incubated in anti-IRF-5 antibody (1:100, ab175317, Abcam) or anit-CD206 antibody

(1:100, ab64693, Abcam) over night at 4 degrees in a humidified container. Slides were then

incubated in ALEXA Fluor 568 donkey anti-rabbit secondary antibody (Life Technologies,

Eugene, OR) in normal blocking serum for 30 mints at room temperature in the dark, followed

by VECTASTAIN ABC reagent containing avidin and biotinylated horseradish peroxidase

solution for 30 minutes at room temperature in the dark. Slides were blocked for 60 minutes in

normal anti-mouse blocking serum in the dark, and then incubated in anti-CD68 antibody (1:25,

ab955, Abcam) over night at 4 degrees in a dark humidified container. Slides were then

incubated in ALEXA Fluor 488 donkey anti-mouse secondary antibody (Life Technologies,

Eugene, OR) in normal anti-mouse blocking serum for 30 mints at room temperature in the dark,

followed by VECTASTAIN ABC reagent containing avidin and biotinylated horseradish

peroxidase solution for 30 minutes at room temperature. Slides were wet mounted with

VECTASHIELD mounting media with DAPI (Vector Laboratories Inc., Burlingame, CA) and

sealed with clear nail polish.

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A.4.3. Cellular ROS production

DHE

Cells were grown on 6-well poly-d lysine coated plates in DMEM +Glutamax 4.5g/L

glucose media with 10% FBS and 1% penicillin and streptomycin until 50-60% confluent,

appropriate wells were grown with or without the addition of 5 mM βHB to the media for 72

hours. After 72 hours, they were then serum starved for 18 hrs with DMEM phenol red free 4.5

g/L glucose + L-Glutamine media with 0.2% FBS and 1% penicillin and streptomycin. At the

end of the 18 hours, cells were subjected to either 100uM tBOOH (Sigma Chemical Co, St.

Louis, MO) in serum starve media for three hours or media only. At the end of three hours, the

media was taken off and the cells were treated with 5 µM Dihydroethidium (DHE) in DMSO

(Life Technologies, Eugene, OR) dissolved in serum starve media for 30min. The cells were put

into Hank’s BSS and fluorescence was read on the plate reader at [528, 617] excitation and

emission. 3 wells in each grouping did not receive the DHE and the average RFUs of these wells

were used as a blank to normalize the readings. The control values have an N=3 (total N=9) and

the 100 µM has an N=6 (total N=18). Graph and statistics were created using GraphPad

PRISM™ for Mac OSX version 6.0a, statistical significance was determined using a one-way

ANOVA with Tukey’s post-hoc analysis.

Mitosox Red

Cells were grown on 6-well poly-d lysine coated plates in DMEM +Glutamax 4.5g/L

glucose media with 10% FBS and 1% penicillin and streptomycin until 50-60% confluent,

191

appropriate wells were grown with or without the addition of 5 mM βHB to the media for 72

hours. After 72 hours, they were then serum starved for 18 hrs with DMEM phenol red free 4.5

g/L glucose + L-Glutamine media with 0.2% FBS and 1% penicillin and streptomycin. Cells

were subjected to 100uM tBOOH (Sigma Chemical Co, St. Louis, MO) in serum starve media

for three hours or media only. At the end of three hours, the media was taken off and the cells

were treated with 1 µM Mitosox Red (Life Technologies, Eugene, OR) in Hank’s Balanced Salt

Solution (with magnesium and calcium) for 10 min. The cells were put into fresh Hank’s BSS

and fluorescence was read on the plate reader at [510, 580] excitation and emission. 3 wells in

each grouping did not receive the Mitosox Red and the average RFUs of these wells were used

as a blank to normalize the readings. The control values have an N=3 (total N=12) and the 100

uM has an N=6 (total N=24). Graph and statistics were created using GraphPad PRISM™ for

Mac OSX version 6.0a, statistical significance was determined using a one-way ANOVA with

Tukey’s post-hoc analysis.

A.4.4. Western Blot Analysis for in vitro and ex vivo SOD2 and NQO1

In Vitro

Cells were grown on 100 mm poly-d lysine coated petri dishes in DMEM +Glutamax 4.5

g/L glucose media with 10% FBS and 1% penicillin and streptomycin until cells were around 50-

60% confluent. Cells were changed to DMEM phenol red free 5mM glucose + L-Glutamine

media with 10% FBS and 1% penicillin and streptomycin and appropriate cells were treated with

5mM βHB for 72 hours. Media was replaced every 24 hours. After 72 hours, appropriate cells

were subjected to 100uM tBOOH (Sigma Chemical Co, St. Louis, MO) or media only. At the

192

end of three hours, the media was taken off, cells were washed with 1mL 1x cold PBS, then

incubated with 300 uL per well of Triton X-100 Lysis Buffer [containing 50mM Tris-HCl pH

7.4,150 mM NaCl, 5mM EDTA and 1% Triton-X 100], with 10 uL/ mL of Halt protease and

phosphatase inhibitor cocktail (Pierce, Rockford, IL) for 30 minutes at 4° Celsius. The cells were

scrape-harvested, and three wells were pooled for each sample. The lysates were sonicated at low

power setting and centrifuged at 16,000xg for 10 minutes. The supernatant of the lysates was

frozen at -80° for future use. Protein concentration of the lysate supernatant was determined

using Pierce BCA protein Assay Kit (Rockford, IL) reading absorbance at 540 nanometers. 20

µg of samples were separated on a 4-20% SDS gel and transferred to nitrocellulose membrane.

Membrane was blocked with 5% Milk in TBS-T (50 mM Tris HCl, 150 mM NaCl and 0.1%

Tween 20, pH 7.5) and then probed with anti-SOD2 antibody (1:2500, ab13533, Abcam), anti-

NQO1 antibody (1:500, ab34173, Abcam) followed by anti Pan-Actin antibody (1:3000, Cell

Signaling, Boston, MA) as a loading control and Anti-Rabbit IgG, HRP-linked (1:3000, Cell

Signaling, Boston, MA) as secondary antibody. Membrane was developed using Pierce ECL

Western Blotting Substrate (Rockford, IL). Quantitative densitometry was determined using

Image J software version 1.45s and graph and statistics were determined using GraphPad

PRISM™ for Mac OSX version 6.0a. Data in graph is represented as an N=3 for each group.

Ex vivo

After 3 days and 7 days post-wounding, young and aged Fischer rats were humanely

euthanized and per-wound tissue was harvested and preserved in liquid nitrogen. 500 mg of

periwound tissue was measured and razor blade minced on a petri dish in 400 µL of RIPA Lysis

193

Buffer (Sigma Chemical Co, St. Louis, MO) with 10 µL / mL of Halt protease and phosphatase

inhibitor cocktail (Pierce, Rockford, IL). Minced tissue with liquid was placed into lysing

matrix-A tubes (MP Biomedical, Santa Ana, CA). An additional 400 µL of RIPA Lysis Buffer

was used to wash petri dish and added to the tube. Tissues were homogenized for 20 seconds

using FastPrep®-24 Instrument (MP Biomedical, Santa Ana, CA). Tubes were centrifuged at

16,000xg for 10 minutes, supernatants were transferred to a clean 1.5 mL conical tube, and

lysates were sonicated at low power setting and centrifuged at 16,000xg for 10 minutes. The

supernatant of the lysates was frozen at -80° for future use. Protein concentration of the lysate

supernatant was determined using Pierce BCA protein Assay Kit (Rockford, IL) reading

absorbance at 540 nanometers. 20 µg of samples were separated on a 4-20% SDS gel and

transferred to nitrocellulose membrane. Membrane was blocked with 5% Milk in TBS-T (50 mM

Tris HCl, 150 mM NaCl and 0.1% Tween 20, pH 7.5) and then probed with anti-SOD2 antibody

(1:2500, ab13533, Abcam), anti-NQO1 antibody (1:500, ab34173, Abcam) followed by anti Pan-

Actin antibody (1:3000, Cell Signaling, Boston, MA) as a loading control and Anti-Rabbit IgG,

HRP-linked (1:3000, Cell Signaling, Boston, MA) as secondary antibody. Membrane was

developed using Pierce ECL Western Blotting Substrate (Rockford, IL). Quantitative

densitometry was determined using Image J software version 1.45s and graph and statistics were

determined using GraphPad PRISM™ for Mac OSX version 6.0a. Data in graph is represented

as an N=6 for each group.

194

A.4.5 Immunohistochemistry Analysis of Periwound Tissue Vascularization

After 3 days and 7 seven days post-wounding, young and aged Fischer 344 rats were

humanely euthanized and peri-wound tissue, livers, and spleen were harvested and preserved in

10% neutral, phosphate buffered formalin. The peri-wound tissue was paraffin-embedded, cut

into 5-micron sections, and mounted onto microscope slides at the USF Morsani College of

Medicine Histology Core. One set of slides was stained with hematoxylin and eosin and mounted

with cover slips for morphological analysis. Another set of slides was probed for the endothelial

cell marker CD31 to visualize blood vessels. These slides were rehydrated through a series of

xylene and ethanol washes, and antigen was retrieved via standard heat-mediated citrate buffer

method. Slides were incubated in 0.3% H2O2 in dH2O to inhibit endogenous peroxide activity.

Immunohistochemical analysis of the slides was performed using the VECTASTAIN Elite ABC

Kit (Vector Laboratories Inc., Burlingame, CA). Slides were blocked for 60 minutes in normal

blocking serum, and then incubated in anti-CD31 antibody (1:25, ab28364, Abcam) over night at

4 degrees Celsius in a humidified container. Slides were then incubated in biotinylated anti-

rabbit secondary antibody in normal blocking serum for 30 mints at room temperature, followed

by VECTASTAIN ABC reagent containing avidin and biotinylated horseradish peroxidase

solution for 30 minutes at room temperature. Slides were developed using the VECTOR

NovaRED Peroxidase (HRP) Substrate Kit (Vector Laboratories Inc., Burlingame, CA). Slides

were counterstained with hematoxylin, washed, mounted with coverslips, sealed with clear nail

polish, and visualized and scanned with light microscope via Moffitt Core Facilities. The entire

periwound tissue was analyzed for blood vessels. The number of CD31+ blood vessels in the

peri wound tissue were counted between control and treated groups as a measure of wound

195

vascularization. Statistical analysis was performed via one-way ANOVA with Tukey’s post-hoc

analysis. Results were considered significant when p<0.05.

A.4.6 Migration Analysis of young and aged HDFs

Cells were grown on 6-well poly-d lysine coated plates in DMEM +Glutamax 4.5g/L

glucose media with 10% FBS and 1% penicillin and streptomycin until 75-80% confluent. Cells

were changed to DMEM phenol red free 5mM glucose + L-Glutamine media with 10% FBS and

1% penicillin and streptomycin and appropriate cells were treated with 5mM βHB for 72 hours.

Media was replaced every 24 hours. After 72 hours, cells were then serum starved for 18 hrs

with DMEM phenol red free 4.5 g/L glucose + L-Glutamine media with 0.2% FBS and 1%

penicillin and streptomycin. There was not a need to use mytomycin to stop proliferation since

used the serum free media. After 18 hours, two scratches were created per well using a 200 µL

pipette tip. Wells were rinsed with 1mL RT Hank’s BBS to remove floating and loose cells.

Wells were visualized using phase contrast microscopy and a picture was taken for baseline

comparison. After 18 hours, cells were fixed with 4% paraformaldehyde for 15 minutes, rinsed

with RT 1x PBS, and a cover slip was placed in the well using VECTASHIELD mounting media

with DAPI (Vector Laboratories Inc., Burlingame, CA) and sealed with clear nail polish. Wells

were visualized using phase contrast and fluorescent microscopy, and number of cells that

migrated into scratch area was counted. Statistical analysis was performed via one-way ANOVA

with Tukey’s post-hoc analysis. Results were considered significant when p<0.05.

196

A.4.7. Proliferation Analysis of young and aged HDFs

75,000 young and aged HDF cells were seeded on 6-well poly-d lysine coated plates in

DMEM phenol red free 5mM glucose + L-Glutamine media with 10% FBS and 1% penicillin

and streptomycin with or without the presence of 5 mM βHB. Media was replaced every 24

hours. Total cell number was counted at 24, 48, 72, 96, and 120 hours post seeding. At given

time point, media was removed and cells were washed with 1 mL sterile RT 1x PBS, cells were

suspended using 250 µL (0.25% Trypsin in Hank’s Phenol Red BBS) and 750 µL of appropriate

media (with or with out βHB). Total cell density was measured using hemocytometry. Two-way

ANOVA with Tukey’s post-hoc analysis, results were considered significant when p<0.05. Error

Bars Represent mean (SD).

A.4.8. Lactate Quantification of young and aged HDFs

Young and aged primary HDFs were seeded with 250,000 cells on 6-well poly-d lysine

coated plates in DMEM phenol red free 5mM glucose + L-Glutamine media with 10% FBS and

1% penicillin and streptomycin with or without the presence of 5 mM βHB. Extracellular lactate

was measured in 10 µL of cellular media at 24, 48 and 72 hours post-seeding using Nova

Biomedical© Lactate Plus (Nova Biomedical, Waltham, MA) blood lactate measuring meter.

Two-way ANOVA with Tukey’s post-hoc analysis, results were considered significant when

p<0.05. Error Bars Represent mean (SD).

197

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2. WarnickG,KnoppR,FitzpatrickV,BransonL:Estimatinglow-densitylipoproteincholesterolbytheFriedewaldequationisadequateforclassifyingpatientsonthe basis of nationally recommended cutpoints. Clinical chemistry 1990,36(1):15-19.

3. FriedewaldW, Levy R, FredricksonD:Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparativeultracentrifuge.Clinicalchemistry1972,18(6):499-502.

4. Moor AN, Tummel E, Prather JL, Jung M, Lopez JJ, Connors S, Gould LJ:Consequencesof ageon ischemicwoundhealing in rats: alteredantioxidantactivity and delayed wound closure. Age (Dordrecht, Netherlands) 2014,36(2):733-748.

5. Gould L, Leong M, Sonstein J, Wilson S: Optimization and validation of anischemicwoundmodel.Woundrepairandregeneration:officialpublicationoftheWoundHealing Society [and] the EuropeanTissue Repair Society2005,13(6):576-582.

6. Trujillo AN, Kesl SL, Sherwood J, Wu M, Gould LJ: Demonstration of the ratischemicskinwoundmodel.Journalofvisualizedexperiments:JoVE2014(98).

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APPENDIX B: COPYRIGHT PERMISSIONS

B.1 Copyright Permissions for: Trujilio AN*, Kesl SL*, Sherwood J, Wu M, Gould LJ. (2014)

Demonstration of the Rat Ischemic Wound Model. Journal of Visualized Experiments: JoVE ”.

*Indicates that both authors contributed equally in the work.

Permission for inclusion of the aforementioned publication was granted via email (see below).

B.2 Copy Right Permissions for: Kesl SL, Poff AM, Ward NP, Fiorelli TN, Ari C, Van Putten

AJ, Sherwood JW, Arnold P, D’Agostino DP. Effects of Exogenous Ketone Supplementation on

Blood Ketone, Glucose, Triglyceride, and Lipoprotein Levels in Sprague-Dawley Rats. Nutrition

and Metabolism (2016).

199

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APPENDIX C: PUBLISHED MANUSCRIPTS

This appendix contains the original publications for the following references:

Trujilio AN*, Kesl SL*, Sherwood J, Wu M, Gould LJ. (2014) Demonstration of the Rat

Ischemic Wound Model. Journal of Visualized Experiments: JoVE. * Indicates that both authors

contributed equally to the work.

Kesl SL, Poff AM, Ward NP, Fiorelli TN, Ari C, Van Putten AJ, Sherwood JW, Arnold P,

D’Agostino DP. Effects of Exogenous Ketone Supplementation on Blood Ketone, Glucose,

Triglyceride, and Lipoprotein Levels in Sprague-Dawley Rats. Nutrition and Metabolism. 2016.

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RESEARCH Open Access

Effects of exogenous ketonesupplementation on blood ketone, glucose,triglyceride, and lipoprotein levels inSprague–Dawley ratsShannon L. Kesl1*, Angela M. Poff1, Nathan P. Ward1, Tina N. Fiorelli1, Csilla Ari1, Ashley J. Van Putten1,Jacob W. Sherwood1, Patrick Arnold2 and Dominic P. D’Agostino1

Abstract

Background: Nutritional ketosis induced by the ketogenic diet (KD) has therapeutic applications for many diseasestates. We hypothesized that oral administration of exogenous ketone supplements could produce sustainednutritional ketosis (>0.5 mM) without carbohydrate restriction.

Methods: We tested the effects of 28-day administration of five ketone supplements on blood glucose, ketones,and lipids in male Sprague–Dawley rats. The supplements included: 1,3-butanediol (BD), a sodium/potassium β-hydroxybutyrate (βHB) mineral salt (BMS), medium chain triglyceride oil (MCT), BMS + MCT 1:1 mixture, and 1,3butanediol acetoacetate diester (KE). Rats received a daily 5–10 g/kg dose of their respective ketone supplement viaintragastric gavage during treatment. Weekly whole blood samples were taken for analysis of glucose and βHB atbaseline and, 0.5, 1, 4, 8, and 12 h post-gavage, or until βHB returned to baseline. At 28 days, triglycerides, totalcholesterol and high-density lipoprotein (HDL) were measured.

Results: Exogenous ketone supplementation caused a rapid and sustained elevation of βHB, reduction of glucose,and little change to lipid biomarkers compared to control animals.

Conclusions: This study demonstrates the efficacy and tolerability of oral exogenous ketone supplementation ininducing nutritional ketosis independent of dietary restriction.

Keywords: Ketogenic diet, Ketone ester, Ketone supplement, Appetite, β-hydroxybutyrate, Hyperketonemia,Triglycerides

BackgroundEmerging evidence supports the therapeutic potential ofthe ketogenic diet (KD) for a variety of disease states,leading investigators to research methods of harnessingthe benefits of nutritional ketosis without the dietaryrestrictions. The KD has been used as an effective non-pharmacological therapy for pediatric intractable sei-zures since the 1920s [1–3]. In addition to epilepsy, theketogenic diet has elicited significant therapeutic effects

for weight loss and type-2 diabetes (T2D) [4]. Severalstudies have shown significant weight loss on a high fat,low carbohydrate diet without significant elevations ofserum cholesterol [5–12]. Another study demonstratedthe safety and benefits of long-term application of theKD in T2D patients. Patients exhibited significant weightloss, reduction of blood glucose, and improvement oflipid markers after eating a well-formulated KD for56 weeks [13]. Recently, researchers have begun toinvestigate the use of the KD as a treatment for acne,polycystic ovary syndrome (PCOS), cancer, amyotrophiclateral sclerosis (ALS), traumatic brain injury (TBI) andAlzheimer’s disease (AD) with promising preliminaryresults [14–26].

* Correspondence: skesl@health.usf.edu1Department of Molecular Pharmacology and Physiology, Morsani College ofMedicine, University of South Florida, 12901 Bruce B. Downs Blvd. MDC8,Tampa, FL 33612, USAFull list of author information is available at the end of the article

© 2016 Kesl et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Kesl et al. Nutrition & Metabolism (2016) 13:9 DOI 10.1186/s12986-016-0069-y

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The classical KD consists of a 4:1 ratio of fat to pro-tein and carbohydrate, with 80–90 % of total calories de-rived from fat [27]. The macronutrient ratio of the KDinduces a metabolic shift towards fatty acid oxidationand hepatic ketogenesis, elevating the ketone bodiesacetoacetate (AcAc) and β-hydroxybutyrate (βHB) in theblood. Acetone, generated by decarboxylation of AcAc, hasbeen shown to have anticonvulsant properties [28–32].Ketone bodies are naturally elevated to serve as alternativemetabolic substrates for extra-hepatic tissues during theprolonged reduction of glucose availability, suppression ofinsulin, and depletion of liver glycogen, such as occursduring starvation, fasting, vigorous exercise, calorierestriction, or the KD. Although the KD has cleartherapeutic potential, several factors limit the efficacyand utility of this metabolic therapy for widespreadclinical use. Patient compliance to the KD can be lowdue to the severe dietary restriction - the diet beinggenerally perceived as unpalatable - and intolerance tohigh-fat ingestion. Maintaining ketosis can be difficultas consumption of even a small quantity of carbohydratesor excess protein can rapidly inhibit ketogenesis [33, 34].Furthermore, enhanced ketone body production and tissueutilization by the tissues can take several weeks (keto-adap-tation), and patients may experience mild hypoglycemicsymptoms during this transitional period [35].Recent studies suggest that many of the benefits of the

KD are due to the effects of ketone body metabolism.Interestingly, in studies on T2D patients, improved gly-cemic control, improved lipid markers, and retraction ofinsulin and other medications occurred before weightloss became significant. Both βHB and AcAc have beenshown to decrease mitochondrial reactive oxygen species(ROS) production [36–39]. Veech et al. have summarizedthe potential therapeutic uses for ketone bodies [28, 40].They have demonstrated that exogenous ketones favorablyalter mitochondrial bioenergetics to reduce the mitochon-drial NAD couple, oxidize the co-enzyme Q, and increasethe ΔG’ (free enthalpy) of ATP hydrolysis [41]. Ketonebodies have been shown to increase the hydraulic effi-ciency of the heart by 28 %, simultaneously decreasingoxygen consumption while increasing ATP production[42]. Thus, elevated ketone bodies increase metabolic effi-ciency and as a consequence, reduce superoxide productionand increase reduced glutathione [28]. Sullivan et al. demon-strated that mice fed a KD for 10–12 days showed increasedhippocampal uncoupling proteins, indicative of decreasedmitochondrial-produced ROS [43]. Bough et al. showed anincrease of mitochondrial biogenesis in rats maintained on aKD for 4–6 weeks [44, 45]. Recently, Shimazu et al. reportedthat βHB is an exogenous and specific inhibitor of class Ihistone deacetylases (HDACs), which confers protectionagainst oxidative stress [38]. Ketone bodies have also beenshown to suppress inflammation by decreasing the

inflammatory markers TNF-a, IL-6, IL-8, MCP-1, E-selectin,I-CAM, and PAI-1 [8, 46, 47]. Therefore, it is thought thatketone bodies themselves confer many of the benefits asso-ciated with the KD.Considering both the broad therapeutic potential and

limitations of the KD, an oral exogenous ketone supple-ment capable of inducing sustained therapeutic ketosiswithout the need for dietary restriction would serve as apractical alternative. Several natural and synthetic ketonesupplements capable of inducing nutritional ketosis havebeen identified. Desrochers et al. elevated ketone bodiesin the blood of pigs (>0.5 mM) using exogenous ketonesupplements: (R, S)-1,3 butanediol and (R, S)-1,3butanediol-acetoacetate monoesters and diester [48]. In2012, Clarke et al. demonstrated the safety and efficacyof chronic oral administration of a ketone monoester ofR-βHB in rats and humans [49, 50]. Subjects maintainedelevated blood ketones without dietary restriction andexperienced little to no adverse side effects, demonstratingthe potential to circumvent the restrictive diet typicallyneeded to achieve therapeutic ketosis. We hypothesized thatexogenous ketone supplements could produce sustainedhyperketonemia (>0.5 mM) without dietary restriction andwithout negatively influencing metabolic biomarkers, suchas blood glucose, total cholesterol, HDL, LDL, and triglycer-ides. Thus, we measured these biomarkers during a 28-dayadministration of the following ketone supplements in rats:naturally-derived ketogenic supplements included mediumchain triglyceride oil (MCT), sodium/potassium -βHBmineral salt (BMS), and sodium/potassium -βHB mineralsalt +medium chain triglyceride oil 1:1 mixture (BMS +MCT) and synthetically produced ketogenic supplementsincluded 1, 3-butanediol (BD), 1, 3-butanediol acetoace-tate diester/ ketone ester (KE).

MethodsSynthesis and formulation of ketone supplementsKE was synthesized as previously described [29]. BMS isa novel agent (sodium/potassium- βHB mineral salt)supplied as a 50 % solution containing approximately375 mg/g of pure βHB and 125 mg/g of sodium/potas-sium. Both KE and BMS were developed and synthesizedin collaboration with Savind Inc. Pharmaceutical gradeMCT oil (~65 % caprylic triglyceride; 45 % capric trigly-ceride) was purchased from Now Foods (Bloomingdale,IL). BMS was formulated in a 1:1 ratio with MCT at theUniversity of South Florida (USF), yielding a final mix-ture of 25 % water, 25 % pure βHB mineral salt and50 % MCT. BD was purchased from Sigma-Aldrich(Prod # B84785, Milwaukee, WI).

Daily gavage to induce dietary ketosisAnimal procedures were performed in accordance withthe University of South Florida Institutional Animal

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Care and Use Committee (IACUC) guidelines (Protocol#0006R). Juvenile male Sprague–Dawley rats (275–325 g,Harlan Laboratories) were randomly assigned to one ofsix study groups: control (water, n = 11), BD (n = 11), KE(n = 11), MCT (n = 10), BMS (n = 11), or BMS +MCT(n = 12). Caloric density of standard rodent chow anddose of ketone supplements are listed in Table 1. Ondays 1–14, rats received a 5 g/kg body weight dose oftheir respective treatments via intragastric gavage.Dosage was increased to 10 g/kg body weight for thesecond half of the study (days 15–28) for all groupsexcept BD and KE to prevent excessive hyperketone-mia (ketoacidosis). Each daily dose of BMS would equal~1000–1500 mg of βHB, depending on the weight of theanimal. Intragastric gavage was performed at the same timedaily, and animals had ad libitum access to standardrodent chow 2018 (Harlan Teklad) for the duration ofthe study. The macronutrient ratio the standard rodentchow was 62.2, 23.8 and 14 % of carbohydrates, proteinand fat respectively.

Measurement and analysis of blood glucose, ketones,and lipidsEvery 7 days, animals were briefly fasted (4 h, wateravailable) prior to intragastric gavage to standardizelevels of blood metabolites prior to glucose and βHBmeasurements at baseline. Baseline (time 0) was imme-diately prior to gavage. Whole blood samples (10 μL)were taken from the saphenous vein for analysis ofglucose and βHB levels with the commercially avail-able glucose and ketone monitoring system PrecisionXtra™ (Abbott Laboratories, Abbott Park, IL). Bloodglucose and βHB were measured at 0, 0.5, 1, 4, 8,and 12 h after test substance administration, or untilβHB returned to baseline levels. Food was returned toanimals after blood analysis at time 0 and gavage. Atbaseline and week 4, whole blood samples (10 μL)were taken from the saphenous vein immediatelyprior to gavage (time 0) for analysis of total choles-terol, high-density lipoprotein (HDL), and triglycerideswith the commercially available CardioChek™ bloodlipid analyzer (Polymer Technology Systems, Inc., In-dianapolis, IN). Low-density lipoprotein (LDL)

cholesterol was calculated from the three measuredlipid levels using the Friedewald equation: (LDL Choles-terol = Total Cholesterol - HDL - (Triglycerides/5)) [51,52]. Animals were weighed once per week to trackchanges in body weight associated with hyperketonemia.

Organ weight and collectionOn day 29, rats were sacrificed via deep isofluraneanesthesia, exsanguination by cardiac puncture, and de-capitation 4–8 h after intragastric gavage, which correlatedto the time range where the most significantly elevatedblood βHB levels were observed. Brain, lungs, liver,kidneys, spleen and heart were harvested, weighed (AWS-1000 1 kg portable digital scale (AWS, Charleston, SC)),and flash-frozen in liquid nitrogen or preserved in 4 %paraformaldehyde for future analysis.

StatisticsAll data are presented as the mean ± standard deviation(SD). Data analysis was performed using GraphPadPRISM™ version 6.0a and IBM SPSS Statistics 22.0. Re-sults were considered significant when p < 0.05. Trigly-ceride and lipoprotein profile data were analyzed usingOne-Way ANOVA. Blood ketone and blood glucosewere compared to control at the applicable time pointsusing a Two-Way ANOVA. Correlation between bloodβHB and glucose levels in ketone supplemented rats wascompared to controls using ANCOVA analysis. Organand body weights were analyzed using One-WayANOVA. Basal blood ketone and blood glucose levelswere analyzed using Two-Way ANOVA. All mean com-parisons were carried out using Tukey’s multiple com-parisons post-hoc test.

ResultsEffect of ketone supplementation on triglycerides andlipoproteinsBaseline measurements showed no significant changesin triglycerides or the lipoproteins (data not shown).Data represent triglyceride and lipoprotein concentra-tions measured after 4 weeks of daily exogenous ketonesupplementation. No significant change in total choles-terol was observed at 4 weeks for any of the ketonetreatment groups compared to control. (Fig. 1a). No

Table 1 Caloric density and dose of ketone supplementsMacronutrient Information Standard Diet Water BMS + MCT BMS MCT KE BD

% Cal from Fat 18.0 0.0 50.0 N/A 100.0 N/A N/A

% Cal from Protein 24.0 0.0 N/A N/A 0.0 N/A N/A

% Cal from Carbohydrates 58.0 0.0 N/A N/A 0.0 N/A N/A

Total Caloric Density (Kcal/g) 3.1 0.0 5.1 1.9 8.3 5.6 6.0

Dose 0–14 Days (g/kg) ad libitum N/A 5.0 5.0 5.0 5.0 5.0

Dose 15–28 Days (g/kg) ad libitum N/A 10.0 10.0 10.0 5.0 5.0

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significant difference was detected in triglycerides forany ketone supplement compared to control (Fig. 1b).MCT supplemented animals had a significant reductionin HDL blood levels compared to control (p < 0.001)(Fig. 1c). LDL levels in ketone-supplemented animals didnot significantly differ from controls (Fig. 1d).

Ketone supplementation causes rapid and sustainedelevation of βHBOver the 28-day experiment, ketone supplements ad-ministered daily significantly elevated blood ketonelevels without dietary restriction (Fig. 2a, b). Naturallyderived ketogenic supplements including MCT (5 g/kg)elicited a significant rapid elevation in blood βHB within30–60 min that was sustained for 8 h. BMS +MCT (5 g/kg) elicited a significant elevation in blood βHB at 4 h,which was no longer significant at 8 h. BMS (5 g/kg) didnot elicit a significant elevation in blood βHB at anytime point. For days 14–28, BMS +MCT (10 g/kg) andMCT (10 g/kg) elevated blood βHB levels within 30 minand remained significantly elevated for up to 12 h. Weobserved a delay in the peak elevation of blood βHB:

BMS +MCT peaked at 8 h instead of at 4 h and MCT at4 h instead of at 1 h. Blood βHB levels in the BMS groupdid not show significant elevation at any time point,even after dose escalation (Fig. 2a). Synthetically derivedketogenic supplements including KE and BD supple-mentation rapidly elevated blood βHB within 30 minand was sustained for 8 h. For the rats receiving ketonesupplementation in the form of BD or the KE, dosagewas kept at 5 g/kg to prevent adverse effects associatedwith hyperketonemia. The Precision Xtra™ ketone moni-toring system measures βHB only; therefore, total bloodketone levels (βHB +AcAc) would be higher than mea-sured. For each of these groups, the blood βHB profileremained consistent following daily ketone supplementa-tion administration over the 4-week duration. (Fig. 2b).

Ketone supplementation causes a significant decrease ofblood glucoseAdministration of ketone supplementation significantlyreduced blood glucose over the course of the study(Fig. 3a, b). MCT (5 g/kg) decreased blood glucose com-pared to control within 30 min which was sustained for

Fig. 1 Effects of ketone supplementation on triglycerides and lipoproteins: Ketone supplementation causes little change in triglycerides andlipoproteins over a 4-week study. Graphs show concentrations at 4-weeks of total cholesterol (a), Triglycerides (b), LDL (c), and HDL (d).MCT supplemented rats had signfiicantly reduced concentration of HDL blood levels compared to control (p < 0.001) (b). One-Way ANOVAwith Tukey’s post hoc test, results considered significant if p < 0.05. Error bars represent mean (SD)

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Fig. 2 Effects of ketone supplementation on blood βHB. a, b Blood βHB levels at times 0, 0.5, 1, 4, 8, and 12 h post intragastric gavage for ketonesupplements tested. a BMS +MCT and MCT supplementation rapidly elevated and sustained significant βHB elevation compared to controls forthe duration of the 4-week dose escalation study. BMS did not significantly elevate βHB at any time point tested compared to controls. b BD andKE supplements, maintained at 5 g/kg, significantly elevated βHB levels for the duration of the 4-week study. Two-Way ANOVA with Tukey’s posthoc test, results considered significant if p < 0.05. Error bars represent mean (SD)

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Fig. 3 Effects of ketone supplementation on blood glucose. a, b Blood glucose levels at times 0, 0.5, 1, 4, 8, and 12 h (for 10 dose) post intragastricgavage for ketone supplements tested. a Ketone supplements BMS +MCT and MCT significantly reduced blood glucose levels compared to controlsfor the duration of the 4-week study. BMS significantly lowered blood glucose only at 8 h/week 1 and 12 h/week 3 (b) KE, maintained at5 g/kg, significantly reduced blood glucose compared to controls from week 1–4. BD did not significantly affect blood glucose levels atany time point during the 4-week study. Two-Way ANOVA with Tukey’s post hoc test, results considered significant if p < 0.05. Error barsrepresent mean (SD)

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8 h at baseline and at week 1. MCT (10 g/kg) likewisedecreased blood glucose within 30 min and lastedthrough the 12 h time point during weeks 2, 3, and 4.BMS +MCT (5 g/kg) lowered blood glucose comparedto control from hours 1–8 only at week 1. BMS +MCT(10 g/kg) lowered blood glucose compared to controlwithin 30 min and remained low through the 12 h timepoint at weeks 2, 3, and 4. Rats supplemented with BMShad lower blood glucose compared to control at 12 h inweek 4 (10) (Fig. 3a). Administration of BD did not sig-nificantly change blood glucose levels at any time pointduring the 4-week study. KE (5 g/kg) significantly low-ered blood glucose levels at 30 min for week 1, 2, 3, and

4 and was sustained through 1 h at weeks 2–4 and sus-tained to 4 h at week 3. (Fig. 3b).

Hyperketonemia suppresses blood glucose levelsAt baseline, 4 h after intragastric gavage, the elevation ofblood ketones was inversely related to the reduction of bloodglucose compared to controls following the administrationof MCT (5 g/kg) (p = 0.008) and BMS+MCT (5 g/kg) (p =0.039) . There was no significant correlation between bloodketone levels and blood glucose levels compared to controlsfor any other ketone supplemented group at baseline(Fig. 4a). At week 4, 4 h after intragastric gavage, there was asignificant correlation between blood ketone levels and

Fig. 4 Relationship between blood ketone and glucose levels: a BMS +MCT (5 g/kg) supplemented rats demonstrated a significant inverse relationshipbetween elevated blood ketone levels and decreased blood ketone levels (r2 = 0.4314, p = 0.0203). b At week 4, BMS +MCT (10 g/kg) and MCT (10 g/kg)showed a significant correlation between blood ketone levels and blood glucose levels (r2 = 0.8619, p < 0.0001; r2 = 0.6365, p = 0.0057). Linear regressionanalysis, results considered significant if p < 0.05

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blood glucose levels compared to controls in MCT (10 g/kg)and BMS+MCT (10 g/kg) (p < 0.0001, p < 0.0001) (Fig. 4b).

Ketone supplementation changes organ weight anddecreases body weightAt day 29 of the study, animals were euthanized andbrain, lungs, liver, kidneys, spleen and heart were har-vested and weighed. Organ weights were normalized tobody weight. Ketone supplementation did not signifi-cantly change brain, lung, kidney, or heart weights

compared to controls (Fig. 5a, b, d, f ). MCT supple-mented animals had significantly larger livers comparedto their body weight (p < 0.05) (Fig. 5c). Ketone supple-ments BMS +MCT, MCT and BD caused a significantreduction in spleen size (BMS +MCT p < 0.05, MCT p< 0.001, BD p < 0.05) (Fig. 5e). Rats administered KEgained significantly less weight over the entire studycompared to controls. BMS +MCT, BMS, and BD sup-plemented rats gained significantly less weight than con-trols during weeks 2 – 4, and MCT animals gained less

Fig. 5 Effects of ketone supplementation on organ weight: Data is represented as a percentage of organ weight to body weight. a, b, d, fKetone supplements did not significantly affect the weight of the brain, lungs, kidneys or heart. c Liver weight was significantly increased ascompared to body weight in response to administered MCT ketone supplement compared to control at the end of the study (day 29) (p < 0.001).e Rats supplemented with BMS +MCT, MCT, and BD had significantly smaller spleen percentage as compared to controls (p < 0.05, p < 0.001, p < 0.05).Two-Way ANOVA with Tukey’s post-hoc test; results considered significant if p < 0.05. Error bars represent mean (SD)

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weight than controls at weeks 3 – 4 (Fig. 6). Increasedgastric motility (increased bowel evacuation and changesto fecal consistency) was visually observed in rats sup-plemented with 10 g/kg MCT, most notably at the 8 and12-h time points. All animals remained in healthy weightrange for their age even though the rate of weight gainchanged with ketone supplementation [53–54]. Food in-take was not measured in this study. However, there wasnot a significant change in basal blood glucose or basalblood ketone levels over the 4 week study in any of therats supplemented with ketones (Fig. 7).

DiscussionNutritional ketosis induced with the KD has proveneffective for the metabolic management of seizures andpotentially other disorders [1–26]. Here we presentevidence that chronic administration of ketone supple-ments can induce a state of nutritional ketosis withoutthe need for dietary carbohydrate restriction and withlittle or no effect on lipid biomarkers. The notion thatwe can produce the therapeutic effects of the KD withexogenous ketone supplementation is supported by ourprevious study which demonstrated that acutely admin-istered KE supplementation delays central nervoussystem (CNS) oxygen toxicity seizures without theneed for dietary restriction [29]. We propose thatexogenous ketone supplementation could provide analternative method of attaining the therapeutic

benefits of nutritional ketosis, and as a means tofurther augment the therapeutic potential of the KD.

Ketone supplementation causes little to no change intriglycerides and lipoproteinsOne common concern regarding the KD is its purportedpotential to increase the risk of atherosclerosis by elevat-ing blood cholesterol and triglyceride levels [55, 56]. Thistopic remains controversial as some, but not all, studieshave demonstrated that the KD elevates blood levels ofcholesterol and triglycerides [57–62]. Kwitervich and col-leagues demonstrated an increase in low-density lipopro-tein (LDL) and a decrease in high-density lipoprotein(HDL) in epileptic children fed the classical KD fortwo years [27]. In this study, total cholesterol in-creased by ~130 %, and stabilized at the elevated levelover the 2-year period. A similar study demonstrated thatthe lipid profile returned to baseline in children whoremained on the KD for six years [63]. Children typicallyremain on the diet for approximately two years then re-turn to a diet of common fat and carbohydrate ingestion[64]. The implications of these findings are unclear, sincethe influence of cholesterol on cardiovascular health iscontroversial and macronutrient sources of the diet varyper study. In contrast to these studies, the majority of re-cent studies have suggested that the KD can actually leadto significant benefits in biomarkers of metabolic health,including blood lipid profiles [65–72]. In these studies, the

Fig. 6 Effects of ketone supplementation on body weight: Rats administered ketone supplements gained less weight over the 4-week period;however, did not lose weight and maintained healthy range for age. KE supplemented rats gained significantly less weight during the entire4-week study compared to controls. BMS +MCT, BMS, and BD supplemented rats gained significantly less weight than controls over weeks 2–4.MCTsupplemented rats gained significantly less weight than controls over weeks 3–4, Two-Way ANOVA with Tukey’s post hoc test, results consideredsignificant if p < 0.05. Error bars represent mean (SD)

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KD positively altered blood lipids, decreasing total triglyc-erides and cholesterol while increasing the ratio of HDLto LDL [68–77]. Although, the KD is well-established inchildren, it has only recently been utilized as a strategy tocontrol seizures in adults. In 2014, Schoeler and col-leagues reported on the feasibility of the KD for adults,concluding that 39 % of individuals achieved > 50 % reduc-tion in seizure frequency, similar to the results reported inpediatric studies. Patients experienced similar gastrointes-tinal adverse advents that have been previously describedin pediatric patients, but they did not lead to discontinu-ation of the diet in any patient [78].With oral ketone supplementation, we observed a sig-

nificant elevation in blood βHB without dietary restric-tion and with little change in lipid biomarkers (Fig. 1).Over the 4 week study, MCT-supplemented rats demon-strated decreased HDL compared to controls. No signifi-cant changes were observed in any of the triglycerides orlipoproteins (HDL, LDL) with any of the remaining ex-ogenously applied ketone supplements. It should benoted that the rats used for this study had not yetreached full adult body size [79]. Their normal growthrate and maturation was likely responsible for the

changes in triglyceride and lipoprotein levels observed inthe control animals over the 4 week study (baseline datanot shown, no significant differences) [80, 81]. Futurestudies are needed to investigate the effect of ketonesupplementation on fully mature and aged animals.Overall, our study suggests that oral ketone supplemen-tation has little effect on the triglyceride or lipoproteinprofile after 4 weeks. However, it is currently unknown ifketone supplementation would affect lipid biomarkersafter a longer duration of consumption. Further studiesare needed to determine the effects of ketone supplementson blood triglyceride and lipoproteins after chronicadministration and as a means to further enhance thehyperketonemia and improve the lipid profile of the clinic-ally implemented (4:1) KD.LDL is the lipoprotein particle that is most often associ-

ated with atherosclerosis. LDL particles exist in differentsizes: large molecules (Pattern A) or small molecules(Pattern B). Recent studies have investigated the im-portance of LDL-particle type and size rather thantotal concentration as being the source for cardiovascularrisk [56]. Patients whose LDL particles are predominantlysmall and dense (Pattern B) have a greater risk of

Fig. 7 Effects of ketone supplementation on basal blood ketone and basal blood glucose levels: Rats administered ketone supplements did nothave a significant change in basal blood ketone levels (a) or basal blood glucose levels (b) for the four week study. Two-Way ANOVA with Tukey’spost-hoc test, results considered significant if p < 0.05. Error bars represent mean (SD)

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cardiovascular disease (CVD). It is thought that small,dense LDL particles are more able to penetrate the endo-thelium and cause in damage and inflammation [82–85].Volek et al. reported that the KD increased the patternand volume of LDL particles, which is considered to re-duce cardiovascular risk [73]. Though we did not show asignificant effect on LDL levels for ketone supplements,future chronic feeding studies will investigate the effectsof ketone supplementation on lipidomic profile and LDLparticle type and size.

Therapeutic levels of hyperketonemia suppress bloodglucose levelsWe demonstrated that therapeutic ketosis could beinduced without dietary (calorie or carbohydrate) restric-tion and that this acute elevation in blood ketones wassignificantly correlated with a reduction in blood glucose(Figs. 2, 3 and 4). The BMS ketone supplement did notsignificantly induce blood hyperketonemia or reducedglucose in the rats. The KE supplemented rats trendedtowards reduced glucose levels; however, the lower doseof this agent did not lower glucose significantly, as re-ported previously in acute response of mice [59]. MCTshave previously been shown to elicit a slight hypoglycemiceffect by enhancing glucose utilization in both diabeticand non-diabetic patients [86–88]. Kashiwaya et al. dem-onstrated that both blood glucose and blood insulindecreased by approximately 50 % in rats fed a diet where30 % of calories from starch were replaced with ketone es-ters for 14 days, suggesting that ketone supplementationincreases insulin sensitivity or reduced hepatic glucoseoutput [89]. This ketone-induced hypoglycemic effect hasbeen previously reported in humans with IV infusions ofketone bodies [90, 91]. Recently, Mikkelsen et al. showedthat a small increase in βHB concentration decreases glu-cose production by 14 % in post-absorptive health males[92]. However, this has not been previously reported withany of the oral exogenous ketone supplements we studied.Ketones are an efficient and sufficient energy substrate forthe brain, and will therefore prevent side effects ofhypoglycemia when blood levels are elevated and the pa-tient is keto-adapted. This was most famously demon-strated by Owen et al. in 1967 wherein keto-adaptedpatients (starvation induced therapeutic ketosis) weregiven 20 IU of insulin. The blood glucose of fasted pa-tients dropped to 1–2 mM, but they exhibited nohypoglycemic symptoms due to brain utilization of ke-tones for energy [93]. Therefore, ketones maintain brainmetabolism and are neuroprotective during severehypoglycemia. The rats in the MCT group had a correl-ation of blood ketone and glucose levels at week 4,whereas the combination of BMS +MCT produced a sig-nificant hypoglycemic correlation both at baseline and atweek 4. No hypoglycemic symptoms were observed in the

rats during this study. Insulin levels were not measured inthis study; however, future ketone supplementation stud-ies should measure the effects of exogenous ketones oninsulin sensitivity with a glucose tolerance test. Anincrease in insulin sensitivity in combination with ourobserved hypoglycemic effect has potential therapy impli-cations for glycemic control in T2D [40]. Furthermore, itshould be noted that the KE metabolizes to both AcAcand βHB in 1:1 ratio [29]. The ketone monitor used in thisstudy only measures βHB as levels of AcAc are more diffi-cult to measure due to spontaneous decarboxylation toacetone; therefore, the total ketone levels (βHB+AcAc)measured were likely higher, specifically for the KE [14].Interestingly, the 10 g/kg dose produced a delayed bloodβHB peak for ketone supplements MCT and BMS +MCT.The higher dose of the ketogenic supplements elevatedblood levels more substantially, and thus reached theirmaximum blood concentration later due to prolongedmetabolic clearance. It must be noted that the dosage usedin this study does not translate to human patients, sincethe metabolic physiology of rats is considerably higher.Future studies will be needed to determine optimal dosingfor human patients.

Effects of ketone supplementation on organ weight andbody weight percentageKetone supplementation did not affect the size of thebrain, lungs, kidneys or heart of rats. As previously men-tioned, the rats were still growing during the experimen-tal time frame; therefore, organ weights were normalizedto body weight to determine if organ weight changed in-dependently to growth. There could be several reasonswhy ketones influenced liver and spleen weight. The ra-tio of liver to body weight was significantly higher in theMCT supplemented animals (Fig. 5). MCTs are readilyabsorbed in the intestinal lumen and transported directlyto the liver via hepatic portal circulation. When given alarge bolus, such as in this study, the amount of MCTsin the liver will likely exceed the β-oxidation rate, caus-ing the MCTs to be deposited in the liver as fat droplets[94]. The accumulated MCT droplets in the liver couldexplain the higher liver weight to body weight percentageobserved with MCT supplemented rats. Future toxicologyand histological studies will be needed to determine thecause of the observed hepatomegaly. It should be empha-sized that the dose in this study is not optimized inhumans. We speculate that an optimized human dosewould be lower and may not cause hepatomegaly or po-tential fat accumulation. Nutritional ketosis achieved withthe KD has been shown to decrease inflammatory markerssuch as TNF-α, IL-6, IL-8, MCP-1, E-selectin, I-CAM, andPAI-1 [8, 46], which may account for the observeddecrease in spleen weight. As previously mentioned,Veech and colleagues demonstrated that exogenous

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supplementation of 5 mM βHB resulted in a 28 % increasein hydraulic work in the working perfused rat heart and asignificant decrease in oxygen consumption [28, 41, 42].Ketone bodies have been shown to increase cerebral bloodflow and perfusion [95]. Also, ketone bodies have beenshown to increase ATP synthesis and enhance the effi-ciency of ATP production [14, 28, 40]. It is possible thatsustained ketosis results in enhanced cardiac efficiencyand O2 consumption. Even though the size of theheart did not change for any of the ketone supple-ments, further analysis of tissues harvested from theketone-supplemented rats will be needed to determineany morphological changes and to understand changes inorgan size. It should be noted that the Harlan standardrodent chow 2018 is nutritionally complete and formu-lated with high-quality ingredients to optimize gestation,lactation, growth, and overall health of the animals. Thesame cannot be said for the standard American diet(SAD). Therefore, we plan to investigate the effects ofketone supplements administered with the SAD to deter-mine if similar effects will be seen when the micronutrientdeficiencies and macronutrient profile mimics what mostAmericans consume.MCT oil has recently been used to induce nutritional

ketosis although it produces dose-dependent gastrointes-tinal (GI) side effects in humans that limit the potentialfor its use to significantly elevate ketones (>0.5 mM).Despite these limitations, Azzam and colleagues pub-lished a case report in which a 43-year-old-man had asignificant decrease in seizure frequency after supple-menting his diet with 4 tablespoons of MCT oil twicedaily [96]. An attempt to increase his dosage to 5 table-spoons twice daily was halted by severe GI intolerance.Henderson et al. observed that 20 % of patients re-ported GI side effects with a 20 g dose of ketogenicagent AC-1202 in a double blind trial in mild to moderateAlzheimer’s patients [24]. We visually observed similargastrointestinal side effects (loose stools) in the ratstreated with MCT oil in our study. Rats were closely mon-itored to avoid dehydration, and gastric motility returnedto normal between 12–24 h. Interestingly, the BMS +MCT supplement elevated βHB similarly to MCT oilalone, without causing the adverse gastrointestinal effectsseen in MCT-supplemented rats. However, this could bedue to the fact in a 10 g/kg dose of BMS +MCT, only 5 g/kg is MCT alone, which is less than the 10 g/kg dose thatelicits the GI side effects. This suggests that this novelcombination may provide a more useful therapeuticoption than MCT oil alone, which is limited in its abilityto elevate ketones in humans.Exogenously delivered ketone supplements signifi-

cantly altered rat weight gain for the duration of thestudy (Fig. 6). However, rats did not lose weight andmaintained a healthy range for their age. Rats have been

shown to effectively balance their caloric intake to preventweight loss/gain [97–99]. Due to the caloric density of theexogenous ketone supplements (Table 1) it is possible forthe rats to eat less of the standard rodent chow and there-fore less carbohydrates while maintaining their caloricintake. Food intake was not measured for this study.However, if there was a significant carbohydrate restric-tion there would be a signifcant change in basal blood ke-tone and blood glucose levels. As the hallmark to the KD,carbohydrate restriction increases blood ketone levels andreduces blood glucose levels. Neither an increase in basalblood ketone levels nor a decrease in basal blood glucoselevels was observed in this study (Fig. 7). Additionally, ifthere were an overall blood glucose decrease due to achange in food intake, this would not explain therapid reduction (within 30 min) in blood glucose cor-related with an elevation of blood ketone levels afteran intragastric bolus of ketone supplement (Figs. 2, 3and 4).

ConclusionsSeveral studies have investigated the safety and efficacyof ketone supplements for disease states such as AD andParkinson’s disease, and well as for parenteral nutrition[40, 48–50, 100–103]. Our research demonstrates thatseveral forms of dietary ketone supplementation can ef-fectively elevate blood ketone levels and achieve deleted:therapeutic nutritional ketosis without the need for diet-ary carbohydrate restriction. We also demonstrated thatketosis achieved with exogenous ketone supplementationcan reduce blood glucose, and this is inversely associatedwith the blood ketone levels. Although preliminary resultsare encouraging, further studies are needed to determineif oral ketone supplementation can produce the sametherapeutic benefits as the classic KD in the broad-spectrum of KD-responsive disease states . Additionally,further experiments need to be conducted to see if the ex-ogenous ketone supplementation affects the same physio-logical features as the KD (i.e. ROS, inflammation, ATPproduction). Ketone supplementation could be used as analternative method for inducing ketosis in patientsuninterested in attempting the KD or those who have pre-viously had difficulty implementing the KD because ofpalatability issues, gall bladder removal, liver abnormal-ities, or intolerance to fat. Additional experiments shouldbe conducted to see if ketone supplementation could beused in conjunction with the KD to assist and ease thetransition to nutrition ketosis and enhance the speed ofketo-adaptation. In this study we have demonstrated theability of several ketone supplements to elevate bloodketone levels, providing multiple options to induce thera-peutic ketosis based on patient need. Though additionalstudies are needed to determine the therapeutic potentialof ketone supplementation, many patients that previously

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were unable to benefit from the KD may now have analternate method of achieving therapeutic ketosis. Ketonesupplementation may also represent a means to furtheraugment ketonemia in those responsive to therapeuticketosis, especially in those individuals where maintaininglow glucose is important.

AbbreviationsAcAc: acetoacetate; ALS: amyotrophic lateral sclerosis; AD: Alzheimer’s;βHB: beta-hydroxybutyrate; BMS: sodium/potassium βHB mineral salt; BMS +MCT: BMS + MCT 1:1 mixture; BD: 1,3-butanediol; CNS: central nervoussystem; HDL: high density lipoprotein; HDACs: histone deacetylases; LDL: lowdensity lipoprotein; KD: ketogenic diet; KE: 1, 3-butanediol acetoacetate dies-ter/ketone ester; MCT: medium chain triglyceride oil; PCOS: polycystic ovarysyndrome; ROS: reactive oxygen species; SAD: standard American diet;TBI: traumatic brain injury; T2D: type-2 diabetes.

Competing interestsInternational Patent # PCT/US2014/031237, University of South Florida, D.P.D’Agostino, S. Kesl, P. Arnold, “Compositions and Methods for ProducingElevated and Sustained Ketosis”. P. Arnold (Savind) has received financialsupport (ONR N000140610105 and N000140910244) from D.P. D’Agostino(USF) to synthesize ketone esters. The remaining authors have no conflicts ofinterest.

Authors’ contributionsConceived and designed the experiments: SK, AP, NW, TF, DP. Performed theexperiments: SK, AP, NW, TF, CA, JS, AVP. Analyzed the data: SK, AP, DP.Contributed reagents/materials/analysis tools: PA. Helped draft themanuscript: SK, AP, NW, CA, DP. All authors read and approved the finalmanuscript.

AcknowledgementsThe authors would like to thank Savind Inc for manufacturing some of theketone supplements, Jay Dean for the use of his Hyperbaric ResearchLaboratory, and the ONR and Scivation Inc. for funding the project.

GrantsThis study was supported by the Office of Naval Research (ONR) GrantN000140610105 (DPD); and a Morsani College of Medicine Department ofMolecular Pharmacology and Physiology departmental grant.

Author details1Department of Molecular Pharmacology and Physiology, Morsani College ofMedicine, University of South Florida, 12901 Bruce B. Downs Blvd. MDC8,Tampa, FL 33612, USA. 2Savind Inc, 205 South Main Street, Seymore, IL61875, USA.

Received: 10 September 2015 Accepted: 28 January 2016

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Kesl et al. Nutrition & Metabolism (2016) 13:9 Page 15 of 15

ABOUT THE AUTHOR

Shannon Kesl was born and raised in Springfield, Ohio. She attended Wittenberg

University in Springfield, Ohio until Spring 2007. In 2007, she moved to Tampa, FL to continue

her studies in Biomedical Sciences at the University of South Florida; she received her BS in fall

of 2008. In 2010, Shannon began her graduate training as a student in the Ph.D. Program in

Integrated Biomedical Sciences at the Morsani College of Medicine, University of South Florida.

In 2014, she married the love of her life and currently shares her heart with her two dogs Bailey

and Isa. Shannon has performed her dissertation research in the Laboratory of Nutritional and

Metabolic Medicine under the co-mentorship of Dr. Dominic D’Agostino and Dr. Mack Wu.

During her time as a graduate student, she presented her research at many national conferences,

won numerous travel awards, and was a member of ASN, AAA, WHS, and APS societies. She

received her Masters of Science in Medical Science in 2013, and successfully defended her

dissertation on December 09, 2015.