Rakesh Jain, MD, MPHAssociate Clinical Professor

Department of Psychiatry & Behavioural SciencesUniversity of Texas Medical School

Why Folate ?

Folate at the Intersection ofHealth & Illness

Basic Science Aspects of Folate

Why? Because Folate as a 1-Carbon Donor is Important at Every Phase of Brain Development

Black MM. Food Nutr Bull. 2008 June; 29(2 Suppl):S126–S131

Why? Because Folate Is Involved in Regulation of Inflammation, Methylation Cycle, and Cognition

(Among Many Other Critical Brain-Body Tasks)

DHF = dihydrofolate; THF = tetrahydrofolate; 5-MTHF = 5-methyltetrahydrofolate;SAMe = S-adenosylmethionine; SAH = S-adenosylhomocysteine; MTR = methionine synthase;

MAT = methionine adenosyltransferase; SAHH = S-adenosylhomocysteine hydrolase.

Bottiglieri T. Psychiatr Clin N Am. 2013;36(1):1-13.

Food source

MethylationCycle

FolateCycle

Homocysteine5-MTHF

Cystathionine

MethionineTHF

DHF

MethyleneTHF

CH3 DNAProteinsPhospholipidsNeurotransmittersSAH

SAMe

MTHFR

MTR

CBS

MAT

SAHH

B12

B6

Why? Because Folate is Important in DNA Methylation, and Its Alternations has Wide Spread Brain-Body Impact

CVDGlier MB et.al Mol. Nutr. Food Res. 2013, 00, 1–11

Specific to:• Tissue• Age• Sex• Ethnicity Altered Gene Expression

5’ 3’

DNA Methylation

GeneCpG Island

Risk Factors

CVD Risk

Altered Gene Expression

• Global hypomethylation

• Gene-specific hyper or hypomethylation

• No change

Tissue-specific

Methyl Nutrient Imbalances

Methionine Cycle

methyionine AdoMet

homocysteine AdoHcy

DNAMethylation

DNA

MTs

Why? Because Methylation of DNA Occurs viathe Folate Pathway – Implications for Epigenetics

Vanhees K, et al. Cell Mol Life Sci. 2013 Jul 27;[Epub ahead of print].

Mother: parental generationIntake of macro- and micronutrients

Gametes:2nd generationExposed to macro- and micronutrientsduring gametogenesis

Fetus:1st generationExposed to macro- and micronutrientsduring fetal development

Maternal NutritionalSUPPLY

Fetal NutritionalDEMAND

FetalAdaptation

Decreasedrisk on

breast andprostate cancer

(Increased)risk on

breast andprostate cancer

THF

5-MTHF

5,10-MTHF

SAM

SAH

Unmethylated DNA

Methylated DNA

B6

B2

Methionine

Homocysteine

B12 DNMTs

Why? Because Maternal Micro and Macro Nutrient Diet Increases Risk of Psychiatric Illness In Offsprings

• First evidence came from the Dutch Hunger Winter of 1944-45because of Nazi blockade

• Calories dropped to 500/ day. Micro and macro nutrients wereseverely hampered

• If this famine occurred during conception or during earlymonths of pregnancy, risk of schizophrenia increased by afactor of 2

• Interestingly, during this same time period, risk of neural tubedefect also went up (thereby implicating folate deprivation asthe cause of both sets of pathologies)

• Results are replicated in tow more studies from two separateChinese provinces. The 1959 Great Chinese Famine wasprecipitated by Mao’s ‘Great Leap Forward’ campaign. Similarresults to the Dutch study.

McClellan, J.M., et al., JAMA 2006. 296, 582–584; Brown AS. Progress in Neurobiology 93 (2011) 23–58

Why? Because Folate Deficiency May Lead to Progressive DNA and Brain/ Body Changes

Fenech M. Mechanisms of Ageing and Development 2010;131:236–241

FOLATE DEFICIENCY

REDUCED DNA METHYLATION

INCREASED HOMOCYSTEINE

INCREASED URACIL IN DNA

INCREASED DNA BREAKS & ABASIC SITES

INCREASED SENSITIVITY TO GENOTOXICITY FROM Aβ42 & ROS

CENTROMERE DYSFUNCTION

EPIGENETIC DRIFT

TELOMERE DYSFUNCTION OR REDUCED TELOMERE LENGTH

INCREASED GENE MUTATION

INCREASED mtDNADELETION

INCREASED CH 17, 21 ANEUPLOIDY

ABNORMAL GENE EXPRESSION

REDUCED REGENERATIVE POTENTIAL

INCREASED CELL DEATH

DEFECTS IN BRAIN DEVELOPMENT

INCREASED BRAIN CANCER RISK

ACCELERATED BRAIN ATROPHY & AGEING

Why? Because MTHFR – T Allele Increases Risk of Higher BMI, & Reduces Weight Reduction Ability

Di Renzo L, et al. European Review for Medical and Pharmacological Sciences.2013;17:2555-2565.

99.2

3

35.5

7

102.

02 115.

28

18.0

104.

39

38.9

1

112.

59

120.

12

26.3

0

20

40

60

80

100

120

140

Weight (kg)

BMI (kg/m )

Waist (cm)

Hip (cm)

tHcy (µmol/l)

Baseline

2

Mea

n Va

lue

*

**

*†

**

83.6

8

32.2

4

95.1

9 109.

03

17.8

95.4

9

35.6

0

104.

79 114.

94

23.9

0

20

40

60

80

100

120

140

Weight (kg)

BMI (kg/m )

Waist (cm)

Hip (cm)

tHcy (µmol/l)

Week 12

2

Mea

n Va

lue

*

**

*

**

T(-) (n=39) T(+) (n=17)

†

†

†† **

* p ≤ 0.01 ** p ≤ 0.05

† p ≤ 0.001

Why? Because the Consequences of FolateDeficiency Impact Both Brain and Body

Bottiglieri T, Reynolds E. In: Bailey LB, ed. Folate in Health and Disease, Second Edition. Boca Raton, FL: Taylor & Francis Group; 2009:355-380.

Folate DeficiencyDietary • Genetic • Drug Induced

Low CNS5-MTHF

CNS Disordersie, Depression, Dementia, Seizures,

Developmental Delay, Neoropathy, Myelopathy

Decreased BH4

DecreasedSAM/SAH Ratio Increased dUMP

IncreasedHomocysteine

ImpairedNeurotransmitter

Metabolism

Impaired MethylationDNA

ProteinsPhospholipids

DNADamage

Endothelial DysfunctionExcitotoxicity

Oxidative Stress

Why? Because Folate—in the Form of L-methyfolate—Is Critical in Monoamine Synthesis

Haroon E, et al. Neuropsychopharmacology. 2012;37(1):137-162.

BH4

BH2

InflammationOxidative Stress XPH2

5-MTHF

PAH

TH

TPH

NOS

phe tyr

arg NO

tyr L-DOPA

tryp 5-HTP

Dopamine

SerotoninMelatonin

NorepinephrineEpinephrine

NMDA Fx

Why? Because Our Understanding of Folate Receptor Advances Care

Wibowo AS et.al PNAS 2013;110(38):15180-15188.

• Folate Receptors FR come in three subtypes

• FR Alpha

• FR Beta

• FR Gamma

Chen C et.al Nature 2013.500:486-489

Why? Because We Now Better Understand the FRα (and its Pathologies)

Serrano M, et.al Drug Discovery Today Volume 17, Numbers 23–24 December 2012

FRα = Folate Receptor Alpha; PCFT = Proton Coupled Folate Transporter

Genetic, or Acquired FRα Pathologies (as in FR auto-antibodies) Lead to low CNS Folate

Why? Emerging Knowledge Regarding FR (Folate Receptor) Auto-antibiotics Reveals Concerns about

Dietary Folate and Folic Acid Absorption

Presence of FR Antibodies lead to low CNE L-methylfolate

Sequeira JM et.al Clin Chem Lab Med 2013; 51(3): 545–554; Frye RE et.al Molecular Psychiatry (2013) 18, 369–381

(n=28)

(n=20)

(n=151)

0

10

20

30

40

1-16 18-35 65+

FR A

uAb

prev

alen

ce, %

Age, years

0

20

40

60

80

0 0.5 1 1.5

CSF

Fola

te (n

mol

/l)

Blocking Folate Autoantibody(pmole/ml)

Average Normal

Lower Limit of Normal

Ramaekers V et.al Clin Chem Lab Med 2013;51(3):497–511

Why? Because Early Onset Cerebral Folate Deficiency (CFD) Is Devastating (But Potentially Entirely Reversible)

1 year

> 50 years

11-14 years

2 years

Birth

Prenatal conditions– Neural tube defect– Malformations ?

Parental FR antibodies and risk of infantile autism

Secondary CFD syndromes– Rett syndrome±MECP-2 gene Defect– Variant of Aicardi-Goutieres syndrome– FOLR-1 and FOLR-2 mutants ?

Infantile CFD Infantile Autism ± neurological deficits Autism spectrum disorders

Dystonia

Spastic-ataxic syndrome

Catatonic schizophrenia

Dementia and myoclonus

Why? Adult Onset Cerebral Folate Deficiency Has Now Been Reported

A 58-year-old woman with progressive memoryloss and myoclonus presented for medical attention

Reported from University of Texas Medical School in Houston

Sadighi Z et.al Arch Neurol. 2012 Jun;69(6):778-779.

Initial Labs:Cerebrospinal fluid (CSF) analysis showed low levels of

• BH4 (8 nmol/L; ref 10-30 nmol/L)

• L-methyfolate (29 nmol/L; ref. 40-120 nmol/L).

• The patient’s serum folate level was normal (20.4 ng/mL; reference,5.4 ng/mL

Serum contained • Folate receptor 1–blocking antibody titer

of 0.41 pmol/mL (ref 0.2 ML/mL) • Folate receptor 1–binding antibody titer

of 0.81 ML IgG/mL (ref 0.5 ML IgG/mL)

6 month treatment with non-folic acid form of folate for 6 months. Full reversal of

symptoms and normalized CSF L-methyfolate

Why? Because MTHFR Status of an Individual Matters

Why? Because the World’s Latest, and Largest Meta-analysis of MTHFR C/T Polymorphism Reveals

Elevated Risk of Depression

Wu, YL et al. Prog Neuro-Psychopharm. & Biological Psychiatry 2013;46:78–85

26 Separate, global studies. including 4992 depression cases and 17,082 controls

Results For TT vs. CC: Odds Ratio was 1.42 (95%CI = 1.16–1.75)

Why? The Presence of a T Allele for MTHFR, Predicts Greater Risk of Metabolic Syndrome With Atypicals

Ellingrod VL, et al. Schizophr Res. 2008;98(1-3):47-54.

The homeostatic modelassessment (HOMA) is amethod used to quantifyinsul in resistance andbeta-cell function

• N = 58.• For T allele participants,

the risk was almost 4 times greater, despite similar antipsychotic exposure.

• For the 677 T allele carriers, 53% met metabolic syndrome criteria, compared to 23% in the CC genotype group, giving an OR = 3.7.

170

35

30

20

0

10

60 80 100 120 140

HO

MA

-IR

Waist (cm)16070 90 110 130 150

25

15

5MTHFR C/C

MTHFR C/T

MTHFR T/T

Why? Because Folate Genetics, in Interaction with Stress, Impact Mood Adversely

Lok A, et al. Transl Psychiatry. 2013;3:e288.

Presence of a T allele with MTHFR (~70% of population has it), means less CNS L-methylfolate

MTHFR C/C

MTHFR C/T

MTHFR T/T

5

35

30

20

0

10

2 4

BD

I (to

tal s

core

)

Childhood Trauma (total score)1 3

25

15

5

Interventional Aspects of Folate

Weaning Wistar rats were fed folate-deficient diet and folate-replete (control) diets for 2 and 4 weeks

Compared to the control counterparts, mtDNA deletions of lymphocytes increased by 3.5-fold (P\ 0.05) after 4 weeks of folate deficiency

Chou YF et.al Eur J Nutr (2009) 48:429–436

Mitochondrial DNA is vulnerable to oxidative stress due to proximity of ROS

and low level of DNA repair

Data reveals that there is a folate dependent accumulation of mtDNA

deletions in lymphocytes

Why? Because Mitochondrial DNA Damage (secondary to oxidative stress) occurs in Folate Deficiency States

Why? Because Two Commonly Mood Stabilizers Negatively Impact 1 Carbon Metabolism

Lamotrigine is a weak inhibitor of DHFR (which can lead to decreases

in functional folate even with normal folate blood levels)

Baek JH et.al Australian & New Zealand Journal of Psychiatry, 2013;00(0):1-6

Sodium valproate inhibits methionine adenosyltransferaseand decreases methionine levels

Why? Folate Has Positive Effects on Oxidative Stress via the eNOS System

Folate Reduced Oxidative Stress &Improves Endothelial Function

Folate promoted eNOSdephosphorylation at

negative regulatory sites

Folate increased phosphorylation at

positive regulatory sites

Mechanism of Folate’s Action on eNOS is through effects on intracellular protein kinase-dependent signaling axes.

Taylor SY et.al European Journal of Pharmacology. 2013;714:193–201

Porcine Aortic Endothelial Cell

in vitro study

Wang LL et.al. Human Reproduction. 2009;24(3):562-579

Frequency and distribution of the observedabnormalities of embryos induced by ethanol

Fetal miRNA in Mouse Mothers Exposed to ETOH

ParameterConcentration of ethanol (mg/ml)

0 2.0 4.0 8.0

Examined embryos 24 22 25 26

Abnormal embryos 0 5* 16** 26**

Microcephaly 0 0 4 7*

Unclosed neural folds 0 5* 9** 15**

branchial arch dysplasia 0 0 7* 13**

Undifferentiated cardiac tube 0 2 5 8*

*P, 0.05; **P, 0.01, compared with control group (0 mg/ml ethanol).

Why? Because Folate Has An Important Positive Impact on Gene Expression and miRNA

Why L-Methyfolate Matters? Because of 2 Issues: 1. BBB Barrier & 2. Bioavailable Form

5-MTHF = 5-methyltetrahydrofolate; DHF = dihydrofolate; MTHFD1 = methylenetetrahydrofolate dehydrogenase 1; THF = tetrahydrofolate.

Willems FF, et al. Br J Pharmacol. 2004;141(5):825-830.

DHF Reductase

MTHFD1

MTHFR

Dihydrofolate

Tetrahydrofolate

10-formyl-THF

5,10 Methenyl THF

5,10 Methylene THF

5-MTHF

5-MTHFBBB

Understanding Blood Brain Barrier and Folate Issues – Only L-Methyfolate Crosses the Blood Brain Barrier

Synergistic Effect of MTHFR/MTR & BMIAdjunctive 15 mg L-methylfolate vs Adjunctive Placebo

-2.7-3.8

-4.7

-8.2-9.9

-14.4-16.0

-14.0

-12.0

-10.0

-8.0

-6.0

-4.0

-2.0

0.0

HAMD 28 P=0.017 P=0.087 P=0.001 P<0.001 0.001 <0.001CGI P=0.01 P=0.024 P<0.001 P<0.001 <0.001 <0.001

L-methylfolate Magnitude of Effect by Biomarker(Treatment Mean Change Less Placebo Mean Change)

TOTAL MTHFR CT/TT BMI >30MTR

AG/GGBMI >30 +MTHFR T

BMI >30+ MTR G

HAM

-D 2

8 M

ean

Cha

nge

Derived from: Papakostas G, Shelton R, Stahl S, et al (May 2012) Society of Biological Psychiatry Annual Meeting. Philadelphia, PA. Poster Presentation.

-2.74

0.07

-4.57

-2.29

-3.61

-0.11

-4.55-5

-4

-3

-2

-1

0

1

Baseline Biomarker Mean Change Treatment Effect Adjunctive 15 mg L-methylfolate vs Adjunctive Placebo

HAM-D 28 P=0.017 P=0.966 P=0.005 P=0.158 P=0.05 P=0.953 P=0.003CGI P=0.01 P=0.466 P=0.002 P=0.109 P=0.019 P=0.07 P=0.03

Treatment Mean Change Less Placebo Mean Change

Total SAM/SAH >med

SAM/SAH <med

hsCRP<med

hsCRP>med=2.2

4HNE<med

4HNE>med

Derived from: Papakostas G, Shelton R, Stahl S, et al (May 2012) Society of Biological Psychiatry Annual Meeting. Philadelphia, PA. Poster Presentation.

-0.41-1.52

-0.97-1.45

-2.83-2.37

-2.00-1.30

-0.17-1.71

-1.03-2.51

-1.14

-3.0 -2.5 -2.0 -1.5 -1.0 -0.5 0.0Average Pooled Effect Size

Effect Size and Multiple Risk Factors

Papkostas G, et al. Effect of L-Methylfolate on Maier Subscale Scores in a Randomized Clinical Trial of Patients with Major Depression. Presented at: Society of Biological Psychiatry 67th Annual Scientific Convention and Program; May 3-5, 2012; Philadelphia, Pennsylvania.

Comparing LM to SGA Utilization and Outcomes Data

Adult, non-pregnant patients with ≥1 pharmacy claim for a SSRI/SNRI from 1/1/2007-12/31/2009 (Index Date) and at least one clinical visit with a 1◦ or 2◦ diagnosis of depression (ICD-9-CM 296.2x, 296.3x, 300.4, 309.1, 311) within the index period were identified.

The propensity score match resulted in 4,053 SGA and 1,351 LM-augmented patients that were closely matched on age, gender, and comorbidities

Meantime on Augmentation Therapy Mean 6-month all-cause unit utilization

LM Superior to SGA (p<.o5) in both data sets

Presented at the College of Psychiatric and Neurologic Pharmacists (CPNP) 16th Annual Meeting, April 21-24, 2013, Colorado Springs, CO, USA

73 days

106 days

0 30 60 90 120 150

SGA

LM n=1,351

n=4,053

SGA LM

Inpatient visits 1.64 0.63

Outpatient visits 18.94 16.43

Emergency room visits 0.38 0.16

LM – L-methylfolate; SGA = Second Generation Atypical

Adult, non-pregnant patients with ≥1 pharmacy claim for a SSRI/SNRI from 1/1/2007-12/31/2009 (Index Date) and at least one clinical visit with a 1◦ or 2◦ diagnosis of depression (ICD-9-CM 296.2x, 296.3x,

300.4, 309.1, 311) within the index period were identified.

Presented at the College of Psychiatric and Neurologic Pharmacists (CPNP) 16th Annual Meeting, April 21-24, 2013, Colorado Springs, CO, USA

The propensity score match resulted in 4,053 SGA and

1,351 LM-augmented patients that were closely matched on

age, gender, and comorbidities

Mean 6-month all-cause and depression-related post-augmentation costs

SGA = Second Generation Atypical

LM – L-methylfolate

$0

$1,000

$2,000

$3,000

$4,000

$5,000

$6,000

$7,000

$8,000

$9,000

All-Cause Costs Depression-Related Costs

SGALM

Comparing LM to SGA Utilization and Outcomes Data (cont’d.)

A ‘Real World” Study – Results from a Naturalistic Study of L-methyfolate

Shelton RC et.al Prim Care Companion CNS Disord 2013;15(4):

Between November 2010 and April 2012, patients who reported being treated for major depressive disorder rated their experiences before and after 3 months on the prescription medical food L-methylfolate 7.5 mg or 15 mg, through an automated telephone system. Survey questions included the 9-item Patient Health Questionnaire (PHQ-9), as well as quality of life and medication satisfaction questions.

554 patients, 502 reported that L-methylfolate was added to their existing antidepressant and 52 were treated with L-methylfolate alone, without an antidepressant

Overall, of the 554 patients, 67.9 % achieved Response and 45.7 % achieved Remission

67.960.2

65.872.0 73.8

0

20

40

60

80

100

Overall 5–9 10–14 15–19 20–27

Trea

tmen

t Res

pons

e R

ate,

%

PHQ-9 Baseline Severity

17

33

44

5310

42 45

0

20

40

60

80

100

ExtremelyDifficult

VeryDifficult

SomewhatDifficult

Not at AllDifficult

Patie

nts,

%

Baseline Posttreatment

Shelton RC et.al Prim Care Companion CNS Disord 2013;15(4):

554 patients, 502 reported that L-methylfolate was added to their existing antidepressant and 52 were treated with L-methylfolate alone, without an antidepressant

Overall, of the 554 patients, 67.9 % achieved Response and 45.7 % achieved Remission

A ‘Real World” Study – Results from a Naturalistic Study of L-methyfolate

45.7

67.2

48.137.8

28.7

0

20

40

60

80

100

Overall 5–9 10–14 15–19 20–27

Rem

issi

on R

ate,

%

PHQ-9 Baseline Severity

-1.1

-1.2

-1.0

-1.1

-0.9

-1.1 -1

.0

-0.6

-0.4

-0.5

-0.5 -0

.4

-0.5

-0.3

-0.5 -0

.4

-0.2 -0

.1

-0.9

-0.9

-0.7

-1.0

-0.6

-0.9 -0

.8

-0.4

-0.2

-1.4

-1.6

-1.3

-1.4

-1.2

-1.4

-1.2

-0.6

-0.4

-1.7

-1.8 -1

.7 -1.6 -1

.5

-1.7 -1

.6

-1.4

-1.1

-2.0-1.8-1.6-1.4-1.2-1.0-0.8-0.6-0.4-0.20.0

Little Interestin Things

FeelingDown

SleepingTroubles

FeelingTired

Poor Appetiteor Overeating

Feeling BadAbout Self

TroubleConcentrating

Too Slow orToo Fidgety

Thoughts ofDying/Hurting

Self

PHQ

-9 S

core

, mea

n (S

D)

PHQ-9 Baseline Severity

Overall 5– 9 10–14 15–19 20–27

Thank you for your attention!

PDFs of these slides and all supporting articles can be accessed at:www.JainFolateInfo.com

(password – NOLA – all upper caps)

In Conclusion: Why Folate ?

Because it matters a great deal in bothhealth and in illness

Because interventional studies, fromvarious sources, compel the clinician to re-consider folate and more specifically

l methyfolate as a therapeutic option