Advanced Nutrition Phytochemicals Soy isoflavones and bone Margi

Advanced Nutrition Phytochemicals Soy isoflavones and bone Margi. Anne Isaia, MD MPH

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

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

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

IFs SOURCES NATURAL very limited sources in nature found exclusively in legumes (soy particularly) SOY FOODS: AGLYCONES (Genistein 50%) Daidzein 40%, Glycitein 10%) GLYCOZIDES (b glycosides) 1 g 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

SOY FOODS 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, (10 x 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 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

EFFECT OF STORAGE, PROCESSING, COOKING 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 gh 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

BONE PHYSIOLOGY IF – ESTROGEN-LIKE PROPERTIES 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. 17 b 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, i IL-6 - enhances osteoblast survival ( inhibition of CASPASE -3) GENISTEIN: enhances differentiation of bone marrow stromal cells to osteoblast lineage.

BONE PHYSIOLOGY 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: i urinary D-Pyridinoline & N-Telopeptide h Serum Osteocalcin, h Alkaline Phosphatase Activity

BONE PHYSIOLOGY 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 i 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

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

IFs METABOLISM 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 g increases solubility in bile/urine g 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)

BIOAVAILABILITY Tmax: Time to reach Cmax (hours) GENISTEIN < GLYCITEIN < DAIDZEIN Cmax (m. MOL / L): DAIDZEIN < GLYCITEIN < GENISTEIN URINARY EXCRETION ( % of intake): GENISTEIN < DAIZEIN < GLYCITEIN ELIMINATION HALF-LIFE (hours): GENISTEIN < DAIZEIN < GLYCITEIN

BIOAVAILABILITY 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 h Genistein bioavailability (i Genistein excretion) Equol – undetectable in human plasma (the highest receptor binging potency from IFs) E>G>D In human plasma: glucuronides > 80%

Total Isoflavone, Daidzein and Genistein Aglycone Content of Selected Foods* (107) Total Isoflavones (mg) Daidzein (mg) Genistein (mg) 102 43 56 Soy protein concentrate, alcohol 3. 5 oz washed 12 7 5 Miso ½ cup 59 22 34 Soybeans, boiled ½ cup 47 23 24 Tempeh 3 ounces 37 15 21 Soybeans, dry roasted 1 ounce 37 15 19 Soy milk 1 cup 30 12 17 Tofu yogurt ½ cup 21 7 12 Tofu 3 ounces 20 8 12 Soybeans, green, boiled (Edamame) ½ cup 12 6 6 Meatless (soy) hot dog 11 3 6 Meatless (soy) sausage 3 links 3 0. 6 2 Soy cheese, mozzarella 1 oz 2 0. 3 1 Food Serving Soy protein concentrate, aqueous washed 3. 5 oz *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.

MECHANISM OF ACTION IFs – Bind to ER (via E Response Elements) – stimulates the transcription of cell specific genes – up regulates the m. RNA 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

GENETIC POLYMORPHISM 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

EPIDEMIOLOGICAL STUDIES 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 DIETARY SOY ISOFLAVONES AND BONE MINERAL DENSITY, RESULTS FROM THE STUDY OF WOMEN’S HEALTH ACROSS THE NATION American Journal of Epidemiology, Vol 155, Nr 8, pp 746 -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 - Caucasian 14 mcg /d - Chinese 3, 511 mcg /d - Japanese 7, 151 mcg /d (h 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 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 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: h BAP significantly in IFs group i DPYR significantly in IFs group These favorable effects (bone formation stimulation and bone formation inhibition) occur even if less than 90 mg /d IFs are consumed or the intervention lasts less than 12 weeks

EPIDEMIOLOGICAL STUDIES 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 (76 mg/d intake) (n=23) - Transdermal progesteron (TDP) (N=22) - Soy milk + TDP - placebo Daily intake of 2 glasses of soy milk (76 mg IFs) prevents lumbar spine loss in post-menopausal women TDP – had bone sparing effects TDP + soy milk – negative interaction – bone loss Placebo –bone loss

CONCLUSION 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 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, k estrogen activity)

PUBLIC HEALTH PERSPECTIVE 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 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.

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

FUTURE RESEARCH 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 (interindividual variation) IFs - CHRONIC INFLAMMATORY BOWEL DISEASE FOOD INDUSTRY – NEW RECIPES FOR WESTERN POPULATION HIGH QUALITY SOY FOOD - AFFORDABLE - ACCESSIBLE

1. XIANGLAN ZHANG et al, Arch Intern Med, 2005; 165: 1890 -1895 REFERENCES Prospective Cohort Study of Soy Food Consumption and Ribk of Bone Fracture Among Post menopausal Woman- 2. 3. 4. 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 Wew ZENG et al: American Journal of Epidemiology, 2005, (62 (11): 1123 - 1131 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-

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