1. MargiAnne Isaia, MD MPH Advanced Nutrition Soy isoflavones and bone Phytochemicals

2. IFs STRUCTURE IFs SOURCES SOY FOODS EFFECT OF PROCESSING, STORAGE, COOKING BONE PHYSIOLOGY – ESTROGENS vs IFs IFs METABOLISM IFs BIOAVAILABILITY IFs MECHANISM OF ACTION EPIDEMIOLOGICAL STUDIES GENETIC POLYMORPHISM CONCLUSIONS PUBLIC HEALTH PERSPECTIVE FUTURE RESEARCH REFERENCES c o n t e n t s :

3. HO ROOH O b GLYCOSIDES GENISTIN DAIDZIN GLYCITIN other…. GL-O OOH O R ISOFLAVONE STRUCTURE AGLYCONES GENISTEIN DAIDZEIN GLYCITEIN

4. IFs = weak E activity [10 -4 activity of bESTRADIOL (E 2 )] GENISTEIN - 5-20x greater affinity to bind ER b than ER a ESTROGEN IFs - ESTROGEN-LIKE CHEMICAL STRUCTURE

5. NATURAL very limited sources in nature found exclusively in legumes (soy particularly) SOY FOODS: AGLYCONES (Genistein 50%) Daidzein 40%, Glycitein 10%) GLYCOZIDES (b glycosides) 1g soy food = approx 3.5 mg IFs 1 serving soy food = approx 25 mg IFs In soy beans: 580-3800 mg/kg fresh product Soy milk: 30-175 mg/L SYNTHETIC: IPRIFLAVONE IFs SOURCES

6. FOUR CLASSES: A) Soy ingredients: unprocessed soy beans, soy flour (Defatted/full-fat), soy concentrate, soy isolates, texturized vegetable soy protein (TVP), hydrolyzed soy protein, soy germ, (10x more than in soybeans) Edamame (green soybean) = 1/5 content of IFs in mature soybeans. B) Traditional soy foods: SOY MILK, TOFU, TEMPEH, NATTO, MISO, SOY SAUCE, KINAKO. C) Second generation soy foods: Soy based burgers, hot dogs, chicken, bacon, meat analogues, soy cheese. D) Food with soy as functional ingredient. Soy flour added to baked goods for accelerating gluten formation in the kneading process; the fried bread products to reduce fat uptake, soy-based infant formula, food in which soy hydrolyzed added to replace mono-sodium glutamate. SOY FOODS

7. SOY FOODS CONTENT OF IFS DEPENDS ON - soybean variety, crop year - type of food. Non fermented – b glycozides Fermented – un-conjugated IFs - greater proportion of IFs intake (digestion is not required) - more readily absorbed form - more efficiently metabolized SOY FOODS

8. ISOFLAVONE content = depends on level, type of processing and food matrix - the highest in soy ingredient class (defatted soy flour, IFs associated with protein fraction) - the lowest in second generation of food (use of soy protein ingredient) - no detectable IFs – soybean oil Processing - Heating - total IFs content the same, IFs not destroyed. IFs forms redistributed. - Toasting - soy flakes, soy flour  acetyl glycozides (the highest source of IFs (KINAKO) - Fermentation (TEMPEH, MISO, NATTO) = microbial hydrolysation of the glycozides – AGLYCONS - Boiling - IFs leach into boiling water (49%) - Soy whey discarded in tofu production – IFs lost - Dehulled soy beans – IFs lost EFFECT OF STORAGE, PROCESSING, COOKING

9. IFs bind to estrogen receptor (ER) a and b ER a and b expressed in many cell types (ERa, ERb, ERab) ERa - endometrium, breast cells, ovarian stromal cells, hypothalamus ERb – kidney, brain, bone, heart, lungs, prostate, endothelial cells. 17b ESTRADIOL binds equally well to both ER ESTRONE and RALOXIFEN - ER a ESTRIOL and GENISTEIN - ER b E action on bone:- inhibits osteoclasts and osteoclasts precursors,  IL-6 - enhances osteoblast survival ( inhibition of CASPASE -3) GENISTEIN: enhances differentiation of bone marrow stromal cells to osteoblast lineage. BONE PHYSIOLOGY IF – ESTROGEN-LIKE PROPERTIES

10. IFS – SELECTIVE ESTROGEN RECEPTOR MODULATORS (SERM) (SERM: Tamoxifen, Raloxifen, Isoflavons!?) They demonstrate differential binding and activity (increased, decreased) to the a and b ERs IFs:- compete with E for the same ER sites: (-) the health risks of excess E (ERa) (antagonist effect) (+) estrogen activity (ERb) (agonist effect) IFs – NON-HORMONAL EFFECTS: - prevent tissue expression of TNFa and the development of skeletal pathology IFs + Soy protein – favorable effects on renal function: inhibit bone turnover in post-menopause W – inhibit development of secondary Hyperparathryoidism:  urinary D-Pyridinoline & N-Telopeptide  Serum Osteocalcin,  Alkaline Phosphatase Activity BONE PHYSIOLOGY

11. The order of E receptor binding potency of soy derived IFs aglycons is Equol > Genistein > Daidzein Their conjugates are less potent (increased level of serum conjugates) Absent or  level of serum aglycones – Equol- the more potent estrogenic metabolite (greater affinity for ERs) - unique anti-androgenic properties -superior anti-oxidant activity NORMAL BONE MATRIX OSTEOPOROSIS BONE PHYSIOLOGY

12. CONJ. IFs (from bile) AGLYCONS TISSUE UPTAKE BLOOD AGLYCONS (from food) GLYCOSIDES (from food) AGLYCONS diffusion LPH hydrolysis reabs Conjugate IFs (glucuronid) Conjugated IFs SI Cell Conjugated IFs Conjugated IF Re-conjugated IFs Conjugated IFs Bile Excretion Blood -- urine Tissue uptake GLY (from food) Intestinal micro flora hydrolysis LI Cell Conj. IFs (from bile) AGLYCONS EQUOL LPH - LACTASE PHLORIZIN HYDROLASE STOMACH CELLS AGLYCONS diffusion GUT LUMEN Liver Cell IFs METABOLISM

13. Role of colonic micro-flora: the glycosides not digested in SI are hydrolyzed microbial metabolites – are absorbed and conjugated with sulfate or glucuronate Metabolic conjugation  increases solubility in bile/urine  excretion In circulation: only conjugated forms Transported bound to albumin Tissue uptake: no correlation – plasma level – tissue levels IFs extensive metabolism – difficult to evaluates plasma metabolites level Un-identified metabolites in plasma (future research) The metabolic profile – may be an important determinant of bioactivity after soy intake (EQUOL) IFs METABOLISM

14. T max : Time to reach C max (hours) GENISTEIN < GLYCITEIN < DAIDZEIN C max (mMOL / L):DAIDZEIN < GLYCITEIN < GENISTEIN URINARY EXCRETION ( % of intake):GENISTEIN < DAIZEIN < GLYCITEIN ELIMINATION HALF-LIFE (hours):GENISTEIN < DAIZEIN < GLYCITEIN BIOAVAILABILITY

15. Depends on dietary interactions IFs associated with soy protein IFs by themselves (supplements) - IFs – from fermented soy food vs. IFs from other soy food - Wheat fiber  Genistein bioavailability (  Genistein excretion) Equol – undetectable in human plasma (the highest receptor binging potency from IFs) E > G > D In human plasma: glucuronides > 80% BIOAVAILABILITY

16. *Isoflavone content of soy foods can vary considerably between brands and between different lots of the same brand; therefore, these values should be viewed only as a guide. Total Isoflavone, Daidzein and Genistein Aglycone Content of Selected Foods* (107)(107) FoodServing Total Isoflavones (mg) Daidzein (mg)Genistein (mg) Soy protein concentrate, aqueous washed 3.5 oz1024356 Soy protein concentrate, alcohol washed 3.5 oz1275 Miso½ cup592234 Soybeans, boiled½ cup472324 Tempeh3 ounces371521 Soybeans, dry roasted 1 ounce371519 Soy milk1 cup301217 Tofu yogurt½ cup21712 Tofu3 ounces20812 Soybeans, green, boiled (Edamame) ½ cup1266 Meatless (soy) hot dog 1 hot dog1136 Meatless (soy) sausage 3 links30.62 Soy cheese, mozzarella 1 oz20.31

17. IFs – Bind to ER (via E Response Elements) – stimulates the transcription of cell specific genes – up regulates the mRNA expression of osteogenic genes for: - ALP (alkaline phosphatase) - Collagen I - Transforming Growth Factor b (TGFb) (role in bone cell regulation) - suppress the activity of cytokine – stimulate bone formation - prevent tissues expression of TNF (a) - binding to ER b – (inhibit tyrosin kinase) - suppress osteoclastic activity MECHANISM OF ACTION

18. INTER-INDIVIDUAL VARIATION OF BACTERIAL METABOLISM OF SOY IFs Daidzein metabolism 30-50% of people produce EQUOL 80-90% of people produce O– DESMETHYLANGOLENSIN (O-DMA) For Equol – 3 DISTINCT DEGRADATION PHENOTYPE - high degradation - moderate degradation - low degradation INTER INDIVIDUAL VARIATION – AFTER CHRONIC ANTIBIOTIC USE in chronic inflammatory bowel diseases GENETIC POLYMORPHISM

19. PROSPECTIVE COHORT STUDY OF SOY FOOD CONSUMPTION AND RISK OF BONE FRACTURE AMONG POSTMENOPAUSAL WOMEN XIANGLAN ZHANG et al. Arch Intern Med 2005; 165:1890-1895 SHANGHAI WOMEN’S HEALTH STUDY 75,000 Chinese woman, aged 40-70 years, 4 years SOY FOOD CONSUMPTION MAY REDUCE THE RISK OF FRACTURE – SIMILAR RESULTS WERE FOUND FOR INTAKE OF ISOFLAVONES Interviews, FFQ: - low consumption of alcohol (1.9%), tobacco (2.4%), fish 40 g/d, soy foods (142 g/d) vegetables, obesity (5.1%) Inverse association was more pronounced among women in early menopause - EPIDEMIOLOGICAL STUDIES

20. DIETARY SOY ISOFLAVONES AND BONE MINERAL DENSITY, RESULTS FROM THE STUDY OF WOMEN’S HEALTH ACROSS THE NATION American Journal of Epidemiology, Vol 155, Nr8, pp746-754 2 years Study. Participants: African American (497), Caucasian (1,003), Chinese (200), Japanese (227); 42-52 years = median intakes of Genistein – micrograms /d - African American 4 mcg /d - Caucasian14 mcg /d - Chinese3,511 mcg /d - Japanese7,151 mcg /d (  fermented soy food intake) BMD – Genistein intake: positive association for J & Ch (premenopausal) Differences between J & Ch; not established if the relationship varies by race/ethnicity or by menopausal status - EPIDEMIOLOGICAL STUDIES

21. SOY ISOFLAVONE INTAKE INCREASES BONE MINERAL DENSITY IN THE SPINE OF MENOPAUSAL WOMAN: META-ANALYSIS OF RCTs Ma DF et al. CLIN NUTR 2008 Feb; 27 (1)57-64 10 RCTs (608 subjects) (China) Soy isoflavone intake vs. placebo – BMD assessed Isoflavone intervention:- more than 90 mg/d IFs consumed - for 6 month can be enough Isoflavone intervention significantly ATTENUATES bone loss of spine in menopausal women EPIDEMIOLOGICAL STUDIES

22. SOY ISOFLAVONE INTAKE INHIBITS BONE RESORPTION AND STIMULATES BONE FORMATION IN MENOPAUSAL WOMEN: META-ANALYSIS OF RCT D-F Ma, et al, European Journal of Clinical Nutrition (2008) 62,155-161 9 Rct (432 subjects) (China) Soy isoflavone intake vs placebo Assessed: - Urinary deoxypyridinoline (DPYR) – bone resorption marker - serum bone specific alkaline phosphatase (BAP) - a bone formation marker Isoflavone intervention:  BAP significantly in IFs group  DPYR significantly in IFs group These favorable effects (bone formation stimulation and bone formation inhibition) occur even if less than 90mg /d IFs are consumed or the intervention lasts less than 12 weeks EPIDEMIOLOGICAL STUDIES

23. SOY MILK OR PROGESTERONE FOR PREVENTION OF BONE LOSS – A 2-YEAR RANDOMIZED PLACEBO-CONTROLLED TRIAL LYDEKING-OLSENE, et al Eur. Journal Nutr 2004 Aug 43 (4) 246-57 Study: 2 years, Caucasian Woman, with established osteoporosis (DEXA) All subjects comparable intake of Ca, minerals, vitamins 4 groups:- Soy milk (76mg/d intake) (n=23) - Transdermal progesteron (TDP) (N=22) - Soy milk + TDP - placebo Daily intake of 2 glasses of soy milk (76mg IFs) prevents lumbar spine loss in post-menopausal women TDP – had bone sparing effects TDP + soy milk – negative interaction – bone loss Placebo –bone loss EPIDEMIOLOGICAL STUDIES

24. EPIDEMIOLOGICAL STUDIES ON SOY & BONE – VARIABLE OUTCOMES - Observational studies – based on insensitive dietary assessment methods - limited databases to estimate the IFs content of foods - Interventional studies in humans – few, differences in dosages of IFs administrated, small sample sizes - Interpretation of the “vitro” biological activity of IFs from “in vitro” data - Data from animals extrapolated to humans (IFs metabolism and bioavailability – quite different in different species). - Serum level of IFs and their metabolites not an indicator of their tissue levels and tissue activity CONCLUSION

25. EPIDEMIOLOGICAL STUDIES ON SOY & BONE – VARIABLE OUTCOMES (Cont) - Some unknown active metabolites (EQUOL) - Bone physiology – under complex “influence” (physical activity, dietary factors, other lifestyle factors) - Not yet established the EAR for IFs intake (25-40 mg /d?) Soy food? IFs supplements? - Risk assessment and safety evaluation – needed (extraction methods – some compounds toxic,  estrogen activity) CONCLUSION

26. Osteoporosis – most prevalent metabolic bone disease in the US and developed countries (Estrogen deficiency – major cause) (1 in 3 W) (US: 25 mil women, 1.5 mil new fracture / year, cost $60 billions (2020) Women’s Health Initiative HRT, RCT 60,000 patients – Started in 2002, stopped after 5.2 years- Benefits for bone health and colon cancer. The risks: breast cancer, CHD, stroke, venous tromboembolic diseases, outweighs its benefits. (HRT only for women with significant risk of osteoporosis) Find natural, safe and effective alternative for women to help maintain bone health after menopause. PUBLIC HEALTH PERSPECTIVE

27. OSTEOPOROSIS – pediatric disorder with elderly outcomes. Early exposure to IFs - - bone protection Pre-pubertal and pubertal exposure to isoflavones – protection against estrogen-deficiency induced pathology. Soy beans – major source of IFs, high quality protein. Soy germ isoflavones – an alternative Dietary intake 25-40 mg/d (like Japan) (5% of Japanese W – 100 mg/d) Soy milk – for girls and women 2-3 caps/day Soy food cannot be recommended at the present time as a substitute for HRT (ERT) but for those who choose not to use estrogen – may be 60-90 mg /d Balanced plant-based diet, active life, healthy life style habits. PUBLIC HEALTH PERSPECTIVE

28.  Role of food MATRIX on IFs METABOLISM and BIOAVAILABILITY  Role of Ethnicity – Genetic variation on - b glucosidase Enzyme - The density of osteoblast estrogen receptor - life style factors  Interactions of IFs with other nutrients (Vit K – Japanese study: Vit K supplementation)  Role of Protein/Ca ratio on bone health (Animal Protein vs. plant protein – soy)  NATURAL IFs suplements vs SYNTHETIC (Ipriflavone)  Physiologically relevant metabolites levels and pattern (appropriate biomarker for assessing IFs exposure) FUTURE RESEARCH

29. IDENTIFY SPECIFIC BACTERIAL SPECIES AND STRAINS CAPABLE OF: - converting DAIDZEIN to EQUOL or increasing EQUOL production IFs – and use of ANTIBIOTICS (AB) – CHRONIC AB EXPOSURE (inter- individual variation) IFs - CHRONIC INFLAMMATORY BOWEL DISEASE FOOD INDUSTRY – NEW RECIPES FOR WESTERN POPULATION HIGH QUALITY SOY FOOD - AFFORDABLE - ACCESSIBLE FUTURE RESEARCH

30. REFERENCES 1.XIANGLAN ZHANG et al, Arch Intern Med, 2005; 165:1890-1895 Prospective Cohort Study of Soy Food Consumption and Ribk of Bone Fracture Among Post menopausal Woman- 2.American Journal of Epidemiology, vol 155, nr 8, pp 746-754 Dietary Soy Isoflavones and bone mineral Density: Results from the study of Women’s Health Across the Nation 3.Wew ZENG et al: American Journal of Epidemiology, 2005,(62 (11): 1123- 1131 4.Ma D.F et al, Clin Nuts 2008 Feb; 27(1)57-64 Soy isoflvone intake increase bone mineral density in the spine of menopausal women; meta analysis of RCTs- 5.D-F Ma et al, European Journal of Clinical Nuts (2008) 62, 155-161 Soy inflammation intake inhibits bone respoption and stimulates bone formation in menopausal women; meta analysis of RCRs- 6.Lydeking – Olsen, E et all- Eur J Nutz 2004 Aug: 43(4):246-57 Soymilk or progesterone for prevention of bone loss- a 2 year randomized, placebo- controlled trial- 7.HEIM M et al, Endocrinology, 2004 Feb; 145(2) 848-59 The phytoestrogen genistein enhances osteogenesis and represses adipogenic differentiation of human primary bone marrow stromal cells- 8.JIAN-PIANG YUAN et al, Molecular Nutrition & Food Research 2007, vol 51, Issue 7, 765-781 Metabolism of dietry soy isoflavones to equal by human intestinal micro flora- implications for health- 9.Liwei Gu et al, 2006, American Society for Nutrition- J. Nutrition. 136: 1215 – 1221 Metabolic Phenotype of Isoflavoens differ among female rats, pigs, monkeys and Women- 10. WILDMAN, R.E.C, 2001 Handbook of nutracentical and Functional Food- 11.MESKIN, M.S et all, 2004 PHYTOCHEMICALS- mechanisms of action- 12. The 5 th I V e, symposia – current issues on soy-

31. QUESTIONS?