Overview of carotenoid bioavailability determinants: from dietary factors to genetic polymorphisms

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1 Overview of carotenoid bioavailability determinants: from dietary factors to genetic polymorphisms Charles Desmarchelier UMR 1062 INSERM/1260 INRA/Aix-Marseille University «Nutrition, Obesity and Risk of Thrombosis» Medicine Faculty Marseille France FBHC September Norwich

2 What are carotenoids? Carotenoids are phytochemicals that belong to the terpenoid class (tetraterpenoids). They are organic pigments with the general formula C 40 H 56 O n (0 n 6). There are over 750 known carotenoids. They are split into 2 classes: - hydrocarbons (n=0) carotenes - oxygenated carotenoids (n 0) xantophylls They can be produced by most plants, bacteria, and fungi, but not by animals or humans, making the diet their sole source. They are lipid micronutrients they share common transport mechanisms and metabolic pathways with lipids. 2

3 Carotenes Xanthophylls Main carotenoids found in human blood and tissues lycopene α-carotene β-carotene β-cryptoxanthin zeaxanthin lutein 3

Interest of carotenoids in human health Consumption of carotenoid-rich foods has been associated

Carotenoids can act as antioxidants by reacting with free radicals, including singlet oxygen and

Light filtering: lutein and zeaxanthin Provitamin A properties: - and β-carotene,

prostate cancer and cardiovascular diseases?

4 Interest of carotenoids in human health Consumption of carotenoid-rich foods has been associated with a decreased risk of disease, including cancer and cardiovascular disease. Carotenoids can act as antioxidants by reacting with free radicals, including singlet oxygen and peroxyl radicals. Light filtering: lutein and zeaxanthin Provitamin A properties: - and β-carotene, β-cryptoxanthin Gene expression regulation Lycopene protective role against the development of prostate cancer and cardiovascular diseases? Lutein, zeaxanthin: high concentrations in the human macula lutea prevention of age-related macular degeneration (ARMD)? β-carotene: 2 µg (supplement)/12 µg (food) 1 µg retinol activity equivalent (RAE) -carotene, β-cryptoxanthin: 24 µg from food 1 µg RAE 4

5 What is the bioavailability of a micronutrient? "Proportion of an ingested micronutrient, or one of its metabolites, that is available for use or storage by the organism." Absorption + Transport + Metabolism Food matrix Dietary fat Portal vein Liver Blood Lymph Enterocyte Target tissue 5

Components of bioavailability + Metabolism!

Blood Other organs Bioaccessibility Uptake

6 Components of bioavailability + Metabolism! Food matrix Mixed micelles Enterocytes Blood Other organs Bioaccessibility Uptake Absorption Bioavailability The bioaccessibility of carotenoids has 2 components: their extraction from the food matrix their solubility into mixed micelles, which depends on the physico-chemical properties of the molecules 6

The bioavailability of carotenoids can be

7 The bioavailability of carotenoids can be low and is very variable to assess the bioavailability of a micronutrient, its blood concentration is measured following the ingestion of a known dose of this micronutrient. Lycopene: 5% β-carotene Lutein: 40% 7

8 S L A M E N G H I Carotenoid bioavailability is affected by numerous factors Species of carotenoid (lutein vs lycopene) Molecular Linkage (esterified vs free lutein) Amount consumed in a meal Matrix in which carotenoids are incorporated (e.g. vegetable oil or supplement) Effectors of absorption and bioconversion (e.g. lipids, dietary fibres, drugs) Nutrient status of the host Genetic factors (e.g. polymorphisms or epigenetic modifications) Host-related factors (e.g. sex, age) Mathematical Interactions between factors West et Castenmiller, Int J Vitam Nutr Res

9 Why improve carotenoid bioavailability? Preventive nutrition to prevent the development of pathologies which these micronutrients seem to protect against. Food industry economic interest, to optimise costs by reducing the quantity of carotenoids incorporated into foods. Marketing interest: to propose a carotenoid form highly bioavailable. Sustainable development to reduce the production and consumption of food by improving carotenoid absorption efficiency. Personalised nutrition to improve carotenoid absorption in low absorbers subjects. 9

10 How to improve carotenoid bioavailability? study the intra-luminal metabolism of carotenoids and their absorption mechanisms. understand how some factors interfer with carotenoid metabolism and absorption mechanisms. 10

11 What is the bioavailability of a micronutrient? "Proportion of an ingested micronutrient, or one of its metabolites, that is available for use or storage by the organism." Absorption + Transport + Metabolism Food matrix Dietary fat Portal vein Liver Blood Lymph Enterocyte Target tissue 11

12 Carotenoid fate in the stomach 10 healthy men fed intragastrically 3 liquid test meals: Tomato purée 10 mg lycopene Chopped spinach 10 mg lutein Carrot purée 10 mg β-carotene β-carotene Lutein Lycopene Vegetable matrix Vegetable matrix Vegetable matrix % Fat phase Aqueous phase (micelles) Fat phase Fat phase Time (min) Tyssandier et al., Am J Physiol Gastrointest Liver Physiol 2003 Time (min) Time (min) the stomach plays a significant role in the bioavailability of carotenoids by initiating their transfer from the vegetable matrix to the fat phase of the meal 12

13 What is the bioavailability of a micronutrient? "Proportion of an ingested micronutrient, or one of its metabolites, that is available for use or storage by the organism." Absorption + Transport + Metabolism Food matrix Dietary fat Portal vein Liver Blood Lymph Enterocyte Target tissue 13

14 Carotenoid fate in the stomach % all-trans carotenoids in the micellar phase Lutein β-carotene Lycopene Time (min) Tyssandier et al., Am J Physiol Gastrointest Liver Physiol 2003 the proportion of carotenoids recovered in the micellar phase of the duodenum is very low (<7%), which probably explains their poor bioavailability. 14

15 What is the bioavailability of a micronutrient? "Proportion of an ingested micronutrient, or one of its metabolites, that is available for use or storage by the organism." Absorption + Transport + Metabolism Food matrix Dietary fat Portal vein Liver Blood Lymph Enterocyte Target tissue 15

16 Intestinal proteins involved in carotenoid absorption Paradigm before 2005: carotenoids are absorbed via passive diffusion only. Yet there were some inconsistencies: - There is a high interindividual variability in the absorption of carotenoids. Micelle - There is a competition between carotenoids regarding their absorption. - The efficieny of carotenoid absorption by enterocytes differs in between molecules. Chylomicron (Apo-B) Carotenoids Carotenoids Cytosolic Transport protein 16

17 Intestinal proteins involved in carotenoid absorption Micelle NPC1L1 CD36 SR-B1? References: Pro-vit A carotenoids Lutein Lycopene Lutein Pro-vit A carotenoids Carotenoids? Reboul et al., Biochem J 2005 Van Bennekum et al., Biochem 2005 Reboul et al., J Biol Chem 2006 Cytosolic transport protein Vit A (CRBPII) Carotenoids? Moussa et al., J Nutr 2008 Sato et al., J Pharm Pharm Sci 2012 Vit A-esters Carotenoids Borel et al., J Nutr 2013 Carotenoids? Carotenoids? ABCA1? Chylomicron (Apo-B) ApoA-I HDL 17

18 How to improve carotenoid bioavailability? study the intra-luminal metabolism of carotenoids and their absorption mechanisms understand how some factors interfer with carotenoid metabolism and absorption mechanisms 18

19 S L A M E N G H I Carotenoid bioavailability is affected by numerous factors Species of carotenoid (lutein vs lycopene) Molecular Linkage (esterified vs free lutein) Amount consumed in a meal Matrix in which carotenoids are incorporated (e.g. vegetable oil or supplement) Effectors of absorption and bioconversion (e.g. lipids, dietary fibres, drugs) Nutrient status of the host Genetic factors (e.g. polymorphisms or epigenetic modifications) Host-related factors (e.g. sex, age) Mathematical Interactions between factors 19

20 Effect of the food matrix on carotenoids bioaccessibility Rationale Paradigm: to be absorbed, carotenoids need to be incorporated into mixed micelles Emulsion Micelle % The extraction of carotenoids from their food matrix is not very efficient, it depends on the matrix and is affected by technological treatments or cooking 20

21 In vitro digestion of a carotenoid-containing food matrix model for bioaccessibility studies food + saliva + pepsin + bile + pancreatin ph 4 ph 6 1. Homogenisation 10 min 2. Buccal phase 10 min 3. Gastric phase 30 min 4. Duodenal phase 30 min Model mimicking gastric and duodenal digestion 21

22 In vitro digestion of a carotenoid-containing food matrix Centrifugation Non hydrolised triglycerides Sampling Digestate 1 h, 3600 rpm, 10 C Intermediate phase Duodenal phase Non-solubilized food debris Bioaccessibility = Micelle content Digestate content Filtration (0,2 µm) Sampling Mixed micelles 22

23 Effect of the food matrix on carotenoids bioaccessibility Reboul et al., J. Agric. Food Chem Bioaccessibility: α-carotene: % β-carotene: % Lycopene: % Lutein: % 23

24 Effect of the food matrix on carotenoids bioaccessibility Reboul et al., J. Agric. Food Chem food processing increases carotenoid bioaccessibility 24

25 S L A M E N G H I Carotenoid bioavailability is affected by numerous factors Species of carotenoid (lutein vs lycopene) Molecular Linkage (esterified vs free lutein) Amount consumed in a meal Matrix in which carotenoids are incorporated (e.g. vegetable oil or supplement) Effectors of absorption and bioconversion (e.g. lipids, dietary fibres, drugs) Nutrient status of the host Genetic factors (e.g. polymorphisms or epigenetic modifications) Host-related factors (e.g. sex, age) Mathematical Interactions between factors 25

26 Effect of 2 nutritional factors: triglycerides and phytosterols Rationale Carotenoids are soluble in triglycerides and hence follow their fate in the digestive tract. Carotenoids use metabolic pathways common to lipids and phytosterols: same intraluminal vehicles (micelles), sometimes common transporters (SR-B1). It is hence reasonable to assume that some interactions (positive or negative) might exist between carotenoids and these 2 nutritional factors. 26

27 Effect of the type of dietary triacylglycerol fatty acids on carotenoids bioaccessibility The type of dietary TG fatty acids affects carotenoid bioaccessibility. The effects differ according to carotenoid types: Saturated fatty acids improve xantophyll bioaccessibility. Olive oil improves carotene bioaccessibility. 27

28 Blood chylomicron β-carotene concentration Effect of phytosterols on β-carotene bioavailability 26 normocholesterolemic men Control beverage Control beverage + 2.2g/d plant free sterols Richelle et al., AJCN 2004 Control beverage + 2.2g/d plant sterol esters Phytosterols inhibit β-carotene bioavailability. But no effect found on β-cryptoxanthin bioavailability following the consumption of a milk-based fruit drink with or without plant free sterols for 28 days (Granado-Lorencio et al., J Agric Food Chem 2011). 28

29 Effect of polyphenols on carotenoid absorption Lutein absorption (% of control) Caco-2 TC7 monolayers (enterocyte cell model) Control: 0.85µM lutein-rich micelles mixture of polyphenols (25µM-naringenin, 25µM-(+)-catechin, 50µM-gallic acid and 50µM-caffeic acid). Reboul et al., Br J Nutr µM naringenin + 150µM naringenin No effect of (+)-catechin, gallic acid, caffeic acid naringenin inhibits lutein uptake by enterocyte cells. Possible mechanism: - competition with SR-B1 (naringenin is the most lipophilic of the tested polyphenols) - interaction with membrane lipids 29

30 S L A M E N G H I Carotenoid bioavailability is affected by numerous factors Species of carotenoid (lutein vs lycopene) Molecular Linkage (esterified vs free lutein) Amount consumed in a meal Matrix in which carotenoids are incorporated (e.g. vegetable oil or supplement) Effectors of absorption and bioconversion (e.g. lipids, dietary fibres, drugs) Nutrient status of the host Genetic factors (e.g. polymorphisms or epigenetic modifications) Host-related factors (e.g. sex, age) Mathematical Interactions between factors 30

31 Feedback regulation of carotenoid uptake and metabolism Vitamin A (retinoic acid) modulates SR-B1 and BCMO1 expression via the transcription factor ISX: Lobo et al., FASEB J

32 S L A M E N G H I Carotenoid bioavailability is affected by numerous factors Species of carotenoid (lutein vs lycopene) Molecular Linkage (esterified vs free lutein) Amount consumed in a meal Matrix in which carotenoids are incorporated (e.g. vegetable oil or supplement) Effectors of absorption and bioconversion (e.g. lipids, dietary fibres, drugs) Nutrient status of the host Genetic factors (e.g. polymorphisms or epigenetic modifications) Host-related factors (e.g. sex, age) Mathematical Interactions between factors 32

Effect of genetic variations on carotenoid bioavailability Rationale: High interindividual variability of carotenoid bioavailability. Apparently an intrinsic characteristic.

33 Effect of genetic variations on carotenoid bioavailability Rationale: High interindividual variability of carotenoid bioavailability. Apparently an intrinsic characteristic. 120 mg beta-carotene (n=16) Some proteins are involved in carotenoid absorption. Borel et al., Journal of Lipid Research (1998) Some genetic variants could explain this variability 33

The Vitagenes study a clinical study aiming at

interindividual variability of lipid micronutrient

Semolina (70 g) Peanut oil (50 g) White bread (40 g) Egg

(supplement) OR - lycopene (tomato paste) OR -

samples, at fast and each hour postprandially (from 0 to

34 The Vitagenes study a clinical study aiming at identifying some genetic variations involved in the interindividual variability of lipid micronutrient bioavailability Genotyping Standardized test meals: Semolina (70 g) Peanut oil (50 g) White bread (40 g) Egg whites (60 g) Water (330 ml) micronutrient: - lutein (supplement) OR - lycopene (tomato paste) OR - beta-carotene (tomato paste) 40 healthy adult men Blood samples, at fast and each hour postprandially (from 0 to 8 h) 3 weeks in between 2 test-meals Determination of chylomicron carotenoid concentration 34

35 Results: interindividual variability High interindividual variability of the postprandial β-carotene response CV = 105% highest responder response > 70 x lowest responder response! 35

36 Association with long term blood β-carotene status Positive correlation between the postprandial βc response and the fasting plasma βc concentration (Pearson s r = 0.78, P < 0.001). the fasting blood βc concentration was associated with its bioavailability, which suggests that βc bioavailability significantly affects the βc, and hence VA, status 36

37 What are the candidate genes for an effect on the beta-carotene bioavailability? SNPs in genes involved in lipid digestion (CEH, PNLIP ) Chylomicrons SNPs in genes involved in the uptake and enterocyte metabolism of beta-carotene (SCARB1, BCMO1 ) SNPs involved in chylomicron metabolism (Desmarchelier et al., J Clin End Metab 2014) Chylomicrons remnants 54 candidate genes 2172 SNPs

38 Results associated SNPs/genes PLS model with 25 SNPs in 12 genes explaining 69% of the variability in β-carotene bioavailability. Chylomicrons APOB LIPC TCF7L2 (chylomicron metabolism) ABCA1 (basolateral efflux) ISX (apical uptake via SCARB1 regulation) BCO1 (βc cleavage) ABCG5 (apical efflux) ELOVL2 (elongase) Chylomicron remnants Unknown role: CXCL8 (inflammatory response) PKD1L2 RPE65 (vision) SOD2 (superoxide dismutase) 38

39 β-carotene Summary: 3 carotenoids Lutein Lycopene Borel, Desmarchelier et al., J Nutr 2015 Borel, Desmarchelier et al., Am J Clin Nutr 2014 Borel, Desmarchelier et al., Free Rad Biol Med

40 Putative mechanism SNPs in ELOVL2 have been associated with increases in plasma EPA and DHA acid proportions after fish oil supplement (AlSaleh et al., Genes Nutr 2014). Mashurabad et al., Arch Biochem Biophy

41 Limits of the Vitagenes study Study carried out on a small group of European men can we apply these results to other populations, e.g. Chinese women? Candidate gene approach some more genes are likely involved. A limited number of SNPs (mostly common variants with MAF>5%) were searched for it is likely some variants with a phenotypic effect were missed out. Only SNPs were studied what about other genetic variations (e.g. CNV) and epigenetic modifications? 40

42 Main conclusions The absorption mechanisms of carotenoids are much more complex than what was thought. Moreover, there are differences between carotenoids. Many factors affect their absorption (SLAMENGHI). There is a high interindividual variabiliy of carotenoid bioavailability, partly due to genetic polymorphisms. The main strategies to improve carotenoid bioavailability are: To modify technological treatments or to provide food preparation advice. To provide nutritional recommandations (e.g. to consume carotenoids with lipids). To create formulations protecting carotenoids or improving their absorption (e.g. nanoencapsulation). Personalised recommandations according to genetic characteristics are promising but not yet ready.

Acknowledgments UMR 1062 INSERM/1260 INRA/Aix-Marseille University «Nutrition, Obesity and Risk of Thrombosis» Patrick Borel (group leader) Emmanuelle Reboul (researcher) Marielle Margier (PhD

43 Acknowledgments UMR 1062 INSERM/1260 INRA/Aix-Marseille University «Nutrition, Obesity and Risk of Thrombosis» Patrick Borel (group leader) Emmanuelle Reboul (researcher) Marielle Margier (PhD student) Marion Nowicki (technician) Charlotte Halimi (technician) Contacts: COST action FA POSITIVe : Interindividual variation in response to consumption of plant food bioactives and determinants involved Collaborations Rachel Kopec (Ohio State University) Torsten Bohn (Luxembourg Institute of Health)

Emmanuelle Reboul. Human Micronutrition team Cardiovascular and Nutrition Center (C2VN) Marseille - FRANCE

Emmanuelle Reboul. Human Micronutrition team Cardiovascular and Nutrition Center (C2VN) Marseille - FRANCE Emmanuelle Reboul Human Micronutrition team Cardiovascular and Nutrition Center (C2VN) Marseille - FRANCE The lipid micronutrients Vitamins are non-energetic molecules present in small amounts in our diet