INNOVATIVE APPROACHES TO STUDY DIETARY ZINC BIOAVAILABILITY, ABSORPTION AND DEFICIENCY STATUS

C-9 Symposium, " The Bridge from Biofortification to Bioavailability“ASA, CSSA & SSSA 2017 annual meeting, Oct. 22-25, Tampa FL

Contact: elad.tako@ ars.usda.govWeb: http://blogs.cornell.edu/traceminnutriunit/dr-elad-tako/

Elad TakoUSDA-ARS, Robert W. Holley Center for Agriculture and Health, Cornell University, Ithaca NY, 14853

The prevalence of (1) dietary Fe and (2) Zn deficiencies

WHO Global Database on Child Growth and Malnutrition. http://www.who.int/gdgm/p-child_pdf/ , 2014

(1) (2)

Zinc (Zn) deficiency is affecting approximately 17 % of the total world’s population. Zninadequacy has been associated with poor growth, depressed immune function, increasedvulnerability to and severity of infection, adverse outcomes of pregnancy, and neurobehavioralabnormalities. Deficiency of Zn is a major cause of early childhood morbidity and mortality.

The World Health Organization estimates that approximately one-thirdof worldwide infant deaths, and one half in developing countries,can be attributed to malnutrition. More specifically, iron (Fe) deficiency isthe most common nutritional deficiency worldwide and a major cause ofinfant mortality.

Worldwide prevalence of dietary Fe deficiency, by severity Worldwide prevalence of dietary Zn deficiency, by severity

To seek innovative solutions for better health. Poor nutrition, specifically Fe and Zndeficiencies are identified as one of the major global health problems to be addressed (WHO)

Low Fe and Zn bioavailability from grain/plant based diets that are low in absorbable Fe and Zn

BiofortificationSupplementationDiversification

To create a full range of optimal, bioavailable nutrients in a single staple plant species

Developing screening tools and biomarkers for Fe and Zn bioavailability and status, and guiding the plant breeding process

Challenge Problem Strategy Goal

Research

• Models (in vitro and in vivo)

• Examples of in vivo screening (beans, pearl millet, wheat)• Physiological (LA:DGLA Ratio), Molecular and Microbial

Biomarkers, as part of the development of Zinc Deficiency Index (ZDI)

Assessment of Fe and Zn bioavailability

FOCUS

Approach: Screening for Fe bioavailability

Tako et al., 2010; Tako and Glahn, 2010; Tako et al., 2011; Tako et al., 2012; Tako et al., 2013; Tako et al., 2014; Tako et al., 2015a,b; Tako et al., 2016

In vivo models

Rattus rattusRattus novergicusMus musculus

Sus scrofa

Long term feeding trialsIntra Amniotic Administration

Gallus gallus

In vivo model (Gallus gallus)

1. Fast growing animal

2. Sensitive for dietary mineral deficiencies

3. Growth period of 4-6 weeks

4. Dietary manipulation

5. Cost effective in vivo bioavailability screening model

6. Broad spectrum of biomarkers that reflect Fe or Zn status, bioavailability and absorption

7. >85% homology between gene sequences of human and chicken intestinal DMT1, DcytB, ZnT1 and Ferroportin

8. There is considerable similarity at the phylum level between the gut microbiota of broilers (Gallus gallus)

and humans, with Bacteroidetes, Firmicutes, Proteobacteria, and Actinobacteria representing the four dominant

bacterial phyla in both

“The broiler chicken (Gallus gallus) model exhibits the appropriate responses to Fe deficiency and can serve as a mode for Fe and other mineral bioavailability (and absorption) ” (Tako et al., 2010; 2011 a-c; 2012 a-c; 2013 a-d; 2014 a-c; 2015a-c; 2016ab; Mahler et al., 2012; Reed et al., 2014; 2015; 2016; Hartono et al., 2015; Siem et al., 2016; Knez et al., 2016; Pacifici et al., 2017; Dias et al., 2017)

Cross experimental analyses (Pearl Millet, Black Beans, Red Mottled Beans, Cream Seeded Carioca Beans)

Tako et al., Review: The combined application of the Caco-2 bioassay coupled with in vivo (Gallus gallus) feeding trial represents an effective approach to predicting Fe bioavailability in humans. Tako et al., Nutrients 2016.

Zinc Status and Bioavailability; The challenge

Source: Kambe et al., Physiological Reviews 2015

• The assessment of marginal zinc status isproblematic (current markers: urine, serum,hair).

• There is no universally accepted singlemeasure to assess zinc status (Lowe et al.,2009).

“The need to develop additional robustindicators of zinc status and to furtherexpand the already known clinicalmarkers, for which limited data ofreliability exists, is evident” (WHO, 2009).

Strategy - Development of Zinc deficiency index

Determination of zinc dietary bioavailability and physiological status

Zinc Deficiency

Index(ZDI)

Gene Expression

Blood LA:DGLA Biomarker

Fecal and Gut Microbial Ecology Profiling

Improved Zn Dietary

Bioavailability and

BiofortificationOutcomes

Novel Zinc Biomarker

Source: Reed et al., Nutrients , 2014

LA:DGLA Ratio A potential Biomarker of Zn status

Initial evaluation of the novel blood LA:DGLA ratio as a potential physiological biomarker of Zn status in vivo (Gallus gallus)

B

0

1

2

3

mR

NA

Exp

ress

ion

(AU

)

Hepatic Δ6

desaturase ***

Zn-Zn+

RBC LA:DGLA ratio: a reactive biomarker of Zn status

Reed et al., 2014

Reed et al., 2015; Tako and Koren, 2015; Tako et al., 2016

Diet and metabolic diseases – is gut microbiota the link?

Micronutrients deficiencies alter the intestinal microbiome composition and function

The microbiome as an indicator of deficiency status and severity

Zinc deficiency and the intestinal microbiome

Association between a speciesand Zn status

Reed et al., 2015

KEGG microbial metabolic pathways database – bacterial profile and disease

Reed et al., 2015

Proposed mechanisms by which Zn deficient gut microbiome, may worsen a Zn deficient phenotype

Reed S, Neuman H, Moscovich S, Glahn RP, Koren O, and Tako E. Chronic Zinc Deficiency Alters Chick Gut Microbiota Composition and Function. Nutrients. 2015 Nov 27;7(12):9768-84. doi: 10.3390/nu7125497.

Conclusions – Development of Zinc Deficiency Index Whole Blood Linoleic Acid: Dihomo–γ–linolenic Acid (LA:DGLA) Ratio, a novel functional indicator ofZn deficiency exploiting erythrocyte fatty acid desaturation.

Fecal and Gastrointestinal Microbial Ecology Phenotyping of defined Zn deficiency stages using nextgeneration sequencing.

mRNA Gene Expression of zinc dependent proteins.

• Indicator of Zn status (moderate to severe deficiency)• Zn nutrition in target populations• Improved Zn biofortification outcomes

LA:DGLA Ratio biomarker validation in human population

“The results are encouraging as they show that concentration of DGLA is decreased and LA: DGLA ratio increased in people with lower dietary Zn intake”

Methodology:• Healthy human volunteers, 25-55 years of age.• The dietary Zn intake was assessed using 24 h recall

questionnaires.

Results:• No significant relationship was found between the

dietary and plasma zinc status.• There was a statistically significant correlation

between DGLA and plasma Zn.• There were statistically significant

difference in DGLA status and LA:DGLA ratio between the Zn formedgroups.

Knez et al., 2016

Zinc Biofortification of staple food crops: Zinc Biofortified Wheat (in collaboration with Dr. James Stangoulis, Flinders University, Australia)

The linoleic acid: dihomo-γ-linolenic acid ratio predicts the efficacy of Zn biofortified wheat in Gallus gallus (Knez et al., Under Review)

Components High Zn flour Low Zn flour

Zn concentration (µg/g) 47.2 ± 0.47 33.5 ± 0.42Fe concentration (µg/g) 58.1 ± 0.45 53.5 ± 0.37Phytate (mg/g) 11 .1 ± 1.5 9.9 ± 0.4Calcium (mg/kg) 340 330

Fatty acids (mg/100g)Total saturated 338.8 330.3Total transaturated 0.4 0.3Total monounsaturated 286.1 274.4Total omega 3 55.2 55.8Total omega 7 19.0 19.1Total omega 9 265.8 254.1Total omega 6 975.8 981.0LA (18:2n-6) 973.1 978.4DGLA (20:3n-6) 0.6 0.5

Table 1. Zn, Fe, phytate, Ca content and fatty acid concentration of wheat flours

Ingredient High-Zn Low-Zn(Biofortified) (Standard)

g/kg (by formulation)High-Zn Wheat (47.2 μg/g Zn) 750 −Low-Zn Wheat (33.5 μg/g Zn) − 750Skim milk, dry 100 100DL- Methionine 2.5 2.5Corn starch 46.75 46.75Corn oil 30 30Choline chloride 0.75 0.75Vitamin/mineral premix (no Fe)1 70 70Total (g) 1000 1000Selected components mean ± SEM, n = 5 (by analysis)Dietary Zn concentration (μg/g)2 46.5 ± 0.99a 32.8 ± 0.17b

Table 2. Composition of the experimental diets – tailoring a specific diet

1 Vitamin and mineral premix provided/kg diet (330002 Chick vitamin mixture; 235001 Salt mix for chick diet; Dyets Inc. Bethlehem, PA). 2 Zn concentrations in the diets were determined by an inductively-coupled argon-plasma/atomic emission spectrophotometer (ICAP 61E Thermal Jarrell Ash Trace Analyzer, Jarrell Ash Co. Franklin, MA) following wet ashing. a,b Within a row, means without a common letter are significantly different (P < 0.05).

Study design; Zn biofortified wheat

Hatch Day 7 Day 14 Day 21 Day 28 Day 35 Day 42

Begin feeding wheat based diets: Std vs. biofortified(n=15)

Daily feed consumption (and Zn uptake) Weekly blood ( ) and BW ( ) sample/data collectionTerminal: duodenum ( ), cecum ( ), liver ( ), nail ( ), feather ( ) tissue collection

Gene expressionHistology

MicrobiomeZn content

Source: PoultryHub. http://www.poultryhub.org/physiology/body-systems/digestive-system/

Day 0 Day 14 Day 28 Day 42

Serum Zn (µg/g)Low Zn group 1.07 ± 0.15a 0.48 ± 0.05a 0.35 ± 0.03a 0.43 ± 0.04a

High Zn group 1.07 ± 0.15a 0.66 ± 0.08b 0.69 ± 0.07b 0.55 ± 0.04b

Feather Zn (µg/g)Low Zn group 89.6 ± 7.7a

High Zn group 117.9 ± 10.2b

Nail Zn (µg/g)Low Zn group 67.9 ± 3.9a

High Zn group 85.8 ± 3.9b

Day 0 Day 14 Day 28 Day 42Body weight (g)

Low Zn group 41.1 ± 0.7a 125.4 ± 4.8a 301.5 ± 11.4a 598.6 ± 22.8a

High Zn group 41.4 ± 0.8a 122.5 ± 3.9a 298.7 ± 12.8a 585.8 ± 29.7a

Feed consumption (kg/d)Low Zn group 0.415±0. 15a 1.135±0.52a 2.125±0.85a

High Zn group 0.405±0. 22a 1.120±0.61a 2.117±0.74a

Zinc intake (g) Low Zn group 13.15±0.6b 36.21±4.8b 69.18±7.5b

High Zn group 19.58±0.8a 49.85±5.1a 100.14±9.8a

Table 3. Body weight, feed consumption and Zn intake of chickens fed a low and a high Zn diet from day 0 to day 42

Values are mean daily feed intakes for the fourteen days preceding the day designated in the column heading. Values are cumulative weekly from day 0. a,b Within a column and for each parameter, means without a common letter are significantly different (n = 15, p < 0.05). Values are means ± SEM.

LA:DGLA ratio reflects Zn status

Index of Zinc

Status

(n=15, p<0.05, means±SEM)

Linoleic Acid: Dihomo–γ–linolenic Acid (LA:DGLA) Ratio

The LA: DGLA ratio responded to dietary Znmanipulations and the consumption of the Znbiofortified wheat lowered the LA: DGLA ratio. TheLA:DGLA ratio can be used as an additional biomarkerof Zn status.

Consumption of Zn biofortified wheat changes gut microbiota composition and function in Zn deficient subjects

Zinc Deficiency Index (ZDI)

Dietary Zinc

Zinc Status

Microbiome

PcoA -Unweighted unifrac Control vs. Zn biofortified, n=11, p=0.02(Knez et al., in preparation )

Fig 1. Characterizing the gut (Gallus gallus) microbiota following the consumption of a zinc biofortified wheat (Triticum aestivum)-based diet

Fig 2. Taxa plot- Genus Level By Treatment control vs. Zn biofortified

Zn biofortifiedControl

Fig 3. LEfSe* method identifying the OTUs with the greatest differences in abundance in the Control and Bio groups

*linear discriminant analysis (LDA) effect size (LEfSe)

Lactobacillaceae phyla 4 fold increase

Morphometric measurements; villus surface area and goblet cells

Treatment Villus surface area (mm2)

Goblet Cells (per villi)

Low Zn groupP>0.05

84.85±𝟑𝟑𝟑𝟑𝟑𝟑𝟑𝐛𝐛

High Zn group 105.94±11.38a

Zn deficiency affects the proliferation of intestinal goblet cells (Liu et al., 2014)

PAS/AB X40

H&E X60

Zinc Deficiency

Index(ZDI)

Gene Expression

Blood LA:DGLA Biomarker

Fecal and Gut Microbial Ecology Profiling

Improved Zn Dietary

Bioavailability and

BiofortificationOutcomes

• Dietary zinc deficiency affects blood linoleic acid: dihomo-γ-linolenic acid (LA:DGLA) ratio; a sensitive physiological marker of zinc status in vivo (Gallus gallus) (Reed et al., 2014)

• Chronic Zinc Deficiency Alters Chick Gut Microbiota Composition and Function. (Reed et al., 2015)• An initial evaluation of newly proposed biomarker of zinc status in humans - linoleic acid: dihomo-γ-linolenic acid (LA:DGLA) ratio

(Knez et al., 2016)• The Linoleic Acid: Dihomo-γ-Linolenic Acid Ratio (LA:DGLA)-An Emerging Biomarker of Zn Status (Knez et al., 2017)• The linoleic acid: dihomo-γ-linolenic acid ratio predicts the efficacy of Zn biofortified wheat in Gallus gallus (Knez et al., Under

Review)• Characterizing the gut (Gallus gallus) microbiota following the consumption of a zinc biofortified wheat (Triticum aestivum)-based

diet (Knez et al., in preparation)

Zinc bioavailability, absorption and deficiency status

Innovative approaches to study dietary iron and zinc bioavailability and physiological status; Conclusions

1. The in vivo (Gallus gallus) screening approach is cost effective, reliable, fast and allows tailoringand assessing Zn bioavailability in a specific diet.

2. This approach with its wide spectrum of parameters (physiological, molecular, microbiome) canpredict human efficacy biofortification studies outcomes.

3. Further development of the “Zn Deficiency Index” and the LA:DGLA ratio as a key biomarker.

Collaborators:Dr. Ray GlahnDr. Omry KorenDr. James StangoulisDr. Tom BrennaDr. Haim BarDr. Ran Ressler

Students and technical support specialists:Spenser ReedNikolai KolbaMarija KnezSarina PacificiAmrutha AnandaramanJaehong SongSybil ShaJulia DeutschCathy ZhangTao HouMary BodisPeipei ChangDr. Hadar NuemanAtara UzanDr. Jon HartDr. Shree Giri

Innovative approaches to study dietary iron and zinc bioavailability, absorption and deficiency status

Thank you

Elad TakoUSDA-ARS, Robert W. Holley Center for Agriculture and Health, Cornell University, Ithaca NY, 14853.

et79@cornell.edu; elad.tako@ ars.usda.gov

The commercial wheat variety, Correll (Triticum aestivum) was grown by Jordan Farms at Murtoa in the West Wimmera Shire of Victoria, Australia. Seeds were sown in the last week of May 2015 and harvested late November to early December. The high Zn grain was obtained by a foliar application of 1.5 L ha-1 ZnSO4 during the mid-vegetative growth stage (mid-August), followed by 2 L ha-1 ZnSO4 two weeks after flowering (mid-October). The lower Zn grain was grown in the same paddock and was treated with one foliar application of ZnSO4 at the rate of 2 L ha-1 one week after flowering and this was a common practise in the region to mitigate any effects of Zn deficiency in the soil. Wheat samples were prepared under trace element-free conditions to avoid contaminant sources of Zn. The samples were dried at 80 °C in a conventional oven, and later on milled using a trace element free mill. The wheat flour was transported to Cornell University, Ithaca, New York in sealed containers for testing in the Gallus gallus model. Zn, Fe, phytate, calcium, fatty acid and protein concentrations were measured in the original grain and in experimental wheat based diets (Table 1).