PLEASE TYPE THE UNIVERSITY OF NEW SOUTH WALES Thesis/Project Report Sheet
Surname or Family name: Knight First name: David Other name/s: Charles Abbreviation for degree as given in the U niversity calendar: MD Sc hool: O&G Faculty: Title: lsoflavone Phytoestrogens in Humans. The biological effects at different ages from the clinical perspective.
Abstract 350 words maximum: (PLEASE TYPE)
In Australia the trend toward dietary change by the general public in an attempt to improve health has resulted in an increased consumption of soy based products containing isoflavones. These compounds are selective oestrogen receptor modulators and have described oestrogen agonist and antagonist effects. The aim of this thesis was to assess the biological effects of isoflavones at different stages of reproductive life in humans. The isoflavone content of different foods, formulas and drinks that may be consumed by infants during their first year of life was investigated by measurement with HPLC in an attempt to define levels of exposure on different feeding regimens. All foods tested contained isoflavones, at varying levels, suggesttng that exposure to these compounds is almost ubiquitous. Given the relatively broad choice of infant foods becoming available, exposure to dietary isoflavones during the first year of life is virtually ubiquitous. The exposure may be higher if soy infant formulas are consumed, however the levels attained appear to fall within normal physiological boundaries. Ovarian follicular fluid was collected during oocyte collection during Assisted Reproduction cycles and examined for the presence of isoflavones by HPLC. Genistein was found to be present in ovarian follicular fluid. This is the first demonstration in humans of the presence of isoflavones of dietary origin in ovarian fluid and the presence of these compounds may effect ovarian function. Supplementation by dietary isoflavones in postmenopausal women was performed to assess the possible effects of isoflavones on estrogen deficiency symptoms and other tissue effects of estrogen deficiency. There was no significant difference in the incidence of vasomotor symptoms between active and placebo groups in three trials conducted. Powdered energy drinks were poorly tolerated and the high withdrawal rate and reporting of side effects suggests that other methods of isoflavone delivery may be more appropriate in the Australian culture. At the doses used no benefit was seen in relief from menopausal symptoms, cardiovascular parameters or bone turnover.
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'-1 AI ,L STATRS Bookbinding Ph. !tl2l ·· ..•. >nt., lsoflavone Phytoestrogens in Humans.
The biological effects at different ages from the clinical perspective.
David C. Knight
MB BS, FRANZCOG, GradCert RepodMed
City West IVF
City West House
12 Caroline St,
Westmead, NSW 2145
A thesis submitted for the degree of
Doctor of Medicine
University of New South Wales
January 2001 UNSW 3 0 OCT ?001 LIBRARY I"
Abstract
In Australia the trend toward dietary change by the general public in an
attempt to improve health has resulted in an increased consumption of soy
based products containing isoflavones. These compounds are selective
oestrogen receptor modulators and have described oestrogen agonist and
antagonist effects. The aim of this thesis was to assess the biological effects of isoflavones at different stages of reproductive life in humans.
The isoflavone content of different foods, formulas and drinks that may be consumed by infants during their first year of life was investigated by measurement with HPLC in an attempt to define levels of exposure on different feeding regimens. All foods tested contained isoflavones, at varying levels, suggesting that exposure to these compounds is almost ubiquitous. Given the relatively broad choice of infant foods becoming available, exposure to dietary isoflavones during the first year of life is virtually ubiquitous. The exposure may be higher if soy infant formulas are consumed, however the levels attained appear to fall within normal physiological boundaries.
2. Ovarian follicular fluid was collected during oocyte collection during
Assisted Reproduction cycles and examined for the presence of
isoflavones by HPLC. Genistein was found to be present in ovarian follicular fluid. This is the first demonstration in humans of the presence of
isoflavones of dietary origin in ovarian fluid and the presence of these compounds may effect ovarian function.
Supplementation by dietary isoflavones in postmenopausal women was performed to assess the possible effects of isoflavones on estrogen deficiency symptoms and other tissue effects of estrogen deficiency.
There was no significant difference in the incidence of vasomotor symptoms between active and placebo groups in three trials conducted.
Powdered energy drinks were poorly tolerated and the high withdrawal rate and reporting of side effects suggests that other methods of isoflavone delivery may be more appropriate in the Australian culture. At the doses used no benefit was seen in relief from menopausal symptoms, cardiovascular parameters or bone turnover.
3 Publications arising from the work related to this thesis
Invited Chapters:
Knight DC. Ch 20. Phytoestrogens. In Walker JJ. (Ed) Current
Concepts for the Management of the Menopause. In press.
Prizes:
Best Clinical Research Paper. To be presented at British Fertility
Society Annual Conference 2001.
Knight DC, Driscoll GL, Tyler JPP. Dietary isoflavones are present in
follicular fluid of women. Combined Conference of The Fertility
Society of Australia and Australian Society of Reproductive Biology
Abs 124, 2000.
4. Papers
Knight DC, Tyler JPP, Driscoll GL. Dietary-derived isoflavones are
present in ovarian follicular fluid of women. Human Reproduction
Submitted
Knight DC, Howes JB, Eden JA, Howes LG. The effects and
acceptance of isoflavone-containing soy powder dietary
supplementation on menopausal symptoms. Climacteric. In press
Knight DC, Howes LG. Phytoestrogens or Dietary SERM 1s? Human
Reproduction Submitted
Hayward CS, Knight DC, Howes JB, Eden JA, Kelly RP. Effect of
supplemental isoflavones on large artery function in postmenopausal
women. J Hypertension Submitted
Knight DC, Howes JB, Eden JA The effect of Promensil™, an
isoflavone extract, on menopausal symptoms. Climacteric 1999;2:79-
84.
5 Knight DC, Eden JA, Huang JL, Waring MA. lsoflavone content of
infant formulas and other food. J Paediatr Child Health 1998;34: 135-
8.
Knight DC, Eden JA. A review of the clinical effects of
phytoestrogens. Obstet Gynecol 1996;87:897-904.
Knight DC, Lyons Wall PM, Eden JA. A review of phytoestrogens
and their effects in relation to menopausal symptoms.[Leading
Article] Aust J Nutr Diet 1996;53:5-11.
Knight DC, Eden JA, Kelly GE. The phytoestrogen content of infant formulas. [letter] Med J Aust 1996;164:575.
Knight DC, Eden JA. Phytoestrogens - a short review. Maturitas
1995;22: 16-75.
6. Abstracts
Knight DC, Driscoll GL, Tyler JPP. Dietary isoflavones are present in
follicular fluid of women. Combined Conference of The Fertility
Society of Australia and Australian Society of Reproductive Biology
Abs 124, 2000.
Husband AJ, Howes JB, Knight DC et al. The correlation between
phenolic estrogen levels and menopause symptoms in women. EEC,
COST-916 Phytoestrogens Meeting, Amsterdam, April, 1998.
Knight DC, Eden JA, Kelly GE. The effects of different doses of
isoflavones in the treatment of menopausal symptoms. 8th
International Congress on the Menopause, Sydney, Nov, 1996.
Haywood CS, Knight DC, Eden JA, Kelly RP. Effect of supplemental
isoflavones on non-invasive cardiovascular haemodynamics in
postmenopausal women. 8th International Congress on the
Menopause, Sydney, Nov, 1996.
7 Knight DC, Eden JA. Dose finding study to assess the effects of
isoflavones in the treatment of menopausal symptoms. Second
International Symposium on The Role of Soy in Preventing and
Treating Chronic Disease. Brussels, Belgium. Sept, 1996.
Knight DC, Eden JA, Huang JL, Waring MA, Kelly GE. lsoflavones
in infant foods. Second International Symposium on The Role of Soy
in Preventing and Treating Chronic Disease. Brussels, Belgium.
Sept, 1996.
Eden J, Knight DC, Mackey R, Rundle F. Hormonal effect of
isoflavones. Second International Symposium on the Role of Soy in
Preventing and Treating Chronic Disease. Brussels, Belgium. Sept,
1996.
Eden JA, Knight DC. Phytoestrogens and the menopause. South
West Pacific Regional Dieticians• Conference, Brisbane, May, 1995.
8. Acknowledgments
This work has proceeded over a number of years, involving input, advice, help and collaboration from many colleagues and friends. Associate
Professor John Eden provided the initial inspiration and enthusiasm to commence this work, and without it this thesis would not have even begun. The formation of his research group resulted in an ongoing friendship with Professor Laurie Howes and my fellow worker Jan Howes.
Without them this work would have been infinitely more difficult and much less fun. Dr Chris Haywood collaborated with the cardiovascular aspects of the thesis. My supervisor, Professor Michael Chapman has been a source of advice and guidance, especially over some difficult times. Dr
John Tyler remains the font of scientific endeavour, a constant reminder of the humble scientist and their ideals. As one of my major sources of motivation, Professor Geoff Driscoll is the mentor that everyone should be lucky enough to find, and I was the fortunate one that did.
Finally, and most importantly, my family. My wife Dianne and children,
Sarah-Jane, Matthew and Benjamin have endured many hardships, moods and boring weekends to complete this thesis. I cannot express my love and appreciation deeply enough. No more playing on the computer!!
Thank you all.
9 Industry Acknowledgments
The study regarding the isoflavone content of infant formulas was funded by a grant from Novogen Ltd. Foods and infant formulas were purchased by Sanitarium Health Food Co and Wyeth-Ayerst (Australia). The isoflavone content of follicular fluid study was funded by City West IVF Pty
Ltd and the isoflavone assays were kindly performed by Novogen Ltd. The
Promensil™ menopause symptom study and the Promensil™ bone marker study were both funded by grants from Novogen Ltd. The
TakeCare™ menopause symptom study was funded by a grant from
Protein Technology Industries.
10. Contents
Page
Abstract 2
Publications arising from the work related to this thesis 4
Acknowledgments 9
Industry Acknowledgments 10
Contents 11
List of Figures 16
List of Tables 17
Abbreviations 20
1 Literature Review 24
1.1 Dietary Sources of Phytoestrogens 28
1.2 Lignans 30
1.3 lsoflavones 31
1.4 Properties of Phytoestrogens 36
1.5 Phytoestrogens and Clinical Practice 41
1.6 Phytoestrogens and the Reproductive Tract 42
11 1.6.1 lsoflavone and infant feeding 42
1.6.2 Phytoestrogens and ovarian function 45
1.6.3 The climacteric 48
1.6.3 The climacteric - current studies 52
1.7 Osteoporosis 55
1.8 Cardiovascular Disease 57
1.9 Summary 63
2 Methodology 65
2.1 Sample Collection and Handling 65
2.1.1 Sample Handling 65
2.1.2 Blood Collection and Storage 65
2.1.3 Urine 66
2.1.4 Follicular Fluid 66
2.2 Assay Techniques 67
2.3 High Performance Liquid Chromatography 67
2.3.1 HPLC Methodology used in this thesis 69
2.3.1.1 HPLC Methodology - Serum, Urine and 69
Follicular Fluid
12. 2.3.1.2 HPLC Methodology - Food Products 71
2.4 Radioimmunoassays 72
2.4.1 History of RIA 73
2.4.2 RIA Methodology 74 .. /. ;·
2.4.3 77 Markers of Bone Turnover
2.4.3.1 Bone resorption markers 77
2.4.3.2 Bone formation markers 79
2.5 Chemiluminescense 80
2.5.1 History of Chemiluminescense 82
2.5.2 Chemiluminescense Methodology used in this 83
thesis
2.6 Applanation Tonometry 84
2.6.1 85 Haemodynamic data acquisition
13 3 lsoflavone content of infant foods and 88
formulas
3.1 Results 89
3.2 Discussion 91
4 Dietary isoflavones are present in 95
follicular fluid of women
4.1 Results 98
4.2 Discussion 99
5 The effect of Promensil™, an isoflavone 103
extract, on menopausal symptoms
5.1 Results 107
5.2 Discussion 109
6 The effect of supplemental isoflavones on 114
large artery function in postmenopausal
women
6.1 Results 117
6.2 Discussion 119
14. 7 The effects and acceptance of isoflavone- 123
containing soy powder dietary
supplementation on menopausal
symptoms
7.1 Results 127
7.2 Discussion 129
8 The effects of an average dietary dose of 134
isoflavones on markers of bone turnover
in postmenopausal women
81 Results 137
8.2 Discussion 138
9 Summary 142
145
10 Future Aspects
Figures 146
Tables 152
References 173
15 List of Figures
Figure 1.2.1: The structure and metabolic pathway of common lignans.
Figure 1.2.2: The physiological enterohepatic circulation of lignans in
humans.
Figure 1.3.1: The structure and metabolic pathway of common
isoflavones.
Figure 1.3.2: The comparative structures of estradiol, diethylstilbestrol
and common isoflavones.
Figure 2.6.1.1: Schematic of carotid waveform.
Figure 5.1: Dose relationship in blinded study between the three groups and 24-hour urinary phytoestrogen levels.
16. List of Tables
Table 1.2.1. Production of Mammalian Lignans from Different Foods
Table 1.3.1. Typical levels of the aglycone isoflavones in different foods.
Table 1.3.2. Total Values of lsoflavones in Foods
Table 1.4.2. Relative potencies of phytoestrogens compared with
estradiol.
Table 3.1. lsoflavone content (mg per 100 ml) of various brands of cow
milk, dairy yoghurts and soy milk drinks available in Australia. Figures in
brackets expressed in ~M.
Table 3.2. Estimated daily exposure to isoflavones of infants consuming different milks and formulas.
Table 4.1. lsoflavone concentrations and follicle volumes of the subjects with detectable isoflavone levels.
17 Table 4.2. A correlation matrix of follicular fluid endocrine and genistein
concentrations.
Table 5.1. Biological and biochemical markers of estrogen activity.
Table 6.1. Demographics of study population
Table 6.2. Results for randomised study of 3 months placebo, 40mg and
160mg supplemental isoflavone, as well as results for 3 months 160mg
isoflavone in a larger subset.
Table 6.2 (cont): Cardiac Indices; Results for randomised study of 3
months placebo, 40mg and 160mg supplemental isoflavone, as well as
results for 3 months 160mg isoflavone in a larger subset.
Table 6.2 (cont): Arterial Indices; Results for randomised study of 3 months placebo, 40mg and 160mg supplemental isoflavone, as well as results for 3 months 160mg isoflavone in a larger subset.
Table 7.1. lsoflavone analysis of TakeCare™.
18. Table 7.2. Biological and biochemical markers of estrogen activity, serum lipid levels, markers of bone turnover data and urinary total isoflavone levels.
Table 8.1. Mean results for bone turnover markers from Week 0 to Week
20. (mean (SEM))
Table 8.2. Simple correlation between percentage change in bone marker and urinary isoflavone excretion.
19 Abbreviations
ART Assisted Reproductive Techniques b-ALP Bone specific alkaline phosphatase
BIO-A Biochanin
BP Blood pressure
BSA Body surface area
DAID Daidzein
OAT Digital applanation tonometry
DELFIA Dissociation enhanced lanthanide fluorescence immunoassay
DHEAS dehydroepiandrostendione sulphate
DNA Deoxyribonucleic acid dP/dtmax> Maximal rate of pressure rise
E2 Estradiol
ECG Electrocardiogram
EDTA Ethylenediamine tetraacetic acid
20. ERT Estrogen replacement therapy
FORM Formononetin
FSH Follicle stimulating hormone
GEN Genistein
GnRH Gonadotrophin releasing hormone hCG Human choriogonadotrophin
HDL High density lipoproteins
HOL-e High density lipoprotein-cholesterol
HERS Heart and estrogen/progestin replacement study
HPLC High performance liquid chromatography
HRT Hormone replacement therapy
ICTP lnterchain C-terminal telopeptide
IGF-1 Insulin-like growth factor-1 lnhib-A lnhibin A lnhib-B lnhibin B
LDL Low density lipoproteins
LH Luteinising hormone
LOQ Limit of analytical quantitation
MV Maturation value
21 OCP Oral contraceptive pill
0-dma 0-desmethylangiolensin
OTC over the counter p Progesterone
PCOS Polycystic ovarian syndrome ppm Parts per million
PRL Prolactin
PWV Pulse wave velocity
RIA Radioimmunoassay
RNA Ribonucleic acid
RSD Relative standard deviation
SEM Standard error of the mean
SERM Selective estrogen receptor modulator
SHBG Sex hormone binding globulin
TAF-1 Tumour activating factor-1
TBG Thyroid binding globulin
TC Total cholesterol
TGFa Transforming growth factor alpha
22. TN Fa Tumour necrosis factor alpha
TSH Thyroid stimulating hormone
TVP Textured vegetable protein
UOR Ultrasound oocyte retrieval u-PCL urinary deoxypyridinoline crosslinks uv Ultraviolet
23 Chapter 1
Introduction
The presence of estrogenic-like activity of certain foods has been known since the 1920's. The use of plants or plant extracts to modulate fertility has occurred in most civilisations over the past 2000 years. However the estrogenic activity of plants was not demonstrated until 1926 in the peduncles of the female willow. (Loewe et al, 1927) Within two decades
(Bennetts et al, 1946) a syndrome of polycystic ovaries, endometriosis and irreversible infertility had been described in grazing ewes (Adams,
1976) and genital and mammary stimulation in wethers (Adams, 1977a,
Adams, 1977b), both secondary to the consumption of estrogenic clovers.
Ewes fed estrogenic forage suffer impaired ovarian function, often accompanied by reduced conception rates and increased embryonic loss.
Male sheep appear to be relatively unaffected, but the mammary glands in females and castrate males undergo hypertrophy of the duct epithelium, accompanied by secretion of clear or milky fluid. In cows, clinical signs resemble those associated with cystic ovaries. The infertility is temporary, normally resolving within a month following removal from the estrogenic feed. However, ewes exposed to estrogen for prolonged periods may suffer a secondary form of inferti'lity that is permanent, caused by developmental actions of estrogen during adult life. The cervix becomes
24. defeminised and loses its ability to store spermatozoa, and conception
rates are reduced, although ovarian function remains normal. Importantly,
both temporary and permanent infertility in ewes occurred without observable signs. The problems were detected only following
measurement of phytoestrogens in the diet of the animal or measurement of these compounds effects on the animal. (Adams, 1995)
During the 1970's, Farnsworth and colleagues listed 300 plants, not all edible, reported to exhibit estrogenic activity and 29 specific compounds that also exhibited this activity. (Farnsworth et al, 1975) These included plants (concentration of estrogenic chemicals) such as Glycyrrhiza glabra
(Liquorice Root) 152567 ppm, Glycine max (Soybean Seed) 22823 ppm,
Trigonella foenum-graecum (Fenugreek Seed) 19000 ppm, Trifolium pratense (Red Clover) 15000 ppm, Scutellaria baicalensis (Chinese
Skullcap Root) 5 000 ppm, Nigella sativa (Black Cumin Seed) 3218 ppm,
Oenothera biennis (Evening-Primrose Seed) 2569 ppm and Salvia officinalis (Sage Leaf) 2496 ppm. (USDA Agricultural Research Service
Phytochemical database.)
Epidemiological evidence suggests a large role for the dietary consumption of estrogenic plants in human health. Diets low in fat and
high in complex carbohydrates from grains, fruits and vegetables are
associated with a lower risk of chronic diseases such as diabetes,
25. cardiovascular disease and some hormone dependent malignancies
including breast and prostate cancers. (National Research Council, 1989)
The observation of the role of diet in reducing or promoting disease has attracted increased interest over the past two decades as we attempt to improve lifestyle through preventative measures. Specific components in the diet are being increasingly studied, from vitamins and anti-oxidants to plant compounds that bind to the estrogen receptor (ER).
Phytoestrogens are naturally occurring compounds found in many foods.
Phytoestrogen is a generic term literally including all plant compounds with estrogenic effects. More appropriate terminology would be
"phytochemical" as these compounds have other properties. Common usage however has perpetuated the term phytoestrogen, with it also generally referring to three classes of compounds; isoflavones, coumestans and lignans. These compounds are able to bind and either activate or inhibit the estrogen receptor, however the subsequent effects are complicated and depend upon the existing hormonal milieu and target tissue. The biological activity of phytoestrogens has been demonstrated in many animals, (Bennett and Dudzinski, 1967) (Leavitt and Wright, 1963)
(Braden et al, 1971) (Lunndh et al, 1988) (Kaldas and Hughes Jnr, 1989) however, their possible actions in humans have been less extensively studied.
26. Over the past decade, lignans and isoflavones have been detected in
humans (Setchell et al, 1980) (Bannwart et al, 1989) (Seely, 1982} and
both have been shown to have biologic activity in man and animals.
(Setchell et al, 1981 a) (Axelson et al, 1984) Coumestans and resorcylic acid lactones (mycoestrogens or fungal estrogens) also have biological activities in animals. (Bennett et al, 1974) (Schoental, 1983) (Price and
Fenwick, 1985)
In the early to mid 1990's an increase in desire within the general population occurred for alternative therapeutic options to mainstream medical treatment and pharmacology. Numerous products appeared on the "Over the Counter" (OTC) market purporting to treat a wide range of symptoms and ailments, and in particular the use of estrogen and progestogen hormone replacement therapy. Most of these claims had no scientific data to support them. Commercialisation without evidence has the potential of significant harm to the individual and society. Millions of dollars are spent annually in Australia on these products, money that may be more appropriately spent on products with proven efficacy. Use of unproven products may also delay onset of appropriately efficacious treatment. This is exemplified in that as of the end of the year 2000, there are still less than ten blinded, placebo-controlled, published clinical trials addressing the clinical effectiveness of isoflavones in the treatment of menopausal symptoms. This is despite approaching a decade on and the
27. enthusiasm of alternative therapists and public for these foods, compounds and approaches.
1.1. Dietary Sources of Phytoestrogens
The human diet provides the precursors of both lignans and isoflavones.
Oilseeds contain the highest detected concentration of lignans, and these compounds are found in lesser concentrations in cereal bran, whole cereals, vegetables, legumes and fruits. (Thompson et al, 1991) A legume is defined as any plant of the family Leguminosae, or alternatively as a dry, dehiscent fruit of the Leguminosae, such as a bean, pea or lentil, that develops from the splitting of a single carpel into two valves, each bearing seeds alternately along the ventral margin. (Academic Press Dictionary of
Science and Technology, 1996} Whilst flavonoids are ubiquitous in photosynthesising plants (Brandi, 1992}, isoflavones are less widely distributed. lsoflavones are largely restricted to members of the
Leguminosae family. lsoflavones occur in high concentrations in soybeans, (Messina and Messina, 1991) chickpea, (Sharma, 1981) toothed medic, bluegrass and clover. (Price and Fenwick, 1985)
Estrogenically active clovers, such as red clover, may contain up to 700 mg of genistein (an isoflavone) per 100 g of dry plant material. (Curnow,
1954) Coumestans are related structurally to isoflavones. They have been reported in highest concentrations in alfalfa and soybean sprouts (Knuckles et al 1976), but also occur in other legumes, clover and fodder
crops.
Phytoestrogens fulfil a number of roles in plants. Lignans are minor constituents required for the formation of lignin, which is ubiquitous in
plant cell walls. (Setchell and Adlercreutz, 1988) Flavones and isoflavones
are phytoalexins, plant defence mechanisms, and have evolved to
modulate the degree of predation by bacteria and animals.
Phytochemicals may act as insecticides, insect anti-feedants and growth
regulators as well as affecting the reproductive capacity of herbivores and birds. (Hughes Jnr, 1988)
These effects are produced in a number of fashions including warding off by producing adverse tastes or odours, direct toxicity or fertility regulation.
The accumulation of isoflavones in legumes secondary to fungal infections is well documented. (Wyman and Van Etten, 1978) lsoflavones and coumestans also accumulate in response to bacterial infections in legumes and the phytoestrogen concentration in plants rises in response to plant stressors such as insect infestations, ultraviolet irradiation, mechanical injury and drought. (Smith and Banks, 1986) These mechanisms may represent effects that aid the survival of the plant species during times where they may be threatened by extinction.
Concentrations can rise up to tenfold in these situations.
29. 1.2. Lignans
The main lignans associated with biological activity in humans are enterolactone and enterodiol. (Setchell et al, 1981 b) The structures of these compounds are illustrated in Figure 1.2.1. Enterolactone and enterodiol are the products of colonic bacterial metabolism of the plant lignans matairesinol and secoisolariciresinol. (Boriello et al, 1985) (Lampe et al, 1994) Enterodiol can be converted to enterolactone, however the reverse does not occur.
Lignans undergo enterohepatic circulation. After bowel absorption they undergo conjugation in the liver and are excreted in the bile where re absorption or excretion occurs. Spill into the blood stream results in excretion in the urine as glucuronides. (Setchell et al, 1982) (See Figure
1.2.2) These compounds have been isolated in human urine, serum, faeces, (Setchell et al, 1980) semen (Dehennin et al, 1982) and bile
(Axelson et al, 1981) with enterolactone, enterodiol and matairesinol being identified in cow's milk. (Setchell and Adlercreutz, 1988) The use of an in vitro fermentation technique with human fecal microbiota to determine the amounts of mammalian lignans produced values that related well to levels of urinary lignan excretion observed in rats and humans. (Thompson et al,
1991) Oilseeds produced the highest amounts of which flaxseed (also known as linseed) accounted for amounts 100 times that of other plants
30. studied. Dried seaweed, whole legumes, cereal bran, legume hulls, whole grain cereals, vegetables and fruit produced decreasing amounts of lignans respectively. The in vitro production values of enterolactone, enterodiol and total lignans from common oilseeds, cereals and vegetables are presented in Table 1.2.1.
1.3. lsoflavones
The relative rarity of isoflavones in botanical families is due to the limited distribution of the enzyme, chalcone isomerase that converts flavones to isoflavone precursors. (Coward et al, 1993) The major isoflavones are genistein and daidzein. These compounds are found in the free form in plants and also with attached sugar moieties (glycosides), called genistin and daidzin. Genistein (GEN) is also produced by the demethylation of biochanin (810). (see Figure 1.3.1) Daidzein (DAID) is produced from its precursor formononetin (FORM). Daidzein may be subsequently metabolised to equol or 0-desmethylangolensin (0-dma). Genistein, daidzein, equol and 0-dma have been detected in human plasma,
(Adlercreutz et al, 1994) formononetin, genistein, daidzein and equol in urine (Adlercreutz et al, 1991) (Bannwart et al, 1987) as well as saliva, breast milk and prostatic fluid. (Franke et al, 1998) Genistein, daidzein and equol have been identified in cow's milk. (Knight et al, 1998) At the time of commencement of this thesis, no literature was available regarding
31. isoflavone and human ovarian follicular fluid, or possible effects on follicular function.
lsoflavones are found as precursors, glucosides, malonyl and acetyl derivatives and in the free form in most plants containing these compounds. They may be demethylated in the stomach, unconjugated in the gut or absorbed unchanged. lsoflavones are readily absorbed from the gastrointestinal tract and the methylated isoflavones, biochanin and formononetin, are rapidly demethylated by the liver. The metabolism of isoflavones to active metabolites by gastrointestinal flora varies between individuals, depending upon the nature of their intestinal flora. (Barnes et al, 1996} More recent pharmacokinetic data suggests that hepatic conversion may be more important due to the rapid appearance of isoflavones in the plasma, whereas colonic production would tend to produce a slower rise in serum levels. (Howes et al, 1997} Peak levels of isoflavones are reached around 4-6 hours after oral administration. The plasma half-life following acute administration is around 6-8 hours. (Kelly et al, 1995} (King and Bursill, 1998)
Following chronic administration, the half-life is significantly prolonged to
12-16 hours, suggesting that daily administration of isoflavone is sufficient to maintain relatively constant plasma levels over a 24 hour period. (Lu et al, 1996) A number of studies have indicated that doses in the range of
32 40-80 mg of isoflavones per day produce plasma levels and urinary excretion rates of isoflavone comparable to those found in communities that consume diets with a high isoflavone content. (Baber et al, 1999)
Approximately 60% of biochanin is metabolised to genistein following absorption, while approximately 60% of formononetin is metabolised to daidzein. In general, the isoflavone phytoestrogens have only about one thousandth of the affinity for the estrogen receptor of estradiol but when consumed in relatively large amounts plasma levels may be up to 1000 times higher than premenopausal or post menopausal estradiol levels.
Comparison of the structures of the common phytoestrogens and estradiol is presented in Figure 1.3.2. A large assumption has been made by the scientific community that the effects of isoflavones are estrogen receptor mediated because binding has been shown to occur. There are no published data comparing the binding kinetics of isoflavones with estrogens in humans. Whilst the assumption that isoflavones are estrogen receptor modulators, other mechanisms may be responsible. This is discussed further later in the chapter.
The isoflavone content of many foods has been elucidated. (Reinli and
Block 1996) Probably the most widely used source of isoflavone phytoestrogens is soy protein, which contains genistein and daidzein. lsoflavones are also present in chickpeas, clovers, beans and alfalfa sprouts. In a number of these sources, including soy, the isoflavones are
33. present in an inactive conjugated form that requires deconjugation, principally in the gut, to the active aglycone form. lsoflavones from other sources, such as red clover, are present in the aglycone form and do not require deconjugation to be active. As the deconjugation process is not always complete, isoflavones from sources such as red clover may be more biologically active on a milligram per milligram basis than those from sources where they are mainly present in the conjugated form, for example soy. However there is no data available yet to confirm this.
Soybeans are composed of approximately 8% hull, 2% hypocotyl and
90% cotyledon, with the isoflavone content of each portion differing. The concentration of phytoestrogens is highest in the hypocotyl, then cotyledon, then hull, with a log fall in concentration respectively. The phytoestrogen content of soybeans varies between varieties and within the same variety variations occur between locations and season of growth. (Eldridge and Kwolek, 1983) lsoflavone levels in legumes apparently increase with time after germination, the rise being more marked after 96 hours. (Sharma, 1981) Soybean protein isolates are common precursors for many meat-substitutes and may be manufactured using different procedures such that the end products have different characteristics in view of taste, texture and flavour. The final isoflavone content is dependent upon the solvent used in the manufacturing process.
When soybean protein isolate is prepared by extracting hexane-defatted
34. soybean meal with aqueous alcohol around 50% of the isoflavone content is lost, whereas aqueous leaching results in isoflavone contents similar to that of soybean flours. (Eldridge, 1982) The commonest effect on isoflavone concentration is cooking. Whilst the form of the isoflavones
(glucoside, malonyl, acetyl or aglycone) may be altered by heat treatment and enzyme reactions during fermentation, the total isoflavone content remains the same. (Anderson and Wolf, 1995) (Coward et al, 1998)
Foods containing isoflavones are principally restricted to soy products, such as tofu, soy drinks, soy flours, soy flakes and foods based on soy protein isolates; other legumes such as peas and beans (of many different varieties); and herbs. Soy flours have been shown to contain between 178 and 305 mg of total isoflavone (including aglycone) per 1OOg. (Eldridge,
1982) Soy protein concentrates contained between 21 and 317 mg of total isoflavone per 1OOg and soy protein isolates, 103 to 145 mg of total isoflavone per 1OOg. The isoflavone content of tofu and soy drinks is considerably less than that of soy flours. (Dwyer et al, 1994) Measurement of a representative range of these products revealed tofu products to contain between 26.0 and 31.3 mg of isoflavone per 1OOg, the isoflavones measured in these studies being daidzein and genistein. No data was available regarding the isoflavone content of soy beverages in Australia
Typical levels of aglycone isoflavones in foods are presented in Table
1.3.1. A study by Wang and Murphy measured isoflavone contents of
35. similar products. The glucoside, malonyl, acetyl and aglycone derivatives
of daidzein, genistein and ~lycetein were assessed and not differentiated
from free daidzein and genistein. (Wang and Murphy, 1994) Total values
of isoflavones in this study are presented in Table 1.3.2.
1.4. Properties of Phytoestrogens
Phytoestrogens have properties consistent with estrogen
agonist/antagonist activity depending upon the prevailing hormonal milieu or tissue. (Adlercreutz, 1990) The activity of compounds in respect to estrogenic action until recently has been confusing, partly as only one estrogen receptor had been recognised and therefore an estrogenic agent was categorised as an estrogen agonist or antagonist. The discrepancies in the effects of estrogenic compounds in different tissues were difficult to explain. New data has suggested that the mechanism of estrogen action
is not the same in all cells and that classification of compounds as estrogen agonists or antagonists is tissue or cell dependent. (Kuiper et al,
1997)
Recently, the term 'selective estrogen receptor modulator' (SERM) has been suggested to characterise those compounds that can bind to and activate the ER but which have tissue specific effects that are distinct from
36. estradiol. (Sato et al, 1994) More recently this has been further complicated by the description of a second estrogen receptor, ERB. It appears that the ERa and ERB, under certain conditions, have opposite effects on gene transcription. Therefore the tissue selectivity of SERM's may at least in part be explained by the different effects on ERa and ERB.
It becomes increasingly clearer that agents cannot be described as agonists or antagonists as a single agent may be an agonist in one tissue and an antagonist in another. (Gustafsson, 1998} While estradiol has equivalent affinity for both estrogen receptors, the active metabolite of tamoxifen, 4-hydroxytamoxifen, and the isoflavone genistein are relatively more selective for ERB. Until further data becomes available however, differences in tissue distribution and molecular affinity for the ERa and
ERB probably only partly explain the tissue selectivity of estrogenic compounds. (Kuiper et al, 1996} It is possible that differences in other factors involved in signal transduction such as second messenger properties and activator protein regulation are also involved in modulation gene transcription. The relative binding affinities of various compounds for the a and B estrogen receptors are presented in Table 1.4.1.
lsoflavones also appear to possess tissue selectivity in the expression of their agonist or antagonist effects. This tissue selectivity is similar to that observed for the SERM's, tamoxifen and raloxifene although there are some important differences. These compounds have agonist activity in
37. bone and antagonist effects in both the breast and hypothalamus. The effects of raloxifene have recently been documented with a proposed role in the treatment of osteoporosis. (Draper et al, 1996) lsoflavones appear to act as agonists at the hypothalamus, with soy protein consumption suppressing post-menopausal flushing. (Aibertazzi et al, 1998) lsoflavones appear to maintain a similar profile of estrogenic and anti estrogenic effects to tamoxifen in breast, bone and cardiovascular tissues.
Phytoestrogens also have several properties that appear to be unrelated to their activity at the level of the ER that may explain their biological effects, such as the ability to inhibit tyrosine kinase. Lignans and flavonoids have been reported to have antiviral, (MacRae et al, 1989) anticarcinogenic, (Hirano et al, 1990) bacteriocidal, (Nairn et al, 1974) and antifungal (Wymen and Van Etten, 1978) effects. Flavonoids also possess antioxidant, (Jha et al, 1985) antimutagenic, (Hartman and Shankel, 1990) antihypertensive, anti-inflammatory, (Wu et al, 1992) and antiproliferative
(Hirano et al, 1989) properties. However most of these effects have only been demonstrated in vitro, and at doses that are substantially higher than those that are obtainable physiologically following dietary intake. The relative estrogenic effects of different phytoestrogens have been assessed using human cell culture bioassays. (Markiewicz et al, 1989) Relative potencies of isoflavones compared with estradiol (E2) were established using concentration-dependent response curves. These compounds are much less potent than E2 and are displayed in Table 1.4.2. The
comparative dissociation constant of genistein for the ER determined in
competitive binding experiments is 100 to 10000 times higher than that of estradiol and diethylstilbestrol. (Messina et al, 1994) (Martin et al, 1978)
Genistein is the most extensively studied isoflavone. It is an inhibitor of tyrosine protein kinases, (Akiyama et al, 1987) DNA topoisomerases I and
II, (Okura et al, 1988) and ribosomal 86 kinase. (Linassier et al, 1990)
Other properties include inhibition of angiogenesis (Fotsis et al, 1993) and differentiation of cell cancer lines. (Constantinou and Huberman et al,
1995). Genistein is reported to inhibit tumour promoter-induced hydrogen peroxide formation, superoxide anion formation, and scavenge exogenously added hydrogen peroxide in human cell culture. (Wei et al,
1993) Other isoflavones, such as daidzein, apigenin, and prunectin, have also been shown to be potent hydrogen peroxide scavengers and antioxidants. (Wei et al, 1995) Tyrosine kinases play a key role in tumour genesis. Many proto-oncogenes encode receptor and non-receptor tyrosine kinases, and they are also involved in growth factor binding to cell receptors. (Cantley et al, 1991) Ligand-dependent activation of receptor associated tyrosine kinase or unregulated synthesis of tyrosine kinase oncoproteins results in tyrosine phosphorylation of cellular substances that have a critical role in the control of mitogenesis, cell-cycle regulation, cell survival, and cellular transformation. (Spinozzi et al, 1994) Growth
39. control factors with cell receptors that are associated with tyrosine kinase
include; epidermal growth factor, (Ushiro and Cohen, 1980) platelet
derived growth factor, (Nishimura et al, 1982) insulin, (Petruzelli et al,
1982) insulin-like growth factors, (Rubin et al, 1983) and transforming
growth factor alpha (TGFa). (Gangrade et al, 1991) These growth factors
have all been implicated in tumour development and propagation.
DNA topoisomerases II and ribosomal 86 kinase inhibition may lead to
protein-linked DNA strand breaks, arrest of tumour cell growth, and differentiation induction of several malignant cell lines. (Pertersen and
Barnes, 1991) Tyrosine kinase mediation of mammary tumour cells to
milk-producing, growth-arrested cells has been reported. (Wen et al, 1992)
Possible mechanisms for the antiproliferative properties of genistein
include prevention of cell mutations by stabilisation of cell DNA and
reduction of cell oxidants, reduction in capacity of malignant cells to metastasise by inhibiting angiogenesis, and subsequent tumour growth as well as inducing cell differentiation.
Most of the above findings were derived from studies stimulated by the discovery of the ability of genistein to inhibit tyrosine kinase. The physiological relevance and relationship with ER interactions are at
present unknown.
40. 1.5. Phytoestrogens and Clinical Practice
Epidemiological studies suggest foodstuffs containing phytoestrogens
may have a beneficial role in protecting against a number of chronic
diseases and conditions. Dietary studies indicate that in women soy and
linseed may reduce the risk of breast cancer and may help to alleviate
postmenopausal symptoms. (Adlercruetz et al, 1988) For osteoporosis, tentative evidence suggests phytoestrogens may have similar effects in maintaining bone density to those of the related pharmaceutical compound ipriflavone. (Brandi, 1992) These findings are supported by animal studies. (Arjmandi et at, 1998) Soy consumption may have beneficial effects on blood lipids and may be implicated in reducing the risk of cardiovascular disease and atherosclerosis. Generally little evidence exists to link these effects directly to phytoestrogens with many other components of soy and linseed being biologically active in various experimental systems and potentially responsible for the observed effects in humans. Current trials will be presented, however at this level of scientific knowledge the evidence is not sufficient to recommend particular dietary practices or changes. (Humfrey, 1998)
41. 1.6. Phytoestrogens and the Reproductive Tract
1.6.1. lsoflavones and infant feeding
Following media attention given in New Zealand in 1995 regarding the outcome of offspring of privately bred parrots fed soy meal, concerns were expressed over the possible biological impact in infants of plant diphenols with estrogenic activity. (Irvine et al 1995) These findings should not necessarily been surprising given previous reports in animals and birds.
Captive cheetahs being fed a commercially prepared feline diet with high levels of plant estrogens (Setchell et al, 1987a) were found to have an increased incidence of reproductive failure. The cheetahs had lesions with similar histopathological findings and liver function studies to those of women taking high dose oral contraceptives. (Alpert, 1976) Similarly, quails become sterile during dry years in California when the phytoestrogen content of available forage is increased. (Leopold et al,
Science 1976) The normal diet is scarce and the remaining fodder has a higher than usual phytoestrogen content secondary to drought conditions.
The estrogenic diphenols are ubiquitous compounds drawn from the chemical groups of flavones, isoflavones, lignans, coumestans, turpenoids, chalcones and acyclics. These compounds have a steric
42 structure closely resembling steroidal estrogens and this theoretically allows these compounds to bind weakly to estrogen receptors and mimic the effect of steroidal estrogens. (Price and Fenwick, 1985) The estrogenic potency of these compounds varies considerably, but in each case is relatively weak, ranging from about 2 x 1o-2 to 2 x 1 o-4 relative to estradiol.
As all plants (cereals, fruits and vegetables) used as human dietary foodstuffs contain at least one of these representative chemical groups, they are a commonplace dietary component. Plants vary considerably in their estrogenic content, depending on the absolute levels and the particular types of estrogenic diphenols present. The typical Asian, Indian and Latin American diets tend to be relatively high in phytoestrogenic activity due to the common usage of dietary legumes and pulses, such as soy, which contain relatively high amounts of the more potent phytoestrogens including coumestans and isoflavones. The typical
Western diet on the other hand contains low to modest phytoestrogenic activity because of the general absence of legumes and the tendency to eat plants containing modest levels of weaker phytoestrogens such as flavones and lignans.
These compounds have been shown to be readily absorbed from the gut, circulate in the blood and may behave as weak estrogens in the body.
43. (Adlercreutz et al, 1987) Additionally, some of the phytoestrogens such as
flavones and isoflavones have demonstrated biological effects that appear
to be independent of their estrogenic potential, in particular, a range of
possible anti-cancer functions currently attracting scientific attention.
(Messina et al, 1994) There is a general consensus in the scientific
literature that dietary phytoestrogens, and isoflavones in particular, have a
beneficial role in adults, perhaps even, one involving routine, homeostatic
mechanisms.
While no adverse effects are known in humans from the long-term intake of normal dietary levels of isoflavones and other phytoestrogens, the potential for dose-related toxicity exists. This is suggested by the previously mentioned hyper-estrogenicity syndrome in adult sheep associated with abnormally high dietary levels (1-9 g daily) of estrogenic isoflavones, Clover Disease of sheep. (Shutt and Cox, 1972)
Studies in laboratory animals show that high doses of estrogenic isoflavones and coumestans in the sexually immature animal can result in dysfunctional development of the reproductive tract. (Whitten et al, 1995)
(Levy et al, 1995) Adult humans are highly unlikely to suffer dose-related toxicity from dietary phytoestrogens given the relatively modest levels in normal human foodstuffs. However, the safety index for phytoestrogens may be lower in infants than in adults given the greater sensitivity of the
44. immature reproductive tract to estrogenic substances, and the possibility of higher dosages relative to body surface area.
The ubiquitous nature of phytoestrogens in foodstuffs and in bodily fluids including serum and breast milk (Finlay et al, 1991) suggests that there is a level of exposure during both foetal and neonatal stages of development. For breast-fed infants or those receiving animal milk substitutes, the level of phytoestrogen exposure is likely to be quite modest up until weaning. However, those infants receiving infant soy formula may be exposed to much higher amounts because of the high levels of estrogenic isoflavones in soy and soy products. The comparative doses on a body weight to surface area ratio between neonates, infants, children and adults exposed to dietary sources of phytoestrogens is unknown.
1.6.2. Phytoestrogens and ovarian function
Estrogens exert various critical actions at the level of the ovaries. Both the granulosa cell and the theca cell appear to be estrogenic sites of action.
At the level of the granulosa cell, estrogens promote cellular division and exert a direct antiatretic effect. Estrogens also play an important role in the promotion of gap junction formation, enhancement of antrum formation
45. and in augmentation of estrogen receptor content. They also act
synergistically with gonadotrophins resulting in promotion of ovarian
growth as well as LH and FSH receptor formation and increased
aromatase activity. It is this ability of estrogens to augment the activity of
the enzyme responsible for their formation that accounts for the pre
ovulatory surge in circulating levels of estradiol. This self-amplification
may play a central role in the process of follicular selection as well as
establishment of follicular dominance.
The follicle destined to ovulate undergoes recruitment, selection and then become dominant around seven days prior to ovulation. This follicle must be both functionally and morphologically dominant with the ability to inhibit the development of competing follicles on both ovaries. The dominant follicle aromatises androstenedione to synthesise estradiol in sufficient quantities resulting in passage of the hormone into the general circulation.
As midcycle approaches there is an LH surge and to a lesser extent an
FSH surge followed by a dramatic rise in estrogen that triggers the dominant follicle to ovulate. Thus estrogen is intrinsically involved in folliculogenesis and ovulation.
Genistein reduces GnRH-induced LH and FSH release from rat pituitaries
in vitro. (Packer et al, 1994) It has also been reported to block oocyte growth and disrupt follicle morphology. Many of the putative intra-ovarian
46. regulators act through binding to their cell membrane receptors that are activated via tyrosine kinase. Treatment of follicular cells with genistein attenuates TGFa-induced estrogen biosynthesis. TGFa has proved to be a potent inhibitor of gonadotrophin-supported granulosa cell differentiation. (Gangrade et al, 1991)
In ovariectomized rats, acute exposure to phytoestrogens alters GnRH induced LH release. (Hughes Jnr et al, 1991) The dose response pattern of enhanced GnRH-induced LH release at lower pre-treatment doses, but inhibited GnRH-induced LH release at higher pre-treatment doses was observed for both estradiol and genistein. (Hughes Jnr, 1987-88) Dietary isoflavone supplementation has been reported to decrease urinary excretion of estradiol, estrone, estriol, and total estrogens. (Xu et al, 1998)
Other studies suggest that consumption of soy diets containing phytoestrogens may reduce circulating ovarian steroids and adrenal androgens and increase menstrual cycle length. (Lu et al, 1996b) (Nagata et al, 1998) Ingestion of flaxseed powder, to produce high concentrations of urinary lignans, was suggested to increase luteal phase lengths in normally cycling women. (Phipps et al, 1993) There were however no significant effects on the concentrations of E2 or estrone during the early follicular, mid-follicular, or luteal phase or progesterone concentrations in the luteal phase. Flaxseed ingestion had no effect on early follicular phase
47. concentrations of dehydroepiandrostendione sulphate (DHEAS), prolactin,
or sex hormone-binding globulin (SHBG).
There is no data available regarding the presence and action of
isoflavones in human ovarian follicular fluid. The potential exists for
isoflavones to exert action at the level of the follicle and disrupt ovarian function in humans. Indeed extrapolating from sheep data, this would
indeed seem likely given high enough doses. The whole concept of estrogen action has undergone a considerable shift with the recognition of a second receptor, and the temptation exists for theories regarding the mechanisms isoflavone action to be squeezed into the status quo regarding estrogen activity.
1.6.3. The climacteric
New strategies in the treatment of the symptoms of estrogen deficiency at the menopause aim to maximise beneficial effects and minimise adverse effects of treatments, ideally increasing ease of therapy and patient acceptance. An improvement in the routes of delivery, and particularly minimisation of oncogenicity, appear to play important roles in increasing acceptance by the general public. For many women the menopause signals a time of change, which may be viewed negatively with connotations of diminishing abilities and aging or conversely as the beginning of a new and promising period of life, relatively free of previous obligations and a time for new choices. This period however, may be marked by the presence of estrogen deficiency symptoms, such as hot flushes, night sweats, formication, poor sleeping and genital atrophy, all of which may cause lifestyle disruption, even with the most positive outlook on life. (Sarrel, 1997)
Menopausal hot flushes are the most common symptom of the climacteric and occur in 60-75% of women undergoing natural menopause with a higher incidence after surgical menopause. (Sturdee and Brincat, 1988)
Flushes may occur at any time and are characterised by a flush usually starting in the face, spreading to the neck, head, chest and rest of the body and can severely disrupt a woman's life. Flushes are often accompanied by the subjective feeling that others notice the occurrence of these symptoms. Flushes also occur at night, awaking women from sleep, with consequences including insomnia, irritability, tiredness and general lethargy.
Whilst hormone replacement therapy (HRT) can alleviate many of these symptoms, contra-indications to estrogen replacement therapy exist and side effects attributed to HRT may preclude its use. Estrogen is a very effective treatment of vasomotor symptoms and other problems associated with estrogen deficiency however, in certain clinical settings,
49. the use of estrogen in some women is undesirable. Other women may
have resolution of symptoms with estrogen replacement but find the side
effects of estrogen more undesirable, even in small amounts. Despite clinical benefits attributed to the use of hormone replacement therapy
(HRT) in postmenopausal women, compliance ranges from 10-50%
(Hahn, 1989) and alternatives to estrogen replacement therapy would be therapeutically valuable in these instances. Varied views on the menopause, including the perception of intervention with medical technology in 'a natural process' and media representation of negative aspects of HRT have prompted many women to search for alternative therapies.
The apparent cultural differences in the incidence of menopausal flushing
(Boulet et al, 1994) (McCarthy, 1994) have been attributed to differences in dietary intake of isoflavones.(Adlercreutz et al, 1992) Much of the evidence regarding the effects of isoflavones on menopausal symptoms is epidemiological and has been linked to the consumption of soy products in different populations. This reflects soy products as a major source of isoflavones. Soybean and its products appear to have high levels of
isoflavones, the extent depending on the type and manner of preparation.
(Price and Fenwick, 1985) (Axelson et al, 1984) Other reasons for the difference in cultural reporting of symptoms of estrogen deficiency should be considered. These also include the role of the female in a particular
50. society, the effect of religious practices as well as the importance of vasomotor symptomatology, especially when the majority of a population dies prior to reaching the age of the menopause.
Intervention by dietary modification, with foods containing isoflavones, has been shown to alter biochemical markers associated with reproductive function. (Morton, 1994) The consumption of soy products is estimated to be highest in Japanese populations with levels in the diet up to 2GG mg per day. (Cassidy et al, 1994) There is a gradation in consumption of isoflavones in the diet through Asia, estimated from 25 to
45 mg of total measurable isoflavones per day, to Western countries where less than 5 mg per day is consumed. (Coward et al, 1993) A
Western-type diet elevates plasma levels of sex hormones and decreases the sex hormone binding globulin (SHBG) concentration, increasing the bioavailability of the sex steroids. (Adlercreutz, 199Gb) The same diet also results in low formation of isoflavone. (Adlercreutz, 199Gb)
The use of isoflavones found in soy products (Reinli and Block, 1996) to modify the symptoms of estrogen deficiency has been addressed in small studies. (Delais et al, 1998) (Baber et al, 1999) (Murkies et al, 1995)
Conflicting results regarding the efficacy of isoflavones in the treatment of menopausal symptoms have been produced. These studies also used different products to deliver the isoflavones. A single study using soy
51. supplementation has shown a decrease in flushing frequency compared
with placebo. (Aibertazzi et al, 1998) The presence of isoflavones in soy
products has been well demonstrated. However, that the presence of weak estrogenic compounds such as isoflavones in foods and consumption of these compounds may reduce menopausal symptoms has epidemiological and biological credence.
Menopausal women appear to be the highest users of alternative therapies (MacLennan et al, 1996) in an industry estimated to be worth in excess of $14 billion annually in the United States and increasing.
(Eisenburg et al, 1993) The non-prescription supplementation however appears to revolve around tablet formulations more than dietary or lifestyle change.
1.6.3.1. The climacteric - current studies
It must be re-emphasised in respect to isoflavone treatment of menopausal symptoms that the majority of the current evidence proposing beneficial effects accredited to phytoestrogens has been obtained from epidemiological studies. These data need to be interpreted with caution given the potential for confounding factors and bias. The results of prospective randomised clinical trials are beginning to appear and in conjunction with an increasing body of preclinical data regarding the effects of isoflavones.
52 The incidence of hot flushes, the most common symptom of the climacteric, varies from 70-80% of menopausal women in Europe,
(Rekers, 1991) to 57% in Malaysia, (lsmael, 1994) and 18% and 14% in
China and Singapore respectively.(Tang, 1994) Substantial dietary differences exist between these populations, especially relating to the consumption of soy products. The estrogenic effects of phytoestrogens have been proposed as a modifier in the frequency and severity of vasomotor symptoms in these different populations. The largest published clinical trial thus far investigating the possible effects of isoflavone consumption on vasomotor symptoms was performed by Albertazzi and colleagues. (Aibertazzi et al, 1998) They randomised 104 postmenopausal women to receive either 60 g of soy protein (containing approximately 76 mg of isoflavones) or 60 g of placebo (casein protein containing no isoflavones) for 12 weeks. This showed a 45% reduction in severe flushing frequency associated with the daily consumption isoflavone, compared with a 30% reduction in the placebo group. Clinically this type of response is only marginally better than placebo and does not approach the efficacy of estrogen replacement therapy (ERT). The clinical outcomes of the small trials assessing the effects of isoflavones on the frequency and severity of menopausal symptoms do not show statistically or clinically significant differences between placebo and isoflavone treatment. (Knight and Eden, 1996) A recent Australian study reported an
53. inverse relationship between changes in urinary daidzein excretion and changes in menopausal symptoms following three months of dietary supplementation with a concentrated extract of 40 mg of isoflavones from red clover in postmenopausal women. (Husband et al, 1998)
Estrogenic effects after dietary supplementation with soy flour
(isoflavones) and linseed (lignans) were shown in a pilot study of postmenopausal women by maturation of vaginal epithelium on cytology.
(Wilcox et al, 1990) This has not been confirmed in subsequent studies.
(Baird et al, 1995) Macaque monkeys are a well-characterised primate model of postmenopause after oophorectomy. In these animals fed soy protein containing isoflavones the cytological pattern of the vagina did not differ from that of controls. (Cline et al, 1996) The effects of E2 were antagonised by the addition of soy protein containing isoflavones in the endometrium and mammary gland in further studies using these animals.
In this non-human primate model, treatment with isoflavones alone did not induce proliferation in endometrial and mammary tissue. (Foth and Cline,
1998)
These studies confirm anecdotal experience that dietary supplementation with isoflavone phytoestrogens from a reliable source, affects factors associated with estrogen deficiency. However the studies published to
54. date do not support the equal efficacy of estradiol and isoflavones, and dose efficacies for isoflavones have not been determined.
1.7. Osteoporosis
Osteoporosis risk increases with increasing length of estrogen deficiency.
Rates of osteoporosis differ within populations in different geographic regions, with a lower incidence in Asian women than their Western counterparts. (Report of a WHO Study Group, 1994, Cooper et al 1992)
Japanese women also have a lower risk of hip fracture than Caucasian women. Postulated factors for these differences include body habitus
(including the effects of balance, strength, and stature); exercise; and diet, including phytoestrogen and calcium consumption. Population studies have not demonstrated a strong relationship between calcium intake and peak bone mass, bone loss, or fracture rates. (Kanis et al, 1989, Valente et al, 1994) Recent analysis of long-term urinary excretion of phytoestrogens as a marker of habitual dietary intake, compared with postmenopausal bone loss did not support a preventive effect of low, unsupplemented dietary intake of phytoestrogens on postmenopausal cortical bone loss. The authors stated also that no conclusions could be drawn concerning effects of higher doses of phytoestrogens. (Kardinaal et al, 1998)
55. lpriflavone, a synthetic isoflavone, maintains bone density when given to
premenopausal women taking GnRH agonists (Gambacciani et al, 1994)
as well as maintaining or increasing bone density in postmen'opausal
women. (Agnusdei et al, 1995) The usual clinical dose of ipriflavone is 600
mg daily, with one of the primary metabolites being daidzein. This forms
around 10% of the breakdown products and exposes patients to around
60 mg of daidzein daily. (Brandi, 1992)
Administration of genistein in oophorectomised rats was associated with
higher bone formation rate per tissue volume and a trend toward a higher
number of osteoblasts per bone perimeter. (Fanti et al, 1998) Genistein
did not affect parameters of bone resorption with serum osteocalcin
concentration and urinary deoxypyridinoline excretion providing
corroborating results. The effect of genistein on lipopolysaccharide
induced in vitro production of tumour necrosis factor alpha (TNFa) was
tested as production of pro-inflammatory cytokines is intimately involved in the pathogenesis of postmenopausal osteoporosis. Production of TNFa
was blocked by genistein. This study suggests that genistein reduces
trabecular and compact bone loss after oophorectomy and the protective
effect observed differs from that of estrogen, as it depends on stimulation
of bone formation rather than suppression of bone resorption. Modulation
of cytokine production may be involved in the effect of genistein on bone.
56. Postmenopausal women recruited to receive dietary soy protein or casein during a 6-month, parallel-group, double-blind trial were assessed for total and regional bone mineral content and density. Significant increases occurred in both bone mineral content and density in the lumbar spine but not elsewhere in women consuming soy protein containing 90mg isoflavones on a daily basis compared with the control group (p<0.05).
(Potter et al, 1998) Lower doses of soy protein consumed during this study did not show any difference compared with placebo in bone mineral content or density.
1.8. Cardiovascular Disease.
Heart disease is a hormone-dependent disease, illustrated by the lower age-related incidence in premenopausal women compared with males; an incidence that rises postmenopausally to approach that of males, and is decreased by hormone replacement therapy. Estrogens are beneficial in reducing the risk of cardiovascular disease. A review of the literature
(Stampfer and Colditz. 1991) concerning hormone replacement therapy and ischaemic heart disease showed an overall relative risk for estrogen use of 0.56 (95%CI 0.50-0.61 ). This data should be interpreted conservatively, as it is retrospective. The recently published HERS study found an excess of deaths in patients with cardiovascular disease treated with HRT. (Hulley et al, 1998) However differences between assumptions and actuality suggest this trial did not achieve sufficient statistical power to
57. provide confident conclusions. Late recruitment may also have affected final results. A primary protective effect of estrogen is likely and may be
manifested through lipid changes, with decreases in low-density
lipoprotein cholesterol and increases in high-density lipoprotein
cholesterol, and vascular effects, in both vasomotor tone and vessel wall compliance. It has long been recognized that coronary heart disease rates
are lower in Japan, where soy consumption and other lifestyle factors are
important, than in Western countries. Nonetheless it is possible that in
postmenopausal women phytoestrogens may act as estrogen agonists, and may produce similar effects to estrogen.
Because of the similarity in chemical structure between phytoestrogens and animal estrogens, it has been suggested that phytoestrogens mediate their effect by interaction with the both classical (Miksicek, 1995) and estrogen receptor ~ (Kuiper et al, 1998}. Animal studies suggest that phytoestrogens and conventional hormone replacement therapy have similar effects on cholesterol uptake and metabolism (Wagner et al, 1997).
Similar to an apparent epidemiological benefit for hormone replacement therapy, it has been suggested that phytoestrogens may confer cardiovascular protection as shown in population studies (Hertog et al,
1995). Of concern, however, is the same populations that have a high dietary intake of phytoestrogens, may also have a slightly higher rate of hypertension and stroke (Menotti et al, 1990}. Although this has been
sa attributed to a possible deleterious effect of a low fat diet on stroke risk
(Takeya et al, 1984), it could equally relate to the higher phytoestrogen
intake. Notwithstanding these epidemiological associations, a number of
isoflavones have been shown to have significant anti-hypertensive effects.
Some have shown ~-blocking properties (Wang et al, 1994), whereas others induce smooth muscle relaxation, similar to natural estrogen (Wu et al, 1992). As menopause is associated with a significant decrease in arterial distensibility (Laogun and Gosling, 1982, Karpanou et al, 1996), such smooth muscle relaxation may be beneficial.
In experimental studies, atherosclerosis was reduced in animals fed diets containing soy protein compared with those fed diets with animal protein and the consumption of soy protein in human studies has shown alterations in lipid levels. (Carroll, 1991) A meta-analysis published in the mid 1990s reviewing the effects of soy consumption on lipid levels concluded that three servings of soy products daily significantly reduce total cholesterol, low-density lipoprotein cholesterol, and triglyceride levels. (Anderson et al, 1995) It also suggests that phytoestrogens account for 60-70% of the effects seen. While there is convincing evidence that soy extracts improve lipid profiles in monkeys {Anthony et al, 1996), the effects of soy protein in humans is less certain and may be restricted to small reductions which principally occur in patients with relatively high plasma cholesterol. (Potter, 1996)
59. Although cholesterol levels are used as predictors of ischaemic heart
disease, apolipoprotein A is now also acknowledged as an important
predictor of heart disease. (Kim et al, 1994) Apolipoprotein A levels are
primarily genetically determined. The only compounds found to alter these
levels are estrogens and other sex steroids, which may decrease
apolipoprotein A levels by 35%. (Shewmon et al, 1994)
Two recent studies address the clinical effects of isoflavones on lipid and apoprotein levels. The first assessed the effects of soy protein containing various amounts of isoflavones on lipoproteins and other selected cardiovascular risk factors in sixty-six hypercholesterolaemic, postmenopausal women over a six month period. (Baum et al, 1998)
These women were supplied different diets, the active soy protein diets containing 56 mg and 90 mg of isoflavones daily. Non-HDL cholesterol in both the soy protein containing groups was reduced compared with the control group (p<0.05), whereas total cholesterol was not changed. HDL cholesterol increased in both soy protein groups (p<0.05), whereas the ratio of total to HDL cholesterol decreased significantly in both groups compared with the control (p<0.05). Mononuclear cell LDL receptor messenger RNA concentrations increased in subjects consuming soy protein compared with the control (p<0.05). The authors concluded that soy protein, with different amounts of isoflavones, may decrease the risk
60. of cardiovascular disease via improved blood lipid profiles. They also
suggested that the mechanism by which apolipoprotein 8-containing
lipoproteins was depressed possibly via alterations in LDL receptor
quantity or activity.
The second study used an isoflavone supplement in tablet form. This
study did not find any improvement in serum lipids, at least in subjects with average serum cholesterol concentrations. (Hodgson et al, 1998)
After adjustment for baseline values in this study, no significant
differences in post-intervention serum lipid and apolipoprotein A concentrations between study and control groups were identified. A single crossover study has been performed addressing the effects of flaxseed ingestion on serum lipids, oxidative measures and ex vivo androgen and
progestin activity. (Jenkins et al, 1999) Partially defatted flaxseed reduced total cholesterol (4.6+/-1.2%; p=0.001), LDL cholesterol (7.6+/-1.8%;
p<0.001), apolipoprotein 8 (5.4+/-1.4%; p=0.001), and apolipoprotein A-1
(5.8+/-1.9%; p=0.005), but had no effect on serum lipoprotein ratios at week 3 compared with the control. There were no significant effects on serum HDL cholesterol, serum protein carbonyl content, or in vivo
androgen or progestin activity after either treatment.
Soy isoflavones, particularly genistein, improve impaired acetylcholine
induced dilator responses of atherosclerotic coronary arteries. (Williams
61. and Clarkson, 1998) Genistein reduces platelet accumulati<;>n of serotonin, whose constrictor properties are augmented by dyslipoproteinaemia and atherosclerosis. Studies were performed on female rhesus monkey to assess vasomotor responses in vivo using quantitative coronary angiography and intramuscular Doppler measurements of blood flow velocity. (Williams and Clarkson, 1998) Soy isoflavones improved impaired dilator responses to acetylcholine. In response to activation of platelets by intracoronary infusion of collagen, reductions in blood flow through the left circumflex coronary artery, but not large artery constriction, were less in the isoflavone containing soy fed monkeys than in the isoflavone free soy fed monkeys (35 +1- 6%, p=0.02). In-vitro platelet aggregation to thrombin and serotonin were less in the isoflavone fed than in the isoflavone free monkeys (p<0.05). The authors concluded that components of the soy protein removed by alcohol extraction, probably the isoflavones, promoted endothelium-modulated dilation and inhibited constrictor responses to collagen infusion by inhibiting platelet aggregation or platelet release of vasoconstrictors, or both. However it is possible that other proteins extracted by alcohol may have vasoactive activity.
Vascular responses to isoflavones were tested in 21 women in a placebo controlled crossover trial addressing the effects of 80mg of isoflavones daily over 5 to 10 week periods. (Nestel et al, 1997) Systemic arterial
62 compliance (arterial elasticity}, which declined with age in this group, improved 26% (p<0.001) compared with placebo however arterial pressure and plasma lipids were unaffected. The improvement in systemic arterial compliance in perimenopausal and menopausal women taking soy isoflavones in this study was to about the same extent as that achieved with conventional hormone replacement therapy.
Thus a number of potential mechanisms exist by which isoflavones might prevent atherosclerosis. These include effects on plasma lipid concentrations, antioxidant effects, antiproliferative and antimigratory effects on smooth muscle cells, effects on thrombus formation and maintenance of normal vascular reactivity. (Anthony et al, 1998} It is important however that further confirmation of the above studies is obtained, particularly in other relevant vascular beds such as the coronary circulation.
1.9. Summary
Clinical applications for phytoestrogens are still very much in their infancy.
Application in hypercholesterolaemia and heart disease is reasonable in a conjunctive dietary approach to these problems however dietary means of influencing menopausal symptoms and osteoporosis are not currently scientifically proven alternatives to current pharmaceutical measures. In humans, there are obvious, potentially important health benefits that may
63. be associated with the consumption of foods containing phytoestrogens.
Adopting a vegetarian style diet inevitably leads to an increased exposure
to phytoestrogens as well as other beneficial dietary effects.
By their nature, isoflavones have captured the popular press as a possible treatment of menopausal symptoms. However, the benefits of this dietary therapy remain to be established. The use of any dietary intervention treatment requires the same scrutiny as for the assessment of a
pharmaceutical product expected to produce a defined clinical endpoint.
These criteria include product stability, pharmacological data,
reproducibility of effects, side effect profile and long term effects.
Unfortunately at this stage satisfactory scientific data is not yet available to
enable firm conclusions or recommendations for phytoestrogen use in the treatment of problems associated with estrogen deficiency after the menopause.
64. Chapter 2
Methodology
2.1. Sample Collection and Handling
2.1.1. Sample Handling
The sample collection was performed at St George Hospital, Liverpool
Hospital and City West IVF. Collection occurred after verbal
acknowledgment of identity and all samples were subsequently labelled
with two identifiers.
2.1.2. Blood Collection and Storage
Blood samples were collected from the left or right antecubital fossa after
application of a tourniquet and occlusion of venous return. Samples were
collected in EDTA or plain glass tubes. Where serum was required the samples were allowed to stand for clot formation prior to centrifugation at
2000 rpm for 5 minutes. The serum obtained was aliquoted into plastic tubes and stored at -20° Celsius until batched assays were performed.
65. 2.1.3. Urine
Urine samples were collected using a standard twenty four hour collection
container. Samples were received the following day. The urine was
measured for volume, specific gravity and then everted within the
container a number of times to ensure that any sedimentation was
admixed in the fluid. One hundred millilitre aliquots were then removed.
The urine obtained was aliquoted into plastic containers and stored at M
20° Celsius until batched assays were performed.
2.1.4. Follicular Fluid
Follicular fluid was collected from two to three of the most accessible
follicles in each woman at transvaginal oocyte collection. Any collection
that did not appear to be a "clean" aspirate of a single follicle, or was
contaminated with blood, was disregarded. After identification and
removal of any oocyte, follicular aspirate volumes were measured,
centrifuged to remove extraneous cellular debris, and the supernatant frozen and stored at M20o Celsius for subsequent analysis
66. 2.2. Assay Techniques
2.3. High Performance Liquid Chromatography
Prior to 1970, few reliable chromatographic methods were commercially
available to the laboratory scientist. During the 1970's, a variety of techniques were available to perform chemical separations. However, these chromatographic techniques were inadequate for either
quantification of compounds or resolution between similar compounds.
During this time, pressure liquid chromatography was developed to decrease flowthrough time, reducing the purification times of compounds isolated by column chromatography. Flow rates were inconsistent, and the issue of constant flow rate versus constant pressure was debated. (Stuting and Krull, 1990) This resulted in the development of high pressure liquid chromatography in the mid-1970's. With rapid improvement in column packing materials, the additional convenience of on-line detectors and new methods by the late 1970's, including reverse phase liquid chromatography, allowed for improved separation between compounds of very similar structure.
HPLC was in common use by 1980 for the separation of chemical compounds. Technique development improved separation, identification,
67. purification and quantification. Automation using computer analysis
improved the convenience of HPLC. Evolution of columns such as micro
columns and affinity columns enhanced reproducibility of results, and Fast
HPLC began to emerge.
The past decade has seen a vast undertaking in the development of the
micro-columns, and other specialised columns. The dimensions of current
commercially available HPLC column range from 30 mm to 250 mm in
length, with an internal diameter between 2 and 20 mm. (Thomson
BIOAdvantage™, 2000.) The usual diameter of micro-columns, or
capillary columns, ranges from 3 J.Jm to 200 J.Jm. Fast HPLC utilises a
column that is shorter than the typical column, with a length of about 3 mm
long, and packed with smaller particles.
HPLC is a widely utilised technique in biotechnological, biomedical and
biochemical research especially in the pharmaceutical industry. These fields currently comprise of about 50% of HPLC users. (Stuting and Krull,
1990) The technique is now used in variety of other fields including
cosmetics, energy, food and environmental industries.
aa 2.3.1 HPLC Methodology used in this thesis
The genistein and daidzein assays were performed by Novogen Ltd
(Sydney, Australia) using high performance liquid chromatography
(HPLC) with UV detection using a modification of the methods described
by Satchell and Franke (Satchell et al, 1987, Franke et al, 1995). All
samples were run in duplicate. Synthesised isoflavonoids used as
standards were obtained from the Department of Organic Chemistry,
University of Helsinki.
2.3.1.1 HPLC Methodology - Serum, Urine and Follicular Fluid
The HPLC system consisted of a 25 em, 51-JM, C-18 stationary phase
column (Symmetry, Waters, Sydney, Australia) and a gradient acetonitrile
water mobile phase. The analytical method was validated by determining the LOQ, linearity, range and specificity. Validation samples were
prepared by spiking blank plasma obtained from patients on isoflavone free diets, at a level of 0, 5, 10, 50 and 1OOng/ml for each of the four
isoflavones. The isoflavones were quantitated at a wavelength of 283nM.
The limit of quantitation for each of the four isoflavones was determined as
5ng/ml (minimum signal to noise ratio of 3:1). The method was found to be
69. linear over the concentration range 5-100ng/ml for all four isoflavones R2
>0.95.
The standard analysis of unknown samples consisted of running a system
suitability standard prior to each run The system suitability standard was
prepared by dissolving flavone in mobile phase at a concentration of
60ng/ml. The flavone solution was then injected six times. The system
was deemed operating suitably when the %RSD of the retention time and
peak area were less than 2% for all six injections. Following a system
suitability run, a calibration curve was generated from a minimum of 4
concentration levels of isoflavones prepared in isopropanol:water (1 :1).
Linear regression was performed on the calibration curve with a minimum
acceptable level of R2 >0.95. A series of 10-15 unknown samples were
then analysed followed by a single injection of standard. This analysis
sequence was followed to the end of the run. The entire analysis was only
passed when the %RSD of both peak area and retention time for the
standards throughout the entire run was <2%.
A series of in study validation samples were run in each analysis. The in
study validation samples were prepared by spiking blank plasma with the four isoflavones at 5ng/ml 50ng/ml and 1OOng/ml then performing the
normal sample preparation methodology. A series of six in study validation samples were prepared at each level. The isoflavones were
70. then quantitated based on the standard calibration curve. The coefficients
of variation at 5ng/ml were 5.65% (daidzein) 13.73% (genistein), 14.21%
(formononetin) and 3.92% (biochanin A) at 50ng/ml they were 1.91.%,
2.32%, 0.99% add 1.93% respectively and at 1OOng/ml they were 1.1 0%,
7 .54%, 0.54% and 5.63% respectively. This data falls within the
acceptable level of 20% at LOQ and 15% at medium and high levels. The
assay was modified from previously published HPLC methods (Satchell et
al, 1987b), (Franke et al, 1995) and is subject to patent.
Aliquots of urine, follicular fluid or plasma were mixed with glucuronidase
(Sigma, St. Louis, MO, USA) and incubated for 24 hours at 37°C. Total
isoflavones were extracted using diethyl ether to separate at the organic
solvent phase. The mixture was centrifuged at 3000 rpm for 10 minutes at
20°C and the organic phase collected and dried. The extract was
reconstituted with ethanol and 101.1L samples injected into the HPLC
system. 10 ml aliquots of urine were mixed with 100 1.1L of glucuronidase
and the mixture was incubated for 24 hours at 37°C.
2.3.1.2 HPLC Methodology - Food Products
For analysis, 2 g of food was diluted in 20 ml water and exposed to glucosidase (0.4 U/ml Megazyme) and incubated for 24 hours at 37Q C.
The aglycone forms of the isoflavonoids then were extracted in absolute
71. ethanol, and filtered through a 0.45 uM cartridge filter prior to injection
onto the HPLC. The reproducibility of this method has been previously
reported. (Franke et al, 1995) Synthesised isoflavonoids for use as
standards were obtained from the Department of Organic Chemistry,
University of Helsinki. The lower limit of detection with this procedure for
daidzein, genistein and equol were 54, 48 and 94 ng/ml, respectively.
The within batch precision of the method, as determined from replicate
analyses (n=6) of the same soy formula, ranged from 0. 7 to 3.0%
(coefficient of variation, CV) for the major glycoside conjugates.
2.4. Radioimmunoassays
Development of the technique of radioimmunoassay has made an immense impact on many areas of medicine. Its sensitivity and specificity allow accurate quantitation of a wide variety of biologically important compounds, such as peptides, hormones, vitamins and drugs, which may occur in biological fluids or tissues at low concentrations. The technique or variations of the technique based on the same principles have been used to measure hundreds of different substances, some of which occur in the blood in ng/ml or pg/ml amounts. Before the development of radioimmunoassay, many of these substances could be assayed only with great difficulty, and in some cases no practical assay was available.
72 2.4.1. History of RIA
The technology was initially developed by Rosalyn S. Yalow and her
colleague, Solomon A. Berson. In the 1950's Berson and Yalow, while
studying the behaviour of 1131 -labeled insulin, made several observations
that led to the development of the radioimmunoassay for plasma insulin.
They found that when patients with diabetes mellitus were treated with
insulin, insulin-binding antibodies were formed to this injected insulin.
Subsequently, Berson and Yalow succeeded in producing an insulin
antibody in animals. In addition, they observed, using an in vitro system,
that unlabelled insulin displaced radioactively labelled insulin from insulin
antibody. They found that when the antibody concentration was kept fixed, the binding of label was a quantitative function of the amount of unlabelled insulin present (Yalow and Berson, 1971). This work formed the basis of
radioimmunoassay. They first published their technique in 1960. (Yalow and Berson, 1960) Yalow won the Nobel Prize in physiology or medicine for "the development of radioimmunoassays of peptide hormones" in
1977.
A similar methodology was reported by Ekins for the identification of thyroxine. (Ekins, 1960) The technique was simplified by Hunter and
Greenwood. (Hunter and Greenwood, 1962) The development in technique for protein iodination increased the practicality of the assays.
73. Through the chloramine T method, initially used for growth hormone
assays, the preparation of labelled polypeptide hormones of high
specificity became possible.
2.4.2. RIA Methodology
In a typical RIA, the substance to be measured, the unlabelled sample
antigen competes with radiolabelled antigen for a limited number of
antibody binding sites. Although several different isotopic tags can be
125 used, a radioactive isotope of iodine (1 ), which emits gamma radiation
as it decays, is the most common. The antibody-antigen complex is
precipitated out of solution, separated from the unbound reagents, and
measured in a gamma counter.
The reagents necessary to perform an assay for a given substance or antigen include an antibody specific for the antigen, labelled antigen, a standard preparation of the antigen, and a system to separate the fraction which is bound to the antibody from that which is unbound or free. An assay for plasma insulin (as per Yalow and Berson above) thus would require;
1. an antibody to insulin,
2. labelled insulin,
3. a preparation of insulin for use as a standard,
74. 4. buffer, and
5. a separation system.
The assay is performed using a series of tubes containing a fixed
concentration of antibody, a fixed amount of label, appropriate amounts of
buffer and an aliquot of either standard or unknown.
A number of standards and controls are determined with each assay,
along with certain parameters of antibody binding and completeness of
separation. The substance to be measured (unlabelled antigen) in the
patient specimen competes with the labelled antigen for the antibody
binding sites. The percentage of antigen bound to the antibody is related
to the total antigen present and is reflected by the distribution of the
radioactive label. With increasing amounts of unlabelled antigen, a
corresponding decreased amount of labelled antigen will be bound to
antibody. The percentage of the total radioactive label that is bound to the
antibody, and that which is free can be monitored after the two fractions
are separated. By comparing the distribution of label obtained with the
unknown to that observed with the standards, the concentration of
unknown can be determined. (Howanitz and Howanitz, 1979)
The distribution of the radioactive label can be expressed in a number of ways, such as the percentage of total counts that are bound or free or the ratio of the counts of the two fractions. A curve is prepared by plotting
75. the percentage or ratio obtained with the standard against the
concentration of standard; unknown values then are determined using the
standard curve. (Howanitz and Howanitz, 1979)
Radioimmunoassay and related techniques are dependent on the degree
of similarity of behaviour of the standard and the unknown, but they do not
fundamentally rely on the use of antibodies or a radioactive label. In
general terms, the principle involves partitioning of the substance to be
measured into two moieties by the reaction with a specific binding reagent
of limited capacity (Ekins, 1974). The ratio of the two moieties depends on
the amount of unknown or standard in the system. For example, the assay
of thyroxine, which was described by Ekins at about the same time as the
insulin assay was developed, was based on principles identical to those
governing the insulin method (Ekins, 1974). However, this assay relied on a naturally occurring protein, thyroxine binding globulin, rather than on an antibody as a specific binding reagent in the system. Since this type of assay uses a binding protein instead of an antibody, it is referred to as a radioassay rather than a radioimmunoassay. Although superficially different, the fundamental principle on which all these assays is based depends on the use of a limited amount of specific binding reagent, which is held constant in the system. The binder may be antibody, as in the insulin assay; specific binding proteins, as in the thyroxine assay; cellular receptors; or even an enzyme, as in the assay of methotrexate. A
76. requirement of the technique is that there must be a means of separating
or identifying the bound and free components.
RIA is used most widely in clinical laboratories. Measurement includes
substances such as pregnancy and growth hormones; drugs ingested,
such as antibiotics, cocaine and steroids; antigens that are characteristic
of autoimmune disorders; and antigens that indicate infection by various
bacteria, parasites and viruses. In research settings, the technique is
used to identify and quantify an even broader range of substances, from
enqogenous opioids to proteins expressed by spliced genes in gene
therapy experiments.
2.4.3. Markers of Bone Turnover
2.4.3.1. Bone resorption markers
The osteoclastic activity (bone resorption) is measured by breakdown
products of collagen. Hydroxyproline correlates with bone resorption, but the test is not sensitive and can only be performed after a special diet.
Newer tests measure collagen cross-links, which are covalent bonds formed between collagen chains after collagen has been secreted. These are therefore not markers of bone formation. Links are formed at both
77. ends of the collagen molecules (N- and C- terminals) and involve a ring
structure called pyridinoline. When bone is resorbed, collagen is broken
down, the cross-linking molecules are released and excreted by the urine.
Currently available assays measure the pyridinoline, the N-terminal cross
link, and the C-terminal cross-link.
These tests have been shown to correlate with bone resorption. There is
some diurnal variation, which probably reflects diurnal variation in bone
resorption itself. The cross-links measurements tend to be high during
adolescence (when there is high bone formation and resorption), dropping
to low levels until menopause, when they increase. Estrogen treatment in
postmenopausal women decreases the cross-link excretion to
premenopausal levels. Anti-resorbing medications also decrease cross
link excretion.
The measurement of collagen cross-links has been promoted as a means of testing for osteoporosis. Excessive bone resorption would be expected to eventually result in low bone mass. There is a correlation between the bone mass measurements and collagen-cross-links excretion. The ability to predict fractures using the cross-link tests is still uncertain with conflicting data amongst studies. In a large prospective cohort of 7598 women older then 75, a high C-telopeptide level was associated with an increased risk of hip fracture with an odds ratio of 2.2. (Garnero et al,
78 1996) During the two years of study, 3% of those with a high C-telopeptide
had a hip fracture.
2.4.3.2. Bone formation markers
Osteoblastic activity is associated with serum osteocalcin, one of the
proteins found in relatively high concentration in bone. Bone specific
alkaline phosphatase is measured by radioimmunoassay, not by iso
enzyme analysis of alkaline phosphatase. It is more sensitive and specific
than alkaline phosphatase.
These tests have been shown to correlate with bone formation as
measured by bone histomorphometry or calcium kinetic studies.
Osteocalcin is excreted via the renal tract. Serum values are increased in
renal failure. This may occur in elderly patients with normal creatinine
levels in the presence of low glomerular filtration rates. Bone specific alkaline phosphatase is not as influenced by renal failure. Osteomalacia may be associated with a high osteocalcin, even though the bone formation rate is low.
In normal individuals the markers of bone formation follow the same pattern as the markers of bone resorption as the two processes are coupled during bone remodelling. The levels are high during childhood
79. and adolescence, low during adult years and increase after menopause.
They decrease following treatment with hormone replacement therapy or
anti-resorptive drugs, but there is a lag between the decrease in the bone
resorption markers and the bone formation markers.
2.5. Chemiluminescence
Energy can be transferred into (and out of) matter in many ways including
heat, light, radiation or by chemical reactions. When energy is released by
matter in the form of light, it is referred to as luminescence. An exception
is usually made for matter that has such a high temperature that it simply
glows, called incandescence. When energy in the form of light is released
from matter because of a chemical reaction the process is called
chemiluminescence.
Chemiluminescence has been adapted for laboratory usage by quantitative measurement of the optical emission from excited chemical species enabling the determination of anolyte concentrations.
Chemiluminescence is usually the result of emission from energised molecules rather than excited atoms. The bands of light determined by this technique emanate from molecular emissions and are therefore broader and more complex then bands originating from atomic spectra.
80. Furthermore, chemiluminescence can take place in either the solution or
gas phase.
Though liquid phase chemiluminescence plays a significant role in
laboratories using this analytical technique (often in conjunction with liquid
chromatography), gas phase chemiluminescence reaction instrumental
components are somewhat simpler. These detectors are also often used
as detectors for gas chromatography.
Like fluorescence spectroscopy, chemiluminescence's strength lies in the
detection of electromagnetic radiation produced in a system with very low
background. And on top of this, because the energy necessary to excite
the anolytes to higher electronic, vibrational, and rotational states (from
which they can decay be emission) does not come from an external light
source like a laser or lamp, the problem of excitation source scattering is
completely avoided. The major limitation to the detection limits achievable
by chemiluminescence involves the dark current of the photomultiplier
necessary to detect the anolyte light emissions.
2.5.1. History of Chemiluminescence
The phenomenon of chemiluminescence or bioluminescence, light emission by organic molecules, has been known for over 100 years.
81. (Radziszewski, 1877) The potential analytical applications of
chemiluminescence or bioluminescence were recognized in 1947 when
firefly luciferase was isolated. (Strahler and Trotter, 1952) Interest in the
use of chemiluminescent and bioluminescent molecules for analysis
heightened, but was overshadowed by the pioneering work of Berson and
Yalow with the hormone insulin. (Yalow and Berson, 1960)
RIA rapidly became a popular and accepted technique for the
quantification of steroids, hormones, and proteins. The precision,
accuracy, simplicity, and overall performance of RIA was unrivalled by any
other analytic technique during the 196o•s. Even though RIA is highly
sensitive, accurate and precise, there are several disadvantages unique to this method. These include the short half-life of the radioisotopes used as
labels and problems related to user safety and disposal of radioactive compounds. In response to the need for non-isotopic labels, interest was
renewed in chemiluminescent molecules as an indicator of the antigen antibody reaction.
Luminol was among the first chemiluminescent compounds to be oxidised by hydrogen peroxide in an alkaline environment in the presence of a catalyst. (Woodhead et al, 1983) While most chemiluminescent compounds emit light via an oxidation reaction, some compounds do not require the addition of a catalyst. In 1978, a chemiluminescent technology
82 for binding assays was described, using isoluminol as the indicator and
requiring the addition of a catalyst for the light emitting reaction to occur.
(Schroeder et al, 1976) Considerable interest in another chemiluminescent
molecule, acridinium ester, developed as a result of the work of
Woodhead, McCapra, and others especially as this compound does not
require the addition, of a catalyst for the oxidative reaction to occur.
2.5.2. Chemiluminesent Methodology used in this thesis
Sex hormone assays performed at City West IVF Pty Ltd APA (Sydney,
Australia) were assayed using standard enzymatic colorimetric kits on a
BM/Hitachi 747 analyser. The FSH was analysed with an automated
chemiluminesence system, Bayer Diagnostics ACS:180, and the SHBG
by DELFIA on a Pharmacia Wallac Arcus DELFIA photometer. DELFIA
covers heterogeneous time-resolved fluorometric assay based on
dissociative fluorescence enhancement.
Haematological and biochemical analyses were also performed by
Southpath Laboratories at St George Hospital. Lipid biochemistry was
assayed using standard enzymatic colorimetric kits on a BM/Hitachi 747 analyser. The FSH was analysed with an automated chemiluminesence system, Chiron Diagnostic ACS:180, and the SHBG by chemiluminesent enzyme immunometric assay on an Biomedic lmmulite Automated
83. Analyser. FSH and SHBG assayed by the Endocrine Laboratory at the
Royal Hospital for Women (Sydney, Australia). These analyses were also
performed using the above equipment.
2.6. Applanation Tonometry
The arterial pressure waveform changes with ageing and in response to
different therapeutic agents. Manual techniques showed that these
changes could be detected using applanation tonometry. The arterial
pulse contour could subsequently be analysed to obtain potentially
diagnostic information. Automation of the manual process of arterial
waveform analysis is a recent addition to medical investigation.
(Drzewiecki et al, 1983, Kelly et al, 1989) The development of Digital
Applanation Tonometry (OAT, Specaway Pty Ltd, Sydney, Australia), a
computerised diagnostic tool for the clinical assessment of several pulse
indices in real time, has had the effect of eliminating time-consuming manual analysis. The information collected can assist in the evaluation of the coupling between the systemic circulation and left ventricle, and can substantially enhance the limited information available from the use of a sphygmomanometer.
Blood pressure recorded in a peripheral artery is different from that of the ascending aorta. This difference may be significantly greater in people
84. using vasoactive drugs. DAT enables the measurement of accurate aortic
pressure waveforms from peripheral sites and the production of accurate
aortic pressure waveform from these sites. Using this technique,
investigators found a late systolic peak in human arterial pressure (Kelly et
al 1990), now believed to be caused by reflected pressure waves and
involved in the development and progression of left ventricular
hypertrophy, an important risk factor in cardiovascular mortality.
2.6.1. Haemodynamic data acquisition
Arterial pressure waveforms were recorded using applanation tonometry
acquired using a DAT system. Representative waveforms for each
individual were obtained by averaging a series of waveforms (between 10
and 30 seconds) using the QRS complex from simultaneous ECG as a trigger. Each series of waveforms was assessed and those with significant
movement or respiratory artifact were excluded. Carotid pressure
waveform indices (both pressure and timing) were determined by
automated software using pressure waveform derivatives to define critical timing points including pulse onset, time of initial systolic peak, time of secondary systolic peak and incisura as described by Takazawa and co workers.
85. Baseline measurements and recordings were taken with subjects in a
supine rested position. While supine, subjects gave a short relevant
history before being connected to 3-lead EGG used for pulse timing
purposes. Once a stable EGG recording was available, subjects
underwent recording of radial, then carotid and finally dorsalis pedis
pulses by applanation tonometry. The carotid pulse was taken without a
pillow and the head facing forward as this was found to better expose the
carotid pulse. Two subjects required that the subclavian pulse immediately
above the medial head of the clavicle to be used. The same position was
used for subsequent recordings in those patients. At the completion of the
tonometry recordings, the average of two blood pressures were taken
using a brachial cuff sphygmomanometer after 10 minutes quiet rest.
Systolic and diastolic pressures were defined by Korotkoff phases I and V
respectively. Heart rate was derived automatically by the analysis software
from the average of recorded pulses in the supine position.
Derived parameters from the carotid waveform included augmentation
index (Simkus and Fitchett 1990), ejection period and the maximal rate of
pressure rise (dP/dtmax)· Augmentation index was defined as the ratio of the augmented pressure (pressure at second peak minus pressure at
initial peak) to the pulse pressure for an 'A' type waveform (Murgo et al,
1980}. Maximal rate of pressure rise (dP/dtmax) was obtained after the carotid waveforms had been calibrated to the mean and diastolic pressure
86. of the radial waveform as suggested by Schwid and co-workers. (Schwid et al, 1987} (See Figure 2.6.1.1) The recalibrated carotid waveform then allowed determination of central systolic pressure.
87. Chapter 3
lsoflavone content of infant foods and formulas
Following media exposure in New Zealand of the birth deformities seen in
a breeder's parrot program where a soy based feed was implicated (Irvine
et al, 1995), concerns were raised regarding the possible effects of
phytoestrogens on the developing reproductive system of human
neonates and infants. Although no documented cases of adverse outcomes related to phytoestrogen exposure in human infants were detectable in medical literature, this study was in part an attempt to better
understand the biology of phytoestrogens in infants. In it, quantification occurred of the levels of isoflavones in foodstuffs that are commonly used to feed Australian infants during the first year of life.
Foods tested were purchased from local supermarkets and pharmacies in
Sydney during June, 1995. Milk samples were also purchased in
Melbourne and Brisbane. The soy-based infant formulas were supplied by
Wyeth-Ayerst (Australia) Pty Ltd.
Each food sample was analysed by HPLC for the isoflavonoids - genistein, daidzein, biochanin A, formononetin and equal. All samples were run in duplicate.
aa The relative exposure of infants to isoflavones in different foodstuffs was estimated at three age intervals, namely 14-28 days, 2-4 months, and 5 -
12 months of age. Calculations were based on mean energy intakes for infants at these three age ranges of 120, 100 and 90 kcal/kg/day respectively. (Foman and Bell, 1993.) The equivalent intakes based on
2 body surface area (BSA) are 8, 5 and 5 kcal/m BSA/day. The differential nutrient intake at these ages (Boulton, 1991) suggests that the mean percentage of dietary energy derived from milk is ~ 100% at 14 to 28 days of age, 88% (range 43-1 00) at 2 to 4 months of age and 53% (range 4-
100) at 5 to 12 months of age. Using weighted means and the estimated proportion of dietary energy derived from formula feeding during the above age intervals, an estimated exposure to isoflavones for each product at these ages was determined and expressed as mg isoflavones per day per m2 of body surface area.
Statistical analysis was performed using the Kruskai-Wallis ANOVA Test in the Statistica for the Macintosh TM package.
3.1 Results
The results of the isoflavone analyses are summarised in Tables 3.1 and
3.2. lsoflavones were detected in all food samples. Levels in fresh cow
89. milk and casein-based yoghurts and infant formulas were low (0.01 - 0.07
mg/1 OOml), comprising principally daidzein. The isoflavonoid metabolites,
equol and 0-desmethylangolensin, were not detected in these products.
lsoflavone levels in the different soy foodstuffs varied considerably, from
1.8 - 8.0 mg/1 OOml. Levels in soymilk (2.3 - 8.0 mg/1 OOml) were higher than those in soy-based infant formulas (1.8 - 2.1 mg/1 OOml), when
reconstituted according to manufacturers' specifications.
The estimated daily exposure of infants to isoflavones in the different foodstuffs at three different age groups is presented in Table 3.3.
Statistical analysis showed a significant difference between isoflavone levels of the soy and casein based formulas and dairy products (p=0.032).
3.2. Discussion
Soy is a major source of dietary estrogenic isoflavones for humans. In countries such as Japan with a traditionally high soy intake, the level of exposure of adults to dietary estrogenic isoflavones has been variously estimated to be between approximately 20 and 200 mg per day. (Knight and Eden, 1996) The amount of study done in this area is however limited. Utilising the urinary isoflavonoid levels of typical Japanese adults
(Adlercruetz et al, 1991) and comparing these values to urinary
90. isoflavonoid levels following challenge with known isoflavone levels (Kelly
et al, 1995}, the estimated typical dietary intake of daidzein and genistein
is 40 mg for adults. This equates to 22.7 mg per m2 BSA in an adult age
group. Cord blood analysis has shown placental transfer of isoflavones, to the extent that equilibration of levels occurs between maternal and fetal sera [H. Adlercreutz, personal communication]. Thus isoflavone
distribution is anticipated to be similar in maternal and fetal tissue, and
exposure therefore also similar.
The levels of estrogenic isoflavones in the soy milk-based infant formulas tested in this study suggest exposures of 6.62 - 14.05 mg of estrogenic isoflavones per m2 BSA for infants under the age of 12 months who are fed such formulas. These levels are within the range of isoflavone levels described in traditional soy-eating adults, as well as soy consuming pregnant women and their fetuses, suggesting that the levels determined in infants are within normal physiological boundaries. This does not however take into consideration the greater sensitivity of the infant reproductive tract to estrogens compared with older children and adults.
Nevertheless, the available epidemiological evidence suggests that such exposure of infants is safe. A MEDLINE search covering the thirty three year period from 1962-1995, did not detect any reports detailing adverse effects of isoflavones in humans. Soy infant formulas have been in common use for approximately 40 years and no adverse effects on
91. endocrine and reproductive development are reported in the scientific literature. Noteworthy is the observation that infants in soy-consuming communities such as Japan, China and Seventh-Day Adventists, are typically weaned onto soy products between 6-12 months of age. These children subsequently then receive isoflavone-containing foodstuffs (for example, tofu, miso, tempeh) on a long-term basis from that time, a period which embraces the bulk of their reproductive development. The lack of any apparent reproductive and endocrinological dysfunction in these communities compared to Western communities argues against detrimental impact of these compounds. Indeed, it is quite possible that these normal dietary components exert beneficial biological effects at the formative neonatal stage including the establishment of certain metabolic patterns such as lipoprotein metabolism. (Cruz et al, 1994) In laboratory animals, neonatal exposure to estrogenic isoflavones has been shown to decrease the susceptibility to breast carcinogenesis in adult life.
(Lamartiniere et al, 1995)
Toxicological studies of estrogenic isoflavones in animals, and in particular those claiming to show a detrimental effect of reproductive tract development in infant animals must be viewed cautiously. Cross species differences in the effects of estrogenic compounds are well known. For example, tamoxifen is estrogenic in mice, anti-estrogenic in frogs and chickens and may be both estrogenic and anti-estrogenic in humans,
92 depending on tissue specificity. (Jordan et al, 1985} The considerably different timetable of developmental milestones in laboratory animals versus humans also suggests circumspection. An approximate seventy fold increase in the maturation rate of the rodent reproductive tract compared with human, suggests a far greater degree of susceptibility of the immature reproductive tissues of rodents to possible adverse effects of hyper-estrogenicity.
The presence of isoflavones in breast milk is suspected however not yet confirmed in publication. This study attempted to establish the presence of isoflavones in the breast milk of lactating women who consumed soy products as part of the analysis. This data was not included as there were major difficulties in establishing the level of maternal exposure to isoflavones. The majority of samples were collected in a hospital setting where the diet provided often varied markedly from the patients' normal diets and contained little soy product.
A further salient point in this matter is the likelihood that infants are exposed to dietary compounds with far greater estrogenic potency than isoflavones. Cow milk, particularly that obtained from pregnant cows, is likely to contain substantial quantities of steroidal estrogens at levels which could easily impart a dietary estrogenic potency exceeding that of dietary isoflavones. This area currently is under study. Lactating women
93. who also take an oral contraceptive preparation (OCP) expose the breastfeeding infant to higher doses of more potent contraceptive steroids. Despite concern of the potential hazard no adverse effects have thus far been identified. (Speroff, 1996) In an 8-year follow-up study on children breastfed by mothers using oral contraceptives containing 50 micrograms of ethinylestradiol, the authors concluded that no detectable
11 effect of the ingested steroid could be found on a panorama of diseases II, psychological behaviour or intelligence. (Nilsson et al, 1986)
In summary, a range of prepared foodstuffs, commercially available in
Australia, and used widely for infant nutrition contain the estrogenic isoflavones - genistein and daidzein. The levels of these compounds in dairy products (fresh cow milk, dairy yoghurts and casein-based formulas) appear to be biologically insignificant. Whilst levels in soy-based infant formulas are likely to be biologically significant, the total daily intakes by infants where these formulas are the primary nutritional source are similar on a BSA basis to those of older children in soy-consuming communities.
Infants may be exposed to far more potent estrogenic compounds, with no apparent longer term effects. Given the weak estrogenic potential of isoflavones and exposures within cultural physiological ranges, theoretical adverse outcomes appear to be unlikely.
94. Chapter 4
Dietary isoflavones are present in follicular fluid of women
lsoflavones are dietary-derived SERM's, described as both estrogen agonists and antagonists in animals, depending upon the prevailing hormonal milieu in the individual (Kaldas and Hughes, 1989). Interest in isoflavones occurred during the 1940's when they were found to cause
Clover Disease in sheep (Bennetts et al, 1946), a polycystic ovarian syndrome-like problem manifesting as infertility and endometriosis. More recent animal research has confirmed their role in the modification of reproductive function (Hughes CL Jr, et al 1991, Hughes CL Jr, 1988).
Genistein, daidzein and equol have been detected in human plasma
(Adlercreutz et al, 1994}, urine (Adlercreutz et al, 1991 }, saliva, breast milk and prostatic fluid. (Franke et al, 1998} In human females, it is not known if isoflavones perfuse into follicular fluid. If found within the ovary, this presence may have the potential to acutely perturb reproductive function at a cellular and gamete level. This preliminary study aimed to assess whether isoflavones from normal dietary intake could be detected in follicular fluid in order to assess the requirement for formal dietary history taking prior to women undergoing controlled ovarian hyperstimulation as part of assisted reproductive techniques (ART).
95. The study was performed by recruiting women undergoing ART in a tertiary reproductive technology program (City West IVF, Sydney,
Australia). After Research and Ethics Committee approval, women undergoing ART were screened with an lsoflavone Food Frequency
Questionnaire to assess whether exposure to dietary isoflavones was likely. This preliminary screening was performed due to the relative rarity of consumption of foods containing appreciable amounts of isoflavones in the Australian population diet and the expensive nature of isoflavone analysis. Those with dietary exposure to isoflavone containing foods on a more than weekly basis were entered into the study.
Demographic data including age, weight, height, duration and cause of infertility was collected for all patients and pre-ART endocrine profiles were performed. Pituitary desensitisation was achieved in all patients with long down regulation with the GnRH antagonist, nafarelin acetate
(Synarel, Searle Pharmaceutical Products, Sydney, Australia) from Day 22 of the menstrual cycle. Controlled hyperstimulation with follitropin alpha
(Gonai-F, Sereno Australia Pty Ltd, Sydney, Australia) or follitropin beta
(Puregon, Organon (Australia) Pty Ltd, Sydney, Australia) was commenced after the E2 level fell below 100 pmol/1. The dose of FSH was flexible depending upon patient age, requirement and response.
Stimulation tracking was performed with serial E2 and ultrasound
96. measurement assessments of follicular size. Maturation of oocytes was triggered with hCG (Profasi, Sereno Australia Pty Ltd, Sydney, Australia) and 38 hours later, oocyte retrieval was performed by transvaginal ultrasound-directed needle aspiration.
Women with dietary exposure to isoflavones had follicular fluid samples collected at transvaginal ultrasound-directed oocyte retrieval (UOR).
Between two and three of the most accessible follicles in the first ovary needled were collected. Samples were collected from individual follicles.
Any collection that did not appear to be a "clean" aspirate of a single follicle or contaminated by blood was excluded from the analysis. After removal of any oocyte, aspirate volumes were measured, centrifuged to remove extraneous cellular debris, and the supernatant frozen for later analysis.
Concentrations of oestradiol (E2), progesterone, testosterone, FSH, LH and prolactin were measured using a commercially available system
(ACS180, Bayer, PO Box 158, Ferntree Gully, VIC 3156. Australia) after suitable dilution to allow reading from the most sensitive part of the assay curve. The inter and intra-assay variation for all analytes was <8%. SHBG was determined using DELFIA (Australian Laboratory Services, Rockdale,
Australia). Similarly lnhibin-A dimer and lnhibin-B dimer were measured using a commercially available ELISA technique (Serotec Ltd, , Kidlington,
97. UK). The inter- and intra-assay variation for each dimer at a concentration of 15pg/ml was 7%.
The genistein and daidzein assays were performed by Novogen Ltd
(Sydney, Australia), as described in Chapter 2. Data is presented in the format, mean ± standard deviation with p values included when ~ 0.05.
4.1. Results
Ninety two samples of follicular fluid were collected from 38 women. Three subjects had identifiable levels of genistein in the follicular fluid {Table
4.1). However not all follicles collected from these women had detectable levels of isoflavones. One woman had detectable levels of genistein in both her serum (8.03 ng/ml) and follicular fluids (15.58 and 14.65 ng/ml from 4.5 and 3.5 ml volume follicles respectively). Daidzein was not detected in the follicular fluid of this, or the other subjects. There were no statistically significant differences between subjects in respect to hormone assays. This data is not presented however is available upon request to the authors. As the number of follicles with isoflavones detected within the follicles was low, further correlation analysis in regard to follicular hormone levels was not performed. One of the follicles with detectable isoflavone was later associated with a pregnancy.
98 4.2. Discussion
Considerable difficulty was encountered on designing this study. Issues
impacting upon trial design included; the relative rarity of consumption of foods containing isoflavones in the Australian population diet, the expensive nature of isoflavone analysis and supplementation of a woman's usual diet with isoflavones during the or an ART cycle was deemed to be inappropriate. Follicular fluid is difficult to obtain outside of an ART program as women are generally likely to decline participation in ovulation tracking and transvaginal follicular aspiration to assess follicular fluid. lsoflavone supplementation of women undergoing ART invokes a number of ethical issues, especially given some of the animal data suggesting that high doses cause infertility (Bennetts et al, 1946). Infertile couples have considerable emotional and financial investments in individual ART cycles and isoflavone supplementation was determined to be inappropriate during ART as possible effects are unknown. lsoflavone supplementation also does not address the issue as to whether normal dietary intake results in the presence of isoflavones in follicular fluid.
This study shows that isoflavones are detectable in ovarian follicular fluid.
The findings reflect the general absence of foods containing isoflavones in the average Australian diet as only three of the 39 patients studied had detectable levels. The low rate of consumption in the population is the
99. likely cause of the low detection rate in the follicular fluid of the subjects studied. The low detection rates in this study preclude the ability to form any conclusion regarding isoflavones other than the presence in ovarian follicular fluid.
The detection does however raise a number of issues. One patient in whom serum and follicular fluid isoflavone levels were performed showed the follicular fluid isoflavone concentration was double the level in the serum. Biologically it is conceivable that isoflavones concentrate in follicular fluid. Estrogens are concentrated within follicles, aiding development of the dominant follicle (Richards et al, 1987), and selective estrogen receptor modulators (SERM's) such as clomiphene citrate also concentrate within follicles. (Oelsner et al, 1986) If this reflects the norm in regard to pharmacology, intriguing ramifications are a hypothetical possibility.
While estradiol has equivalent affinity for both alpha- and beta- estrogen receptors (ERa and ERB), genistein is more selective for ERB receptors.
(Kuiper et al, 1997) Human granulosa cells from the ovarian follicle contain only ERB m-RNA. (Enmark et al, 1997) As the regulatory domain of ERB lacks transcription activation function-1 (TAF-1), estrogen agonist antagonists (such as genistein) may be pure antagonists in cells that
100. respond only to the ERB, for example granulosa cells. (Enmark et al,
1997)
lsoflavones are known to cause a polycystic ovary-like condition in animals (Bennetts et al, 1946), and changes in menstrual cycle length have been reported in women on a soy diet. (Baird et al 1995, Cassidy et al, 1994) Other reproductive effects in animals have been reported.
(Leopold et al, 1976) (Setchell et al 1987) In oophorectomised rats, acute exposure to phytoestrogens alters GnRH-induced LH release. A dose response pattern of enhanced GnRH-induced LH release at lower pre treatment doses, but inhibited GnRH-induced LH release at higher pre treatment doses was observed for both estradiol and genistein. (Hughes
CL Jr, 1987-88} The potency of genistein appears to be approximately 0.1 that of estradiol in this system.
Genistein is a selective tyrosine kinase inhibitor and has been shown to reduce GnRH-induced LH and FSH release from rat pituitaries in vitro, block oocyte growth and disrupt follicle morphology. (Packer et al, 1994)
Many of the putative intra-ovarian regulators act through binding to their cell membrane receptors that are activated via tyrosine kinase. Insulin-like growth factor-1 (IGF-1) activity is tyrosine kinase mediated. IGF-1 is important in the granulosa for the formation of and increase in numbers of
FSH and LH receptors, steroidogenesis, the secretion of inhibin and
101. oocyte maturation. Furthermore, treatment of follicular cells with genistein has been shown to attenuate transforming growth factor-a (TGF-a) induced estrogen biosynthesis. (Gangrade et al, 1991) TGF-a has proved to be a potent inhibitor of gonadotrophin-supported granulosa cell differentiation.
Polycystic ovary syndrome (PCOS) is associated with anovulation and with hypersecretion of androgen, LH, insulin, and abnormal insulin-like growth factor metabolism. The cellular activity of insulin and insulin-like growth factor are both mediated by tyrosine kinases, which may be affected by isoflavones. Nutrition appears to have a significant role in modulation of PCOS, often with resumption of ovulation after weight loss.
The possibility of dietary isoflavones causing or affecting the course of
PCOS in humans has not been explored but offers fascinating possibilities.
This study reports the presence for the first time of dietary derived isoflavones in the ovarian follicular fluid of women. These compounds have proven biological effects in animals that disrupt reproductive function and effect fertility. Given the presence of these compounds in the ovary, effects on human ovarian function are possible. Further research to ascertain bioavailability and possible effects on human reproductive function are appropriate.
102 Chapter 5
The effect of Promensil™, an isoflavone extract, on menopausal symptoms.
The use of isoflavones found in soy products to modify the symptoms of estrogen deficiency has been addressed in small studies producing conflicting results regarding the efficacy of isoflavones in the treatment of menopausal symptoms. These studies also used different products to deliver the isoflavones. This is the first of two studies in this thesis addressing the issue of isoflavones and their possible effects on estrogen deficiency. This study used a tablet supplement.
A double blinded, randomised, placebo controlled trial was performed consisting of three arms; placebo, 1 tablet (40 mg) of Promensil™ and 4 tablets ( 160 mg) of Promensil™. Promensil™ is a standardised isoflavone supplement prepared from red clover extract in tablet form. Each tablet contained 40 mg of total isoflavones comprising the four primary isoflavones, genistein (4.0 mg), daidzein (3.5 mg) and their methylated precursors biochanin (24.5 mg) and formononetin (8.0 mg).
Thirty seven (37) subjects were recruited through the University
Department of Obstetrics and Gynaecology at St George Hospital,
103. Sydney, Australia. The inclusion criteria for the trial were postmenopausal women who were symptomatic, having at least three flushes per day.
Menopause was defined by bilateral oophorectomy or amenorrhoea for at least 6 months with typical symptoms of the menopause and a serum follicle stimulating hormone (FSH) greater than 40 IU/1. Age criteria was restricted to 40-65 years, upon trial entry. Participants were instructed not to alter their usual diet for the duration of the study. The exclusion criteria included HRT use within the previous six (6) weeks, allergy to foodstuffs known to contain isoflavones; current history of active bowel, liver or gallbladder disease; diabetics requiring drug therapy; and malignancy
(excluding skin cancers). Women with contraindications to HRT use, vegetarians and/or regular soy product users and those receiving medications that result in liver enzyme induction were also excluded.
Pre-trial flushing was assessed using a daily flush diary for the week prior to trial entry. The severity of menopausal symptoms was assessed during this period, using the Greene Menopause Scale. A twenty four hour urine collection for isoflavone measurement was performed during this week.
Upon fulfilment of inclusion criteria, subjects were entered into the study.
The randomisation procedure was performed by an external statistician.
The allocation schedule was produced in random permuted blocks of six generated using a computer random number generator. Subsequently tablets were packed in daily sachets and supplied in individual subject
104. containers to the investigators. Packaged subject containers were
received prior to trial recruitment. There was no contact between
generator of randomisation and executor of trial, prior to, during or after the trial.
After screening, the subjects were randomly assigned to placebo or one
of the active treatment groups. Physical and vaginal examination was
performed. A vaginal wall smear for determination of maturation value
(MV) (Koss, 1979) and pH was performed at trial entry. The vaginal wall smear was performed using a speculum and wooden spatula, with a single pass along each lateral vaginal wall. Blood was collected in a non fasting state for haematological profile, liver function tests and serum
levels of FSH, and sex hormone binding globulin (SHBG).
A 24 hour urine collection for isoflavone measurement preceded trial entry. Urinary isoflavone assays were performed by Novogen Ltd.
The trial was twelve weeks in length and the subjects were seen every four weeks for clinical assessment, compliance checks and assessment of flush count and Greene Score. Contact between appointments was provided if required by individual subjects. In the final week of the study physical and vaginal examinations, the vaginal smear, urine and blood collections were repeated. Compliance was assessed by return of tablet
105. containers and urinary isoflavone levels. All subjects were included in the analysis on an intention to treat basis. On a post hoc basis, the stored serum was analysed for serum total cholesterol (TC) and high density lipoprotein-cholesterol (HOL-e) levels.
The Greene Menopause Score is a validated menopause symptom self assessment form and was completed weekly. For analysis, the Greene
Score was calculated as a total and in the accompanying subgroups, the
Psychological Scale, Somatic Scale and Vasomotor Scale. Data was entered into Statistica™ (StatSoft™ Inc). Nonparametric data was analysed using the Kruskai-Wallis nonparametric analysis of variance.
Normally distributed data was analysed using one way analysis of variance. Any differences between individual groups was re-analysed using the Newman-Keuls test provided the F value from the analysis of variance was significant. This test was preferred to the Bonferoni modification, a corrected student's t-test, as the Newman-Keuls is based on the least significant difference between the means of each group and is a "protected' test taking into account the risk of spacious results due to multiple comparisons. Data is presented in the format, mean ± standard deviation with p values included when $; 0.05.
All subjects consumed four tablets daily. Placebo and active tablets were of the same appearance (in colour and size) and taste. Each day's tablets
106. were in an individually marked sachet, containing four placebo tablets, one active and three placebo tablets (40 mg group) or four active tablets
(160 mg group). The code was broken only after trial completion, analysis of serum and urine samples, database entry with subsequent checking and locking of the database.
5.1. Results
Thirty seven subjects were randomised. One subject withdrew for personal reasons before commencing any treatment and a further subject was recruited using the same randomisation position. Twelve subjects were randomised to each of the placebo and 40 mg groups and 13 to the
160 mg group. Two patients were subsequently withdrawn from the 160 mg group because of intervention by their general practitioners. All packages containing tablets were returned. In only two patients were tablets not taken, with missed tablets constituting less than 7 days.
The ages of the women participating in the study were 53.1 ± 2.5, 54.5 ±
4.4 and 56.1 ± 3.9 years in the placebo, 40 mg and 160 mg groups respectively. The age at menopause in these groups was 47.7 ± 8.0, 48.5
± 3.6 and 51.1 ± 8.8 years. There was no statistically significant difference in age, weight and age at menopause between the groups. Weight was
107. monitored throughout the study and there was no difference in these values within each group, between trial entry and exit.
Flushing frequency decreased in all groups over the 12 week period of the trial. There was no difference in flushing frequency between the active and placebo groups. This data and that of other measures of estrogen activity are presented in Table 5.1. Analysis of urinary isoflavone levels showed a dose-dependent increase between Week 0 and Week 12 in the groups receiving active tablets. A smaller but significant increase was also observed in the placebo group, indicating that subjects in this group may have adopted altered dietary patterns to include foods containing isoflavones. This occurrence resulted despite inclusion criteria requests that dietary patterns not be altered throughout the course of the trial.
None of the biological parameters of estrogen activity measured, including
FSH, SHBG and climacteric symptom scores showed any change with time compared with placebo. Analysis of the vaginal smears showed no difference between the groups in vaginal MV, although 20% of the smears were unable to be evaluated due to severe atrophy. There was no increase in vaginal acidity associated with isoflavone use.
Serum HDL-cholesterol levels increased significantly by 18% (p=0.038) in subjects on the 40 mg dose of isoflavones. The results for 160 mg did not
108. differ from placebo. Total cholesterol and triglyceride levels could not be accurately evaluated as the samples were collected in a non-fasting state.
5.2. Discussion
The paucity of published data concerning the effects of isoflavones as an intervention in the treatment of menopausal symptoms, in the presence of epidemiological data suggesting these compounds may modify many physiological processes including the menopause formed the basis for this study. A recent larger study has suggested that isoflavones from soy protein may modify flushing frequency in women with severe flushing to a mild degree (45% reduction in flushes compared to 30% reduction in the placebo group). (Aibertazzi et al, 1998)
A similar pattern was observed in the trial reported here. An overall decrease in flushing frequency in the treatment groups was matched by a large fall also in the control group. The data in this study suggested that a trend towards improvement on active medication may have been obscured by the large placebo effect and the highly variable responses to treatment apparent for both the placebo and active treatments. The decrease in flushing frequency in the placebo group should be interpreted with regard to the increase in urinary isoflavone excretion in this group at trial exit. These effects may also be consistent with the data of small
109. intervention trials already published and represent placebo response and time effects. (Murkies et al, 1995)
Closer scrutiny of individual data revealed other difficulties within the control group that may have altered the final analysis. Two subjects were suspected by the investigators of changing the criteria on which flushing frequency was based when completing the diary cards, after enrolment into the trial. Both subjects initiated lengthy discussions at the enrolment visit, and after completion of the weekly pre-trial flush count. Information and education of these patients appears to have altered their perception in determining or characterising hot flushes. In these two patients, there was a fall in flushing frequency within a week of enrolment from greater than ten flushes daily, to one or two each day. Another in the placebo group grew alfalfa in her garden and consumed this whilst on the trial, inadvertently increasing her isoflavone exposure. This was reflected by a rise in the urinary isoflavone levels for this patient. The increased overall exposure to isoflavones noted in the urinary levels of the control group may have contributed to the placebo response reported. However the study was performed on an intention to treat basis and therefore all subjects were included in the analysis.
No differences between the control and active groups were observed in the subjective scoring of menopausal symptoms. The Greene Climacteric
110. Scale has been previously validated as an assessment of menopausal symptoms. The Vasomotor Symptom Score does assess severity of vasomotor function as a numerical assessment of occurrence, however it does not address subjective assessment of the intensity of flushes or night sweats. Lack of effect is certainly an explanation, inability to detect an effect is another. In addressing this issue, it is appropriate that questionnaires used for subjective assessment of menopausal symptoms in future studies are capable of detecting changes in intensity of symptoms as well as frequency.
Whilst urinary levels of isoflavones increased in the active groups, there were subjects in both the 40 and 160 mg group who had a fall in urinary isoflavone levels compared with trial entry. Return of packaging suggested good compliance, and while falls in the total urinary isoflavones with time may indicate poor compliance it may also be indicative of individual variations in absorption profiles. There is minimal pharmacokinetic data available for isolated isoflavone use in humans, however preliminary data does suggest individual differences in the metabolism of daidzein to its end-metabolites, equal and 0-DMA. (Kelly et al, 1995) Recent data suggests that isoflavones are readily absorbed via the gut and both serum daidzein and genistein levels reflect, in a positive relationship, the oral dose consumed. (Howes et al, 1997) Of note in this trial is that equal was
111. rarely detected in the urine of the study patients whilst 0-DMA levels appeared to rise in these groups.
This study confirms the results of others in isoflavones having no effect on serum levels of FSH and SHBG. The lack of change from baseline and between groups in haematological parameters, liver enzymes and proteins suggests the doses used in the study cause no gross abnormalities in haematological and liver function in the short or medium term. Debate has also ensued over isoflavone effects on vaginal epithelium and pH, used as biological indicators of estrogen activity. Our data suggests that isoflavones have no effect on vaginal epithelium or vaginal pH.
The hypocholesterolaemic effects of dietary soy protein has been previously demonstrated. In a meta-analysis of dietary soy protein and the effects on serum lipids, Anderson showed a statistically significant decrease in total cholesterol and low density lipoprotein-cholesterol (LDL c) levels in hypercholesterolaemic subjects. (Anderson et al, 1995) In this analysis there was a non-significant trend towards a rise in HOL-e. The authors suggest that 60-70% of the effects on serum lipid levels by soy protein are due to the presence of isoflavones in the soy protein. As the lipid levels were performed on a post-hoc basis in this study and were collected in the non-fasting state, LDL-c levels were not performed. The
112 rise in HOL-e of 18.1% seen in the 40 mg Promensil™ group, may
represent a window effect. There may be a response to low levels of isoflavones, with diminution or loss of the effect as serum levels increase.
This study emphasises many of the problems with pharmaceutical style intervention studies using naturally occurring dietary compounds. There is a need for further larger studies investigating the areas of clinical effectiveness of isoflavone supplementation in the treatment of menopausal symptoms. Initial indications of biological activity require appropriate assessment of pharmacokinetic properties, and dose response relationships. Although these compounds may well obtain a complementary role in the treatment of menopausal symptoms and disease associated with estrogen deficiency, issues concerning dose response relationships, therapeutic variability and side effect profiles at these doses, and effects on long term diseases associated with estrogen deficiency remain to be addressed.
113. Chapter 6
The effect of supplemental isoflavones on large artery function in postmenopausal women
Because of the similarity in chemical structure between phytoestrogens and animal estrogens, it has been suggested that phytoestrogens may mediate their effect by interaction with both the classical estrogen receptor
(Miksicek, 1995) and estrogen receptor ~· (Kuiper et al, 1998) Animal studies suggest that phytoestrogens and conventional hormone replacement therapy have similar effects on cholesterol uptake and metabolism. (Wagner et al, 1997) Similar to an apparent epidemiological benefit for hormone replacement therapy, it has been suggested that phytoestrogens may confer cardiovascular protection as shown in population studies. (Hertog et al, 1995) Of concern, however, is the same populations that have a high dietary intake of phytoestrogens, may also have a slightly higher rate of hypertension and stroke. (Menotti et al, 1990)
Although this has been attributed to a possible deleterious effect of a low fat diet on stroke risk (Takeya et al, 1984), it could equally relate to the higher phytoestrogen intake. Notwithstanding these epidemiological associations, a number of isoflavones have been shown to have significant anti-hypertensive effects. Some have shown ~-blocking
114. properties (Wang et al, 1994), whereas others induce smooth muscle
relaxation, similar to natural estrogen. (Wu et al, 1992) As menopause is
associated with a significant decrease in arterial distensibility (Laogun and
Gosling, 1982, Karpanou et al, 1996), such smooth muscle relaxation may
be beneficial.
While the majority of phytoestrogens in human diets are from soy products and legumes such as lentils or chick peas, other plant products such as
red clover have also been found to have a very high isoflavone content
(Saloniemi et al, 1995) and to have potent estrogenic effect in binding studies. (Zava et al, 1998) The aims of this study, therefore, were to define the effect of oral supplemental isoflavones at typical dietary levels and at doses at the upper border of normal on arterial stiffness and indices of vascular load in postmenopausal women. The primary endpoints in these studies was arterial function as defined by brachial and derived central blood pressure as well as augmentation of the late systolic peak in the central arterial pressure waveform and arterial pulse wave velocity measured by arterial applanation tonometry.
The trial was conducted as a randomised, double blinded, placebo controlled trial and analysed on intention to treat basis. Twenty four women were randomised to placebo (n=9) or one of two doses of active
115. medication (40mg, n=7 and 160mg, n=8) as part of a dose-finding pilot
study. (Knight et al, 1999) At completion of the three months randomised
therapy, 19 of the 24 subjects completed a further 3 months of the higher
dose (160mg) isoflavone in an open label fashion.
Patients were recruited via the Menopause Centres at St George Hospital,
Sydney. Inclusion criteria included postmenopausal women aged 40-
65years with significant vasomotor symptoms (3 hot flushed per day),
seeking treatment for management of those symptoms and agreed to
participate in the study. Menopause was defined by amenorrhoea with typical symptoms of estrogen deficiency and a serum follicle stimulating
hormone (FSH) greater than 40 IU, or bilateral oophorectomy. Exclusion
criteria included hormone replacement therapy usage within the last 6
weeks; any contraindications to hormone replacement therapy; vegetarians or regular soy product users; active bowel, liver, gall bladder
disease or current therapy with liver enzyme inducing medication;
diabetics requiring drug therapy; and malignancy (excluding skin cancers).
Participants were instructed not to alter their usual diet for the duration of the study. Low or absent urinary isoflavone levels confirmed the verbal screening. The St George Hospital Research Ethics Committee approved the study and all subjects gave informed consent. This study represents a subset of a study examining the effect of the same supplemental
isoflavone on menopausal markers and symptoms, (see Chapter 5).
116. A double blinded, randomised, placebo controlled trial was performed consisting of three arms; placebo, 40 mg of Promensil™ and 160 mg of
Promensil™ (Novogen Ltd Sydney, Australia). All subjects consumed four tablets daily. Each day's tablets were in an individually marked packet, containing four placebo tablets, one active and three placebo tablets (40 mg group) or four active tablets (160 mg group). Subject compliance was assessed by return of tablet packets. A 24 hour urine collection for isoflavone measurement preceded trial entry. Arterial pressure waveforms were recorded using applanation tonometry acquired using a OAT system.
(see Chapter 2) Representative waveforms for each individual were obtained, operator Dr Chris Haywood. Each series of waveforms was assessed and those with significant movement or respiratory artefact were excluded. Carotid pressure waveform indices were determined by automated software.
Comparisons were made using paired t-tests against baseline results for each parameter. Significance was set at p<0.05.
6.1. Results
Demographics for the 24 women who completed three months
117. randomised medications are shown in Table 3.4.1. Six women were being treated for hypertension with three receiving angiotensin converting enzyme inhibitors, 2 diuretic therapy and one alpha blockade. After screening and randomisation, nine women received placebo, seven received 40mg, and eight received 160mg isoflavone for the first three months.
Total urinary phytoestrogen levels increased significantly in a dose-related manner (Figure 6.1 ). Urinary phytoestrogens did not differ from baseline after 3 months in the placebo group. There was a 3.3-fold increase in urinary phytoestrogens in the 40mg group (from 3.4 to 11.0ng/mL) and an
8-fold increase in the 160mg group (from 3.7 to 31.5ng/mL). Daidzein and genistein made up the great majority of urinary phytoestrogens at baseline
(53.7% and 38.3% respectively) and following supplementation (54.1% and 25.3% respectively) in the 160mg group.
At 3 months there was no significant effect of isoflavone consumption on blood pressure, heart rate or ejection duration at concentrations of 40mg or 160mg compared to baseline (Table 6.2). Similarly there was no effect on parameters of pulsatile loading or arterial stiffness (Table 6.2). No dose trend was apparent. The only significant difference in the study was between baseline and placebo for systolic augmentation. The 3 month
118. placebo measurement for the group was similar to the measurements for
both doses of active medication, and the difference from baseline most
likely relates to a chance elevation of the baseline augmentation results,
rather than an effect of placebo per se. There was no correlation between change in urinary phytoestrogens and change in any haemodynamic
parameter in those subjects who had received the supplemental isoflavones (40mg and 160mg group).
Following the further 3 months at a dose of 160mg of Promensil™, the previous results were confirmed in an open label continuation of the same study with a greater number of subjects. Results for all women who took the higher dose of isoflavone were compared to their respective baseline measurements. Again, there was no significant effect of 160mg isoflavone on any parameter even at a higher dose (Table 6.2). Even after exclusion of all women who were taking vasoactive medications (n=5), there was no effect of phytoestrogens on any index of arterial function.
6.2. Discussion
This study did not show any significant effect of isoflavonoid phytoestrogens on blood pressure, heart rate, or arterial function as assessed by pulse waveform analysis or pulse wave velocity in health
119. postmenopausal women. Significant dose-related increases in 24-hour
urinary phytoestrogens confirmed both compliance with the regimen of
tablets as well as absorption of the study drugs. The 160mg dose
represents a higher intake of isoflavones than is generally taken even in
national diets thought to be high in phytoestrogens. (Messina, 1995)
Despite the fact that populations associated with high phytoestrogen
intake have a typically lower incidence of coronary vascular disease, the same populations tend to have a greater incidence of stroke and
hypertension. (Menotti et a, 1990) As such, it is reassuring that no
evidence of blood pressure elevation is evident even at these increased
doses. It is of note that some other forms of supplemental sex steroids, such as the modern low-dose oral contraceptive pill, are associated with a slight increased in the incident rate of hypertension. (Chasan-Taber et al,
1996) While current regimens of hormone replacement therapy, which
involve a lower dose of supplemental estrogen and progestins than the contraceptive pill, are not usually associated with a change in brachial blood pressure, (Lip et al, 1994) there remains some reluctance to institute hormone replacement in hypertensive menopausal women.
As in the current study, a number of prospective studies examining cardiovascular effects of phytoestrogens have not shown any effect on blood pressure. (Neste! et al, 1997, Hodgson et al, 1998) Although vegetarians have been shown to have lower blood pressure than non-
120. vegetarians in case control studies, a causative role for phytoestrogens
has not been proven. One study which have substituted dairy protein with
soy protein (high in phytoestrogens) for short period (3-6 weeks) did not find any change in blood pressure. (Sacks and Kass, 1988) Similarly, a weight-loss study using isocaloric diets based either on meat proteins
(meat 46%, soy 0% of total protein) or legume proteins (meat 14%, soy
31% of total protein) demonstrated equal improvement in blood pressure, cholesterol and arterial compliance over a 4 month period in both groups.
The authors attributed these changes to weight loss alone. (Yamashita et al, 1998)
One group has found a significant improvement in systemic arterial compliance in subjects receiving phytoestrogen supplementation derived either from soy (Neste! et al, 1997) or from red clover extract. (Neste! et al,
1999) In both of those studies arterial compliance improved by approximately 25%. Neither index of arterial stiffness, pulse wave velocity or systolic augmentation, used in the current study improved, despite using a higher isoflavone dose (160mg compared to 80mg) for longer (3 month compared to 5 weeks). Although the number of subjects in the current study were slightly smaller than Neste! and colleagues' study,
(Neste! et al, 1999) a type 2 error does not appear likely for two reasons.
There was no trend detected in any parameter during the randomised
121. phase of the trial and no haemodynamic effect was apparent after the 3 months at the open-label higher dose in a larger number of subjects.
Studies investigating arterial effects of phytoestrogens are limited.
Protection by dietary phytoestrogens against acetylcholine induced coronary vasospasm has been demonstrated in monkeys with diet induced atherosclerosis. (Honore et al, 1996) These studies also showed.
In the same series, acute reversal of coronary vasoconstriction by intra coronary genistein was also demonstrated. Despite the epidemiological association of phytoestrogens and lower incidence of cardiovascular disease in humans, there are no studies examining coronary vasomotor effects. Neste! and colleagues• study of systemic vasomotor effects,
(Neste! et al, 1997) found that acetylcholine-mediated dilation in the brachial artery was similar with active and placebo treatments.
In summary, this trial suggested that three months of dietary isoflavone supplementation did not significantly affect brachial blood pressure or indices of arterial function in healthy postmenopausal women. Further studies are required before a definite vascular role for phytoestrogens can be confirmed.
122 Chapter 7
The effects and acceptance of isoflavone-containing soy
powder dietary supplementation on menopausal
symptoms.
The use of isoflavones found in soy products to modify the symptoms of
estrogen deficiency has been addressed in small studies producing
conflicting results regarding the efficacy of isoflavones in the treatment of
menopausal symptoms. This is the second of two studies in this thesis
addressing the issue of isoflavones and their possible effects on estrogen
deficiency. This study followed a similar protocol to that reported in
Chapter 5, however a dietary supplement was used to supply the
isoflavones.
A double blinded, randomised, placebo controlled trial was performed
consisting of two arms; placebo and isoflavones in the form of a powder to be made into a beverage. The isoflavone powder, TakeCare™, was
manufactured and supplied by Protein Technology International, (St
Louis, USA). The placebo arm was an isocaloric casein-based beverage
also produced by Protein Technology International. These powders were
not artificially flavoured. Both placebo and TakeCare™ were packaged in
sachets to be used on a daily basis.
123. The daily dose of TakeCare™ was four scoops or 60g. The isoflavone
analysis of the batch is listed in Table 7.1. The total isoflavone concentration consisting of genistein, daidzein and glycetein compounds was 134.4mg. The total isoflavone concentration of the aglycone formulations was 77.4 mg.
Twenty four (24) subjects were recruited through the University
Departments of Obstetrics and Gynaecology at the St George and
Liverpool Hospitals, Sydney, Australia. The inclusion criteria for the trial were postmenopausal women who were symptomatic, having at least three flushes per day. Menopause was defined by amenorrhoea for 6 months with typical symptoms of estrogen deficiency and a serum follicle stimulating hormone (FSH) greater than 40 IU, or bilateral oophorectomy.
Participants were aged between 40-65 years upon trial entry and were instructed not to alter their usual diet for the duration of the study.
Exclusion criteria included HRT use within the previous six weeks, current history of active bowel, liver or gallbladder disease; diabetics requiring drug therapy; and malignancy (excluding skin cancers). Women with contraindications to HRT use, vegetarians and regular soy product users
(defined as consumption greater than once weekly) were also excluded.
124. Urinary isoflavone levels were performed prior to the study to confirm the
low dietary intake of isoflavones suggested by the dietary history.
Pre-trial flushing was assessed using a daily flush diary for the week prior to trial entry. Menopausal symptom severity was assessed using the
Greene Menopause Scale. Upon fulfilment of inclusion criteria, subjects were entered into the study and serum and urine were collected for FSH,
SHBG, TBG, markers of bone turnover (b-ALP, u-PLC and osteocalcin) and isoflavone levels.
After screening, the subjects were randomly assigned to placebo or active treatment groups. Physical and vaginal examination was performed. A vaginal wall smear for determination of maturation value (MV) (Koss,
1979) and pH was performed at trial entry. The vaginal wall smear was performed using a speculum and cytobrush, with a 360° rotation of the brush on each lateral vaginal wall. The isoflavone assays were performed by Novogen Ltd.
The trial was twelve weeks in length and the subjects were seen every four weeks for clinical assessment, compliance checks and assessment of supplement acceptability, flush count and Greene Score. Contact between appointments was provided if required by individual subjects. In the final week of the study physical and vaginal examinations, vaginal
125. smear, urine and blood collections were repeated. Compliance was
assessed by empty sachets return and measurements of urinary
isoflavone excretion. All subjects were included in the analysis on an
intention to treat basis.
The Greene Menopause Score is a validated menopause symptom self assessment form (Greene, 1976) and was completed weekly. For analysis, the Greene Score was calculated as a total and also in the accompanying subgroups, the Psychological Scale, Somatic Scale and
Vasomotor Scale.
Data was entered into Statistica™ (StatSott™ Inc). Nonparametric data was analysed using the Kruskai-Wallis nonparametric analysis of variance. Normally distributed data was analysed using one way analysis of variance. The differences between groups were analysed using the
Newman-Keuls test provided the F value from the analysis of variance was significant. This test was preferred to the Bonferoni modification, a corrected student's t-test, as the Newman-Keuls is based on the least significant difference between the means of each group and is a
"protected" test taking into account the risk of spacious results due to multiple comparisons. Data is presented in the format, mean ± standard deviation with p values included when :::;; 0.05. Differences between the
126. incidence of patients reporting any side effects was analysed using the
Chi squared test.
An external statistician performed the randomisation procedure. The allocation schedule was produced in random permuted blocks of six generated using a computer random number generator. Subsequently sachets were packed in weekly lots and supplied in individual subject containers to the investigators. Packaged subject containers were received prior to trial recruitment. All subjects consumed one sachet daily.
Placebo and active sachets were of the same appearance (in colour and size). Each day's sachet was individually marked. Only after trial completion, analysis of serum and urine samples, database entry with later checking of database and finally locking of the database, was the code broken.
7.1. Results
Twelve subjects were randomised to each of the placebo and active groups. The ages of the women were 53.8±5.4 and 52.3±3.7 years in the placebo and active treatment groups respectively. The age at menopause in these groups was 49.0±4.9 and 49.8±3.8 years. Weight was monitored throughout the study and there was no significant difference in these values within each group, between trial entry and exit. (Placebo group;
127. 66.1±10.9 kg at entry and 66.8±10.1 kg at exit. Active group; 69.9±14.4
and 70.2±15.0 kg at entry and exit respectively.) There was no statistically
significant difference in age, weight and age at menopause between the
groups.
Adverse side effects reported during the trial included dislike of the taste
(6}, bloating (4}, nausea (3}, weight gain (2) and changes in bowel function (2). The complaints with respect to taste included five (5} the
TakeCare™ and one in the placebo group (p=0.07). The total number of patients reporting side effects in the active group was 9 compared to 2 out of 12 in the placebo group (Chi square = 12.3, p < 0.001 ). Four patients withdrew during the trial, three women from the TakeCare™ arm and one from the placebo arm. The women from the TakeCare™ arm all cited not liking the taste as the reason for withdrawal. The woman who withdrew from the placebo arm was unable to be contacted. In only two patients completing the trial were sachets not completely taken, with missed sachets constituting less than 7 days.
Flushing frequency decreased in both groups over trial duration, but there was no difference in flushing frequency between the active and placebo groups. There was no statistically significant difference in the incidence of flushes between the groups at trial conclusion, with flushing frequency decreasing by 32% in the placebo group and 43% in the active group
128. (p=0.32). There was no difference between the groups in Menopause
Symptom Scores, FSH, SHBG, TBG or bone turnover markers. Analysis of smears showed no difference between the groups in vaginal Maturation
Value (MV). Both groups showed an increase in MV that was not statistically significant. The estrogen biological activity data and bone data are presented in Table 7.2.
Analysis of urinary isoflavone levels showed no increase in mean levels in the placebo group. A greater than 20 fold increase in isoflavone excretion occurred in the active group (table 2), which was a statistically significant increase compared with placebo (p=0.008). Entry levels of total isoflavones in the placebo and active groups were 3.0±1.6 ng/ml and
4.0±2.0 ng/ml with exit values, 3.0±1.1 ng/ml and 93.0±24.1 ng/ml respectively.
7 .2. Discussion
There are few published randomised, placebo-controlled trials assessing the effects of dietary or supplementary isoflavones. The clinical outcomes of the small trials thus far presented assessing the effects of isoflavones on the frequency and severity of menopausal symptoms do not show clinically significant differences between placebo and isoflavone treatment. Studies that compare symptom changes between trial initiation
129. and conclusion are poorly designed to detect effects of treatment on
menopausal symptoms, given the change in estrogen deficiency
symptoms with time, duration and season. In a recently published double
blind crossover trial of 149 breast cancer survivors with hot flushes using
50 mg of isoflavones in soy extract tablets, flushing frequency, intensity
and perceived side effects were recorded. (Quella et al, 2000} The soy tablet in this trial was not more effective than the placebo in reducing hot flushes. Another study using a 50 mg isoflavone extract from soy, also in tablet form, showed no statistically or clinically significant changes in the frequency of hot flushes between active and control groups at trial conclusion. (Upmalis et al, 2000} A further study likewise using a standardised soy extract in tablet form, at the same isoflavone dose (50 mg), studied the effects of isoflavones compared with placebo. There was no clinical significant difference in flushing frequency between the control and active groups after six weeks of use. (Scambia et al, 2000} The comparative isoflavone assessment ended at this stage of the study. No bioavailability data was available regarding the products used in the above trials.
There is only one adequate randomised, placebo-controlled trial in the published peer review medical literature (Medline 1990-current, PubMed
1990-current) that addresses the issue of treatment of hot flushes with dietary derived isoflavones from food sources. This relatively large trial
130. reported a 45% reduction in severe flushing frequency with the daily
consumption of soy protein containing 76 mg of isoflavones (Aibertazzi et
al, 1998) however this was only 15% above the placebo response. Dalais
and colleagues reported no change between a group of women consuming soy bread and a control group in the incidence of vasomotor symptoms. (Delais et al, 1998) This group also showed a significant weight gain from baseline due to inclusion of food products. At the dose
used, our pilot study did not suggest any benefit in relief from menopausal symptoms, although the sample size studied could only have been expected to detect larger differences between treatment and control groups, than have previously been reported. (Aibertazzi et al 1998,
Scambia et al, 2000, Delais et al, 1998) The lack of difference between the groups in Menopause Symptom Scores, FSH, SHBG, TBG and vaginal epithelial changes is supported by previously published data.
(Duncan et al, 1999) Whilst isoflavones have anecdotally been suggested as useful in modifying the symptoms of estrogen deficiency, this has not been borne out in the trials that have been published on the topic.
The primary aim of this study was to assess the suitability and acceptability of the dietary intervention for possible treatment of menopausal symptoms. For food supplements to make the transition to a pharmaceutical intervention, appropriate scientific evaluation is paramount. The hypothesis that these compounds may relieve
131. menopausal symptoms is biologically sound with pharmacokinetic data suggesting absorption occurring in direct proportion to dose. (Aibertazzi et al 1999) Irrespective of the efficacy of the compounds, which still has not been clearly established, the tolerability of foods or beverages containing isoflavones remains an important issue. The side effect profile of any intervention is important as it directly affects compliance with therapy.
The acceptability of any product is subject to large cultural differences between societies, and acceptance in one society should not imply a similar response in others. Powdered energy drinks are not commonly consumed in Australia and were poorly tolerated in this study.
Notwithstanding the high side effect rate reported, the authors involved in clinical contact in this trial used considerable encouragement to ensure a larger withdrawal rate did not occur, as almost all patients expressed some dislike for the beverages. Although the Albertazzi study ((Aibertazzi et al, 1998) demonstrated a modest but significant decrease in menopausal symptoms, there was a marked decrease in daily compliance in the soy group during the second month of treatment. Similar products to those in our study were used. In this study of 104 patients, 25 withdrew
(24%). Gastro-intestinal complaints and food intolerance resulting in constipation, bloating, nausea and vomiting were the most common factors cited.
132 Despite the small size of the present study, a high rate of dissatisfaction
was observed with respect to the tolerability of the formulation of
isoflavone supplementation used. This could theoretically reduce
compliance and lead to a reduction in the relatively small, if any, impact that isoflavone supplementation has on menopausal symptoms. Products for future trials should be appropriate to specific societies addressing the
issues of taste, ease of use·, energy intake, mouth feel and cultural
acceptability. Without attention to these details, statistical analysis of
changes in clinical parameters is likely to be affected by compliance
issues.
133. Chapter 8
The effects of an average dietary dose of isoflavones on markers of bone turnover in postmenopausal women
Osteoporosis is a group of metabolic bone diseases characterised by microarchitectural deterioration of bone tissue leading to an increase in bone fragility and greater propensity for fracture. This disease is related to ageing, gender and hormone deficiency. The incidence of fractures due to bone thinning increases after the age of 60, however this relates not only to bone density but also to the risk of falls and bone strength. The most common entity, postmenopausal osteoporosis, effects elderly women with the incidence increasing with the duration of estrogen deficiency.
The primary aim of this study was to examine possible effects of supplementation in the diet, using 160 mg of isoflavones, on markers of bone turnover. The trial was designed in an open parallel format and was
20 weeks in length. Twenty two (22) subjects were recruited through the
University Department of Obstetrics and Gynaecology at the St George
Hospital, Sydney, Australia.
Postmenopausal women within 10 years of the menopause and aged between 45 and 60 were eligible for trial entry. Menopause was defined by
134. a serum follicle stimulating hormone (FSH) > 40 IU/1, bilateral oophorectomy or amenorrhoea for at least 6 months with typical symptoms of the menopause. The exclusion criteria included HRT use within the previous three (3) months, allergy to foodstuffs known to contain isoflavones, current history of active bowel, liver or gallbladder disease; serious venous or arterial thrombosis; hyperparathyroidism; renal failure; hyperthyroidism; diabetics requiring drug therapy; and malignancy
(excluding skin cancers). Women who were vegetarians and/or regular soy product users were also excluded.
lsoflavone supplementation was achieved using 160 mg of Promensil™
(Novogen Ltd, Sydney, Australia). Supplementation in the diet was given for the first ten (1 0) weeks of the study, followed by no treatment for the following ten (1 0) weeks.
After screening, physical examination was performed. Blood was collected in a non-fasting state for serum levels of FSH, SHBG, thyroid function and renal function. Trial entry investigation of serum calcium, creatinine and
TSH excluded women with hyperparathyroidism, renal failure and hyperthyroidism respectively.
Bone turnover was assessed using specific markers including bone specific alkaline phosphatase, osteocalcin (which is an osteoblast (bone-
135. forming cell) product that is incorporated into bone matrix) as markers of bone formation. Serum interchain C-terminal telopeptide, released after breakdown following the synthesis of type I collagen by osteoblasts and fibroblasts, and urinary deoxypyridinoline crosslinks, a crosslink of bone released during the resorption process, as markers of bone resorption.
Biochemical and sex hormone assays were performed by Southpath
Laboratories at St George Hospital (Sydney, Australia). A 48 hour urine collection for isoflavone measurement preceded trial entry. Urinary isoflavone assays for the isoflavones daidzein, genistein, equol, 0-dma, formononetin and biochanin were performed by Novogen Ltd.
Subjects were subsequently seen at 5 weekly intervals to renew tablets, check blood pressure and weight. Repeat analysis of bone turnover and biochemical parameters described previously were performed at 10 weeks and 20 weeks after trial entry. Compliance was assessed by return of pill containers and 24 urine analysis for isoflavones.
Statistical analysis was performed using Statistica™ (StatSoft™ Inc).
Subgroup analysis, descriptive analysis and multiple regression analysis were performed. Significance was set at p<0.05.
136. 8.1. Results
Twenty two women were recruited into the trial and 18 women completed the ten week period. The ages of the women were 52.8±4.57 years. There was no statistically significant difference in weight from trial entry to end.
There was no difference in serum levels of FSH, SHBG, thyroid function and renal function between trial beginning and completion.
Bone turnover markers (bone-specific alkaline phosphatase, osteocalcin, serum interchain C-terminal telopeptide and urinary deoxypyridinoline crosslinks) also showed no difference between groups and from trial commencement, at the ten week mark and at trial completion. The mean results for bone turnover markers are detailed in Table 8.1. Urinary isoflavones greater than 2 ng/ml were not detected in any of the subjects prior to trial commencement with a mean total isoflavone level of
0.68±0.81 ng/ml. At week 10, urinary levels of all the supplemented isoflavones were detectable with a mean total isoflavone level of
20.29±1 0.67 ng/ml.
As bone loss is individually variable, generally referred to as 11 fastn or
11 SIOW 11 bone-losers, subgroup analysis was performed. A subgroup analysis using the 50% of subjects with the highest change in bone
137. markers was performed. Sample correlations between percentage change in bone markers and urinary isoflavone excretion were performed for each marker and the nine women with the largest change. These results are presented in Table 8.2.
Some significant negative correlations occurred with daidzein secretion, however in respect only in the subgroup analysis and osteocalcin. Positive correlations were identified with 0-dma. No correlations were identified regarding genistein, equol, formononetin and biochanin.
8.2. Discussion
Osteoporosis risk increases with increasing length of estrogen deficiency.
Estrogen replacement therapy forms the mainstay of treatment for this condition, however side effects or other conditions, for example breast cancer, necessitate that other options are desirable for treating this condition. The recent advances in estrogen receptor research and the advent of selective estrogen receptor modulators, such as raloxifene and tibolone, suggest that tissue specific approaches may become available. lpriflavone is a synthetic isoflavone that has been shown to have similar bone-sparing effects to conjugated equine estrogens. (Gambacciani et al,
1993, Gambacciani et al, 1994) The potential use of isoflavones as part of
138. a normal diet to modify the rate of postmenopausal osteoporosis is an attractive proposition, especially in countries where the cost of treatment for osteoporosis is so large.
There are published trials, both in humans and animal models addressing the issue of bone loss and isoflavones. Administration of genistein in oophorectomised rats has been shown to be associated with higher bone formation rate per tissue volume as well as identifying a trend toward a higher number of osteoblasts per bone perimeter. (Fanti et al, 1998)
Genistein did not affect parameters of bone resorption with serum osteocalcin concentration and urinary deoxypyridinoline excretion providing corroborating results. The effect of genistein on lipopolysaccharide-induced in vitro production of tumour necrosis factor alpha (TNF-a) was tested as production of pro-inflammatory cytokines is intimately involved in the pathogenesis of postmenopausal osteoporosis.
Production of TNF-a was blocked by genistein. This study suggests that genistein reduces trabecular and compact bone loss after oophorectomy and the protective effect observed differs from that of estrogen, as it depends on stimulation of bone formation rather than suppression of bone resorption. Modulation of cytokine production may be involved in the effect of genistein on bone.
139. Measurement of bone mineral density is currently regarded as the "Gold standard" of assessing changes in bone over time. Postmenopausal women were recruited in a recent parallel-group, double-blind trial conducted over a 6-month period. (Potter et al, 1998) They received either dietary soy protein (containing isoflavones) or casein (placebo and free of isoflavones). Over the period of the trial, the subjects were assessed for total and regional bone mineral content and density changes. Significant increases occurred in both bone mineral content and density in the lumbar spine but not elsewhere in women consuming soy protein containing
90mg of isoflavones on a daily basis compared with the control group
(p<0.05). Lower doses of soy protein consumed during this study did not show any difference compared with placebo in bone mineral content or density.
A recent analysis of long-term urinary excretion of phytoestrogens as a marker of habitual dietary intake, compared with postmenopausal bone loss does not support a preventive effect due to low, unsupplemented dietary intake of phytoestrogens on postmenopausal cortical bone loss.
The authors summarised by stating that "no conclusions could be drawn concerning effects of higher doses of phytoestrogens". (Kaardinal et al,
1998)
140. The small numbers and the short time frame may have affected the ability of the trial to detect changes in bone markers. Daidzein is one of the main primary metabolites of ipriflavone (Brandi, 1992}, a synthetic isoflavone that has been previously shown to have bone-sparing effects.
(Gambacciani et al, 1994) (Agnusdei et al, 1995) Of interest in this study is the possibility that 0-dma may antagonize any bone-sparing effects of individual isoflavones or perhaps increase bone loss. Whilst suggesting possible effects on bone marker turnover by daidzein and 0-dma, no conclusions could be drawn.
141. Chapter 9
Summary
Clinical applications for phytoestrogens are still very much in their infancy.
In humans, there are obvious, potentially important health benefits that may be associated with the consumption of foods containing phytoestrogens. Leaning toward a vegetarian style diet inevitably leads to an increased exposure to phytoestrogens.
Dietary intervention as a method of preventing chronic disease is attractive and generally cost-efficient in respect to health benefits however appropriate scientific evaluation is required to assess measures of outcome. The use of any dietary intervention as a pharmaceutical-style treatment requires the criteria for assessment of a pharmaceutical product being applied to the dietary treatment. These criteria include product stability, pharmacological data, reproducibility of effects, side effect profile and long term effects. Unfortunately adequate amounts of this type of data is not yet available to enable firm conclusions or recommendations for phytoestrogen use in the treatment of problems associated with estrogen deficiency after the menopause.
142 This thesis has added incremental evidence to the increasing information regarding isoflavones. lsoflavones have been identified in foods and beverages consumed in Australia, including those that result in exposure of neonates and infants to these compounds. This level of exposure however does not appear to occur at levels that have resulted in neurendocrine or reproductive changes as reported in animals.
lsoflavones have been reported for the first time to be present in ovarian follicular fluid. The consequences of this presence have yet to be determined. However the properties of isoflavones, especially with respect to inhibition of tyrosine kinase, implies that those hormones mediated through this mechanism may be affected. This type of activity may affect ovarian function especially in relation to Polycystic Ovarian Syndrome.
Animal data has shown these effects at high doses, similar effects may or may not occur in humans at lower or medium exposure.
In the dietary supplementation with isoflavones, in either food or tablet forms, these compounds do not appear to be useful in the treatment of vasomotor symptoms of estrogen deficiency at the doses studied. This thesis adds to the negative trials investigating low doses of isoflavones in the treatment of menopausal symptoms. There were no effects on serological parameters reflecting bone and cardiovascular function.
143. Whilst lifestyle change reflecting an improvement in diet and exercise is generally accepted by the general public as a positive approach to improving health, fad dieting and dietary supplementation require appropriate scrutiny and study before medical claims are appropriate.
Whilst the future may say otherwise, the evidence is not currently available to support the beneficial claims of soy products and isoflavones in the treatment of estrogen deficiency symptoms and long term effects.
144. Chapter 10
Future Aspects
Despite large amounts of animal and in vitro data regarding the effects of isoflavones, these effects have not been translated into reproducible and efficacious pharmacological therapies. The lack of appropriate pharmacokinetic data hampers research in this field. Studies directed at determining the absorption profiles, areas of metabolism and excretion in the human body are paramount.
Dose finding studies are the most important approach clinically, prior to development and commencement of any further small or large trials. The supposition that isoflavones have effects requires some proof that these compounds in fact have clinical effects. Short term side effects may be similar to those of estrogen, and the possibility of adverse reactions exist.
Cost is a major impediment in isoflavone research. The compounds are expensive to isolate, expensive to measure in body fluids and do not attract the same type of enthusiasm or level of financial support within corporate structures of proprietary or patented compounds.
145. Figures
Figure 1.2.1. The structure and metabolic pathway of common lignans.
(from Borriello et al, 1985)
OH
Secoisolariciresinol-dig lycoside
OH
facultative hydrolysis Matairesinol aerobes dehydroxylation demethylation facultative dehydroxylation aerobes Idemethylation
0 HO HO
oxidation facultative aerobes
Enterodiol Enterolactone
146 Figure 1.2.2. The physiological enterohepatic circulation of lignans in humans. The arrow illustrate enterohepatic circulation occurring and do not indicate exact sites of absorption of lignans.
(modified from Satchell et al, 1982)
Renal Clearance Urine t LIGNAN PRECURSORS GLUCURONIDES
Bacteriav LIGNANS
Fecalt loss UN CONJUGATED LIGNANS
147 Figure 1.3.1. The structure and metabolic pathway of common isoflavones.
(modified from Bingham et al, 1998 and Kurzer and Xu, 1997)
Formononetin HO
HO HO
OH
0 0 I Dihydrogenistein Dihydrodaidzein OH I \
6'-hydroxy-0-dma 0 Equol 0-desmethylangolensin (0-dma)
148 Figure 1.3.2. The comparative structures of estradiol, diethylstilbestrol and common isoflavones.
(modified from Kaldas and Hughes Jr, 1989)
OH
OH HO Coumestrol
OH HO Estradiol
HO
OH Daidzein
OH HO Diethylstilbestrol
HO Equol
149 Figure 2.6.1.1: Schematic of carotid waveform. The timing of the pressure
peaks and onset and completion of ejection are determined automatically from derivatives as described in Methods. Calculation of augmentation
index is shown. dP/dt and ejection duration are shown schematically. max
Systo Ii ~ -~ ~- ______Peak 2
Diastolic B _.,., ___ ------____. ..,_ Ejection Duration
1 Second
(Peak 2 - Peak Aug mentation Index = (Pulse
150 Figure 5.1: Dose relationship in blinded study between the three groups
and 24-hour urinary phytoestrogen levels. A clear dose relationship is
seen.
40 * Placeb 30 D
...J E ~ 40m 20 -0') c ~160m 10
Mean±SEM
0 * p<0.001 3 months
151 Tables
Table 1.2.1. Production of Mammalian Lignans from Different Foods
Foods Enterodiol Enterolactone Total (mg/1 OOg)
Flaxseed meal 59.02 8.52 67.54
Flaxseed flour 40.86 11.82 52.68
Lentil 1.00 0.78 1.78
Dried seaweed 0.98 0.16 1.14
Soybeans 0.17 0.69 0.86
Oat bran 0.39 0.26 0.65
Kidney bean 0.23 0.33 0.56
Wheat 0.08 0.41 0.49
Garlic 0.33 0.08 0.41
Asparagus 0.24 0.14 0.38
Rye 0.09 0.07 0.16
Values in the table are expressed as mg produced by fecal flora from 100 g of sample on the wet basis.
(Modified from Thompson et al, 1991)
152 Table 1.3.1. Typical levels of the aglycone isoflavones in different foods.
Values in the table are expressed as mg per 100 g dry weight.
Foods Daidzein Genistein Total
(mg/1 OOg)
Tofu
-Kikkoman 7.6 21.2 28.8
-Nasoya Soft 7.3 18.7 26.0
-Vitasoy 8.6 20.6 29.2
Soy drink 0.7 2.1 2.8
Soy formula 0.03 0.31 0.34
Soyflour
-Nutrisoy 78 69 127 196
-Centrex 400SL 110 180 290
-unflavoured TVP 114 167 281
Soy concentrate
-Response 14 5 19
-GL-301 87 213 300
Soy isolate
-Edi Pro N 24 89 113
-Supro 710 51 72 123
(Modified from Eldridge, 1982 and Dwyer JT et al, 1994.)
153 Table 1.3.2. Total Values of lsoflavones in Foods
Food Daidzein Genistein Glycetein Total
(mg/100g)
Roasted soybeans 56.3 86.9 19.3 162.5
TVP 47.3 70.7 20.2 138.2
Green soybean 54.6 72.9 7.9 135.4
Soyflour 22.6 81.0 8.8 112.4
Tempeh 27.3 32.0 3.2 62.5
Tofu 14.6 16.2 2.9 33.7
Tofu yogurt 5.7 9.4 1.2 16.4
Soy hot dog 3.4 8.2 3.4 15.0
Soy noodle (dry) 0.9 3.7 3.9 8.5
Values in the table are expressed as mg per 1OOg dry weight.
(Modified from Wang and Murphy, 1994)
154 Table 1.4.1. Relative binding affinities of various compounds for the a and
B estrogen receptors.
Compound ERa ERB
Physiological Estradiol 100 100
estrogens:
Estrone 60 37
Estriol 14 21
Synthetic Diethylstilbestrol 468 295
estrogens:
Tamoxifen 7 6
Raloxifene 570 29
Dietary Genistein 5 36 estrogens:
Coumestrol 94 185
Zearalanol 16 14
(Modified from Gustafsson, 1998)
155 Table 1.4.2: Relative potencies of phytoestrogens compared with estradiol.
Compound Potency (E2=1 00)
coumestrol 0.202
genistein 0.084
equol 0.061
daidzein 0.013
biochanin <0.006
formononetin <0.006
(Modified from Markiewicz et al, 1993)
156 Table 3.1. lsoflavone content (mg per 100 ml) of various brands of cow
milk, dairy yoghurts and soy milk drinks available in Australia. Figures in
brackets expressed in IJM.
Product Daidzein Genistein Total
- Cow's Milk:
Country Milk 0.01 (0.4) nd* 0.01 (0.4)
Farm Fresh 0.03 (1.2) nd 0.03 (1.2)
Perfection 0.09 (3.5) 0.01 (0.4) 0.10 (3.9)
-Yoghurt:
Yo Plait 0.07 (2.7) nd 0.07 (2.7)
Yo Baby 0.02 (0.8) nd 0.02 (0.8)
- Soy Drinks:
Aussie Soy 1.08 (42.4) 1.21 (44.6) 2.29 (87)
So Good 2.17 (85.3) 4.20 (155) 6.37 (240)
So Natural 2.14 (84.1) 2.61 (96.2) 4.75 (180)
Vita Soy 3.15 (124) 4.80 (177) 7.95 (301)
*not detected
157 Table 3.2. lsoflavone content (mg per 1 00 ml) of reconstituted infant formulas.*
Figures in brackets expressed in J..JM.
Product Total lsoflavones
Casein-based infant formulas
Enfelac 0.0001 (0.004)
Karicare 0.003 (0.11)
Pre Nan 0.0001 (0.004)
Soy based infant formulas lnfasoy 2.14 (81.7) lsomil 2.19 (83.6)
Karicare Soy 1.72 (65.7)
ProSobee 1.75 (66.8)
*All products reconstituted in water according to manufacturers' specifications.
158 Table 3.3. Estimated daily exposure to isoflavones of infants in the age
groups 14 to 28 days of age, 2 to 4 months of age and 5 to 12 months of
2 age, consuming different milks and formulas expressed in mg per m body
surface area per day. lsoflavone exposure of an average 70 kg adult
2 consuming 40 mg isoflavone daily is 22.7 mg/m BSA/day.
Product Estimated isoflavone exposure
14 to 28 days of 2 to 4 months of 5 to 12 months of
age age age
Casein-based inf1
Enfelac 0.00064 0.00062 0.00038
Karicare 0.0019 0.0019 0.011
Pre Nan 0.00054 0.00052 0.00031
Soy-based infant
lnfasoy 14.05 13.75 8.27
lsomil 13.88 13.57 8.17
Karicare Soy 11.42 11.17 6.72
ProSobee 11.25 10.9 6.62
Fresh cow milk* 0.10 0.31 0.062
* Mean of three products
159 Table 4.1. lsoflavone concentrations and follicle volumes of the subjects
with detectable isoflavone levels.
Subject Follicle Volume Daidzein Genistein
Number Concentration Concentration
10 7.0 0 0.00
11.0 0 3.02
8.5 0 0
27 13.0 0 7.95
10.0 0 0
8.0 0 11.22
160 Volume P* E2 T FSH SHBG FAI LH Prl lnhibin-8 )> 0 p 0.315 0..... ro a E2 0.299 0.066 a· ::J 3 T 0.164 0.063 0.556 a .....x· FSH -0.032 -0.080 -0.014 -0.146 8. -Q. (5" SHBG 0.244 -0.150 0.268 0.075 -0.125 c .....Sll ::!::! FAI 0.000 0.202 0.208 0.541 0.073 -0.675 c 0.: CD LH -0.020 -0.148 0.146 0.146 0.021 -0.126 0.306 ::J 0.. 0 0 :::!. PrJ -0.193 0.150 -0.141 -0.238 0.130 -0.290 -0.023 -0.108 ::J CD 0 0 lnhinin-8 -0.293 -0.227 -0.031 -0.158 0.124 -0.170 -0.052 -0.116 0.160 ::J 0 CD lnhibin-A 0.410 0.0005 0.227 0.126 -0.052 0.059 0.117 -0.092 0.079 0.068 ::J...... §. a· ::J *Progesterone (P), Oestradiol (E2) ,Testosterone (T), Sex Hormone Binding Globulin (SHBG), Prolactin (PrJ). (/) Table 5.1. Biological and biochemical markers of estrogen activity.
Placebo 40mg 160 mg Promensil™ Promensil™ Flushes -Week 0 8.6±4.6 6.9±2.1 9.0±5.2
(n) -Week 12 5.8±4.5 4.9±4.8 5.9±4.6
Greene Score -Week 0 18.5±11.4 19.9±10.6 19.9±14.4
-Week 12 9.9±5.9 11.2±8.8 14.7±16.8
FSH -Week 0 75.9±26.6 87.7±31.6 69.9±24.9
(IU/1) -Week 12 76.2±29.4 82.2±30.2 58.7±22.3
SHBG -Week 0 66.2±46.0 72.6±28.2 66.7±28.7
(IU/1) -Week 12 64.7±62.2 64.1±26.0 55.8±19.1
MV -Week 0 51.3±1.7 49.4±2.2 51.1±1.7
-Week 12 49.9±7.8 45.8±24.1 51.1±1.9
Vaginal pH -Week 0 5.3±0.8 5.4±0.7 5.1±0.6
-Week 12 5.4±0.7 5.1±0.9 5.0±0.8
Urinary isoflavone-Week 0 2.68±1.92 3.43±2.07 3.70±2.96
(ng/ml) -Week 12 3.67±2.79 9.40±5.67 28.18±17.52
HDL-chol -Week 0 1.08±0.31 1.05±0.40 1.06±0.80*
(mmol/1) -Week 12 1.13±0.28 1.24±0.49 1.19±0.40
*p<0.05
162 Table 6.1: Demographics of study population
n=24 Units Mean SEM
Age years 54.5 0.7
Years post-menopause years 4.7 0.7
Height em 165 1.0
Weight kg 71 2.8
Systolic BP mmHg 129.6 3.4
Diastolic BP mmHg 77.1 1.6
Hypertension n 6/24
Hypercholesterolaemic: n 3/24
History of smoking n 1/24
163 Table 6.2: Results for randomised study of 3 months placebo, 40mg and 160mg supplemental isoflavone, as well as results for
3 months 160mg isoflavone in a larger subset.
Parameter Baseline 3 months Baseline 3 months Baseline 3 months Baseline 3 months 160mg- Placebo 40mg 160mg open label
n 9 7 8 19
Systolic BP 126.1 (4.8) 126.2 (5.8) 135.7 (7.4) 137.1 (1 0.3) 128.1 (6.4) 123.4 (3.9) 130.0 (4.0) 128.8 (4.2)
Diastolic BP 73.9 (2.0) 76.8 (8.6) 79.3 (2.3) 81.7 (2.7) 78.8 (3.8) 78.1 (3.0) 77.7 (1.9) 76.5 (2.0)
Central 123.6 (4.9) 122.9 (6.3) 134.1 (7.7) 133.4 (9.2) 121.2 (4.5) 125.0 (6.8) 127.0 (4.3) 125.0 (4.4)
Systolic BP Table 6.2 (cont): Cardiac Indices; Results for randomised study of 3 months placebo, 40mg and 160mg supplemental isoflavone, as well as results for 3 months 160mg isoflavone in a larger subset.
Parameter Baseline 3 months Baseline 3 months Baseline 3 months Baseline 3 months 160mg- Placebo 40mg 160mg open label
n 9 7 8 19
Heart Rate 66.9 (2.1) 70.3 (4.5) 69.0 (2.4) 72.0 (1.4) 66.4 (4.5) 67.4 (5.0) 68.4 (2.1) 70.7 (2.4)
dP/dtmax 605.8 (55.3) 651.8 (82.3) 711.5 (58.5) 676.2 (80.2) 590.0 (60.3) 575.9 (48.7) 608.8 (32.0) 626.2 (31.5)
Ejection 325.5 (4.1) 323.0 (1 0.1) 326.7 (5.7) 319.0 (4.3) 332.4 (7.9) 324.6 (8.5) 326.3 (3.9) 323.7 (4.7) Duration Table 6.2 (cant): Arterial Indices; Results for randomised study of 3 months placebo, 40mg and 160mg supplemental isoflavone, as well as results for 3 months 160mg isoflavone in a larger subset.
Parameter Baseline 3 months Baseline 3 months Baseline 3 months Baseline 3 months 160mg- Placebo 40mg 160mg open label
n 9 7 8 19
~ugmentatio 30.9 (2.5) 23.5 (2.4)* 27.4 (1.8) 25.4 (2.3) 24.7 (6.6) 23.6 (4.0) 27.4 (4.0) 26.4 (2.1) nlndex
Aortodorsalis 8.8 (1.0) 8.9 (0.7) 10.0 (1.3) 9.6 (1.3) 8.4 {0.5) 8.3 (0.3) 9.0 (0.3) 8.9 (0.3) PWV
Brachial 9.2 (1.3) 9.2 (0.7) 9.5(1.1) 9.5 (1.0) 8.4 (0.3) 8.8 (0.3) 9.0 (0.3) 9.2 (0.3) PWV Table 7.1. lsoflavone analysis of TakeCare™.
All Forms* mg/g mg/g Aglycone mg/g mg/g product protein components product protein Genistein 1.42 2.09 Genistein 0.82 1.21 compounds Daidzein 0.73 1.08 Daidzein 0.42 0.61 compounds Glycetein 0.09 0.13 Glycetein 0.05 0.08 compounds TOTAL 2.24 3.30 Total Aglycone 1.29 1.90 ISOFLAVONES Compounds * Aglycones, Glycosides, Glycoside esters
168 Table 7.2. Biological and biochemical markers of estrogen activity, serum
lipid levels, markers of bone turnover data and urinary total isoflavone
levels.
TakeCare™ (n=9) Placebo (n=11)
WeekO Week 12 WeekO Week 12
Flushes (no.) 50.2±13.6 29.1±42.5 56.2±26.5 45.5±31.3
Greene Score 18.7±8.3 7.7±5.6 19.4±1 0.6 10.7±7.0
FSH (IU/1) 74.7±30.9 67.4±40.7 74.3±24.3 63.9±26.7
SHBG (IU/1) 44.8±18.0 46.3±25.1 65.4±29.8 57.7±25.1
TBG (IU/1) 291.7±55.6 268.7±33.1 254.1±74.3 234.1±77.3
Mat 1n Value 162.7±126.3 208.6±166.0 127.5±69.8 159.8±118.4
ALP 21.9±10.4 25.3±12.6 19.0±4.7 21.7±5.3 x-links (IU/1) 7.5±2.5 7.0±2.3 7.2±3.5 6.4±1.3 gla-protein 6.3±2.5 6.0±2.1 5.7±1.8 6.0±2.0
Total isoflavone 0.7±1.2 18.2±11.5 0.5±0.7 0.3±0.5 (ng/ml)
169 Table 8.1. Mean results for bone turnover markers from Week 0 to Week
20. (mean (SEM))
WeekO Week 10 Week20
b-ALP 19.3 (1.0) 19.8 (1.3) 21.5 (1.7)
Osteocalcin 5.8 (0.3) 6.0 (0.4) 6.2 (0.4) (ng/ml) ICTP 55.2 (5.2) 69.4 (12.4) 67.1 (8.2) u-PCL (IU/1) 54 (4.8) 63 (8.7) 66 (5.9)
170 Table 8.2. Simple correlation between %change in bone marker and
urinary isoflavone excretion. Nine women in each group are presented,
those having the highest initial bone marker levels. Different women are
included in each group as they varied in the bone marker excretion at
presentation.
DAID GEN 0-dma FORM 810-A Total
b-ALP -0.54 -0.15 0.40 -0.30 -0.25 -0.45
Osteocalcin -0.78* -0.42 -0.35 -0.31 -0.20 -0.61 (ng/ml) ICTP 0.28 -0.14 0.69t 0.22 0.14 0.12
u-PCL -0.04 -0.06 0.63t -0.48 -0.53 -0.22
(IU/1)
* p<0.05 t p approaches 0.05
171 References
1. Academic Press Dictionary of Science and Technology, CD-ROM
Version 1.0. Academic Press ISBN: 0122004019, 1996.
2. Adams NR. Pathological changes in the tissues of ewes with clover
disease. J Comp Pathol 1976; 86: 29-35.
3. Adams NR. Cervical mucus and reproductive efficiency in ewes after
exposure to estrogenic pastures. Aust J Agric Res 1977; 28: 481-489.
(a)
4. Adams NR. Morphological changes in the organs of ewes grazing
estrogenic subterranean clover. Res Vet Sci 1977; 22: 216-221. (b)
5. Adams NR. Detection of the effects of phytoestrogens on sheep and
cattle. J Anim Sci 1995 May;73(5):1509-15.
6. Adlercreutz H, Hockerstedt K, Bannwart C, Bloigu S, Hamalainen E,
Fotsis T, Ollus A. Effect of dietary components, including lignans and
phytoestrogens, on enterohepatic circulation and liver metabolism of
estrogens and on sex hormone binding globulin (SHBG). J Steroid
Biochem 1987;27:4-6: 1135-44.
173. 7. Adlercreutz H, Hockerstedt K, Bannwart C. Associations between
dietary fibre, urinary excretion of lignans and isoflavonic
phytoestrogens and plasma non-protein bound sex hormones in
relation to breast cancer. In; King RJB, Upperman ME, Raynaud J-P.
eds Progress in cancer research and therapy: hormones and cancer.
Vol3. New York, Raven Press. 1988,409-412.
8. Adlercreutz H. Diet, breast cancer and sex hormone metabolism. Ann
NY Acad Sci 1990;595:281-90. (a)
9. Adlercreutz H. Western diet and Western diseases: some hormonal
and biochemical mechanisms and associations. Scand J Clin Lab
Invest 1990;201 :83-823. (b)
10. Adlercreutz H, Honjo H, Higashi A, Fotsis T, Hamalainen E, Hasegawa
T, Okada H. Urinary excretion of lignans and isoflavonoid
phytoestrogens in Japanese men and women consuming traditional
Japanese diet. Am J Clin Nutr 1991 ;54:1 093-110.
11.Adlercreutz H Hamalainen E, Gorbach S, Goldin B. Dietary
phytoestrogens and the menopause in Japan. [letter] Lancet
1992;339: 1233.
174. 12. Adlercreutz H, Fotsis T, Watanabe S, Lampe J, Wah ala K, Makela T,
Hase T. Determination of lignans and isoflavonoids in plasma by
isotope dilution gas chromatography-mass spectometry. Cancer Detect
Prevent 1994;18:259-271.
13.Agnusdei D, Gennari C, Bufalino L. Prevention of early
postmenopausal bone loss using low doses of conjugated estrogens
and the non-hormonal, bone-active drug ipriflavone Osteoporos lnt
1995;5:462-6.
14.Akiyama T, Ishida J, Nakagawa S, Ogawara H, Watanabe S, ltoh N,
Shibuya M, Fukami Y. Genistein: a specific inhibitor of tyrosine-specific
protein kinase. J Bioi Chern 1987;262:5592-5.
15.Aibertazzi P, Pansini F, Bonaccorsi G, Zanotti L, Forini E, De Aloysio
D. The effect of dietary soy supplementation on hot flushes. Obstet
Gynecol1998;91:6-11.
16.Aibertazzi P, Pansini F, Bottazzi M, Bonaccorsi G, De Aloysio D,
Morton MS. Dietary soy supplementation and phytoestrogen levels.
Obstet Gynecol 1999;94:229-31.
175. 17.Aipert Ll. Vena-occlusive disease of the liver associated with oral
contraceptives: case report and review of literature. Hum Pathol
1976;7:709-718.
18.Anderson JJ, Ambrose WW, Garner SC. Orally dosed genistein from
soy and prevention of cancellous bone loss in two ovariectomized rat
models [Abs]. J Nutr 1995;125:7998.
19.Anderson JW, Johnstone BM, Cook-Newell ME. Meta-analysis of the
effects of soy protein intake on serum lipids. N Eng J Med
1995;333:276-82.
20. Anderson RL, Wolf JW. Compositional changes in trypsin inhibitors,
phytic acid, saponins and isoflavones related to soybean processing. J
Nutr 1995;125:5818-5888.
21.Anthony MS, Clarkson TB, Hughes CL Jr, Morgan TM, Burke GL.
Soybean isoflavones improve cardiovascular risk factors without
affecting the reproductive system of peripubertal rhesus monkeys. J
Nutr. 1996;126:43-50.
176. 22. Anthony MS, Clarkson TB, Williams JK. Effects of soy isoflavones on
atherosclerosis: potential mechanisms. Am J Clin Nutr 1998;68:13908-
13938.
23.Arjmandi BH, Alekel L, Hollis BW, Amin D, Stacewicz-Sapuntzakis M,
Guo P, Kukreja SC. Dietary soybean protein prevents bone loss in an
ovariectomised rat model of osteoporosis. J Nutr 1996;126:161-7.
24.Arjmandi BH, Getlinger MJ, Goyal NV, Alekel L, Hasler CM, Juma S,
Drum ML, Hollis BW, Kukreja SC. Role of soy protein with normal or
reduced isoflavone content in reversing bone loss induced by ovarian
hormone deficiency in rats. Am J Clin Nutr 1998;68:13588-13638.
25.Axelson M, Setchell KDR. The excretion of lignans in rats -evidence
for an intestinal bacterial source for this new group of compounds.
FEBS Lett 1981 ;123:337-342.
26.Axelson M, Sjovall J, Gustafsson BE, Setchell KDR. Soya- a dietary
source of the non-steroidal estrogen equal in man and animals. J
Endocrinol 1984; 102:49-56.
177. 27. Baber RJ, Templeman C, Moreton T, Kelly GE, West L. Randomised
placebo-controlled trial of an isoflavone supplement and menopausal
symptoms in women. Climacteric 1999;2:85-92.
28. Baird DO, Umbach OM, Lansdell L, Hughes CL, Satchell KD, Weinberg
CR, Haney AF, Wilcox AJ, Mclachlan JA. Dietary intervention study to
assess estrogenicity of dietary soy among postmenopausal women. J
Clin Endocrinol Metab 1995;80: 1685-90.
29. Bannwart C, Adlercreutz H, Wahala K, Brunow G, Hase T. lsoflavonic
phytoestrogens in humans, identification and metabolism. Eur J Cancer
Clin Oncol1987;23:1732.
30. Bannwart C, Adlercreutz H, Wahala K, Brunow G, Hase T. Detection
and identification of the plant lignans lariciresinol, isolariciresinol and
secoisolariciresinol in human urine. Clin Chim Acta 1989;180:293-302.
31. Barnes S, Sfakianos J, Coward L, Kirk M. Soy isoflavonoids and
cancer prevention. Underlying biochemical and phamacological issues.
Adv Exp Med Bio11996;401:87-100.
32. Baum JA, Teng H, Erdman JW Jr, Weigel RM, Klein BP, Persky VW,
Freels S, Surya P, Bakhit RM, RamosE, Shay NF, Potter SM. Long-
178. term intake of soy protein improves blood lipid profiles and increases
mononuclear cell low-density-lipoprotein receptor messenger RNA in
hypercholesterolemic, postmenopausal women. Am J Clin Nutr
1998;68:545-51.
33. Bennett D, Dudzinski ML. Bioassay responses of ewes to legume
swards I. Uterine weight response: variability, calibration, and
prediction. Aust J Agric Res 1967; 18: 485-494.
34. Bennett G, Beaumont WH, Brown PR. Use of the anabolic agent
zearanol (resorcylic acid lactone) as a growth promoter for cattle. Vet
Rec 1974;94:235-9.
35. Bennetts HW, Underwood EJ, Shier FL. A specific breeding problem of
sheep on subterranean clover pastures in western Australia. Aust Vet J
1946; 22: 2-12.
36. Bingham SA, Atkinson C, Liggins J, Bluck L, Coward A. Phyto
estrogens: where are we now? Br J Nutr 1998;79:393-406.
37. Boriello SP, Setchell KDR, Axelson M, Lawson AM. Production and
metabolism by human fecal flora. J Appl Bacteriol1985;58:37-43.
179. 38. Boulet MJ, Oddens BJ, Lehert P, Verner HM, Visser A. Climacteric and
menopause in seven south-east Asian countries. Maturitas
1994;19:157-176.
39. Boulton T J. Nutrition in children. Acta Paed Scan 1991 ;284:S20-S46.
40. Braden AWH, Thain Rl, Schutt DA. Comparison of plasma phyto
estrogen levels in sheep and cattle after feeding on fresh clover. Aust J
Agric Res 1971 ;22:663-70.
41. Brandi ML. Flavonoids: biochemical effects and therapeutic
applications. Bone Miner 1992;19:Suppl:3-14.
42. Cantley LC, Auger KR, Carpenter C, Duckworth B, Graziani A, Kapeller
R, Soltoff S. Oncogenes and signal transduction. Cell 1991 ;64:281-
302.
43. Carroll KK. Review of clinical studies on cholesterol-lowering response
to soy protein. JAm Diet Assoc 1991 ;91 :820-7.
44. Cassidy A, Bingham S, Satchell KDR. Biological effects of a diet of soy
protein rich in isoflavones on the menstrual cycle of premenopausal
women. Am J Clin Nutr 1994;60:333-340.
180. 45. Chasan-Taber L, Willett WC, Manson JE, Spiegelman D, Hunter OJ,
Curhan G, Colditz GA, Stampfer MJ. Prospective study of oral
contraceptives and hypertension among women in the United States.
Circulation 1996;94:483-389.
46. Cline JM, Paschold JC, Anthony MS, Obasanjo 10, Adams MR. Effects
of hormonal therapies and dietary soy phytoestrogens on vaginal
cytology in surgically postmenopausal macaques. Fertil Steril
1996;65:1 031-5.
47. Constantinou A, Huberman E. Genistein as an inducer of tumor cell
differentiation: Possible mechanisms of action. Proc Soc Exp Bioi Med
1995;208: 1 09-15.
48. Cooper C, Campion G, Melton LJ Ill. Hip fractures in the elderly: A
world-wide projection. Osteoporos lnt 1992;2:285-9.
49. Coward L, Barnes NC, Satchell KDR, Barnes S. Genistein, daidzein
and their B-glycoside conjugates: antitumour isoflavones in soybean
foods from American and Asian diets. J Agric Food Chern
1993;41 :1961-1967.
181. 50. Coward L, Smith M, Kirk M, Barnes S. Chemical modification of
isoflavones in soyfoods during cooking and processing. Am J Clin Nutr
1998;68:1486S-1491 s.
51. Cruz ML, Wong WW, Mimouni F, Hachey OL, Satchell KO, Klein PO,
Tsang RC. Effects of infant nutrition on cholesterol synthesis rates.
Pediatr Res 1994;35: 135-40.
52.Cummings SR, Black Om, Rubin SM. Lifetime risks of hip, Colles', or
vertebral fracture and coronary heart disease amongst white
postmenopausal women. Arch Intern Med 1989;149:2445-8.
53. Curnow OH. Estrogenic activity of subterraneum clover. 2. The
isolation of genistein from subterraneum clover and methods of
quantitative estimation. Biochem J 1954;58:283-287.
54. Oehennin L, Reiffsteck A, Joudet M, Thibier M. Identification and
quantitative estimation of a lignan in human and bovine semen. J
Reprod Fert 1982;66:305-309.
55. Oelais FS, Rice GE, Wahlqvist ML et al. Effects of dietary
phytoestrogens in postmenopausal women. Climacteric 1998;1 :124-9.
182 56. Draper MW, Flowers DE, Huster WJ, Neild JA, Harper KD, Arnaud C.
A controlled trial of raloxifene (L Y139481) HCI: Impact on bone
turnover and serum lipid profile in healthy postmenopausal women. J
Bone Miner Res 1996; 11 :835~842.
57. Drzewiecki GM, Melbin J, Noordergraaf A. Arterial tonometry: review
and analysis. J Biomechanics 1983; 16:141 ~ 153.
58. Dwyer JT, Goldin BR, Saul N, Gualtieri I, Barakat S, Adlercreutz H.
Tofu and soy drinks contain phytoestrogens. J Am Diet Assoc
1994;94:739~743.
59. Duncan AM, Underhill KE, Xu X, Lavalleur J, Phipps WR, Kurzer MS.
Modest hormonal effects of soy isoflavones in postmenopausal
women. J Clin Endocrinol Metab. 1999;84:3479~84.
60. Eisenberg DM, Kessler RC, Foster C, Norlock FE, Calkins DR,
Delbanco TL. Unconventional medicine in the United States.
Prevalence, costs and patterns of use. N Engl J Med 1993;328:246~52.
61. Ekins RK. The estimation of thyroxine in human plasma by an
electrophoretic technique. Clin Chim Acta 1960;5:453~459.
183. 62. Ekins RP. Radioimmunoassay and saturation analysis. Basic principles
and theory .Br Med Bull. 1974;30:3-11.
63. Eldridge AC. Determination of isoflavones in soybean flours, protein
concentrates and isolates. J Agric Food Chern 1982;30:353-355.
64. Eldridge AC, Kwolek WF. Soybean lsoflavones: Effect of environment
and variety on composition. J Agric Food Chern 1983;31 :394-396.
65. Fanti P, Monier-Faugere MC, Geng Z, Schmidt J, Morris PE, Cohen D,
Malluche HH. The phytoestrogen genistein reduces bone loss in short
term ovariectomized rats. Osteoporos lnt 1998;8:274-81.
66. Farnsworth NR, Bingel AS, Cordell GA et al. Potential value of plants
as sources of new antifertility agents, II. J Pharm Sci 1975;64:717-54.
67. Finlay EMH, Wilson D, Adlercreutz H, Griffiths K. The identification and
measurement of "phyto-estrogens" in human saliva, plasma, breast
aspirate or cyst fluid, and prostatic fluid using gas chromatography
mass spectrometry. [abs] J Endocrinol1991 ;129:49S.
68. Foman SJ, Bell EF. Chapter 7 Energy, In: Nutrition of Normal Infants,
Ed; Foman SJ Mosby, StLouis, 1993, 103-120.
184. 69. Foth D, Cline JM. Effects of mammalian and plant estrogens on
mammary glands and uteri of macaques. Am J Clin Nutr
1998;68:1413S-1417S.
70. Fotsis T, Pepper M, Adlercreutz H, Fleischmann G, Hase T,
Montesano R, Schweigerer L. Genistein, a dietary-derived inhibitor of
in vitro angiogenesis. Proc Nat Acad Sci USA 1993;90:2690-4.
71. Franke AA, Custer LJ, Cerna CM, Narala K. Rapid HPLC analysis of
dietary phytoestrogens from legumes and from human urine. Proc Soc
Exp Bioi Med 1995;208: 18-26.
72. Franke AA, Custer LJ, Tanaka Y. lsoflavones in human breast milk and
other biological fluids. Am J Clin Nutr 1998;68:1466S-1473S.
73. Gambacciani M, Spinetti A, Cappagli 8, Taponeco F, Felipetto R,
Parrini D, Cappelli N, Fioretti P. Effects of ipriflavone administration on
bone mass and metabolism in ovariectomized women. J Endocrinol
Invest 1993;16:333-337.
74. Gambacciani M, Spinetti A, Piaggesi L, Cappagli B, Taponeco F,
Manetti P, Weiss C, Teti GC, La Commare P, Facchini V. lpriflavone
185. prevents the bone mass reduction in premenopausal women treated
with gonadotropin hormone~releasing hormone agonists. Bone Miner
1994;26: 19~26.
75. Gangrade BK, Davis JS, May JV. A novel mechanism for the induction
of aromatase in ovarian cells in vitro: Role of transforming growth factor
alpha~induced protein tyrosine kinase. Endocrinology 1991; 129:2790~
2.
76. Garnero P, Hausherr E, Chapuy MC, Marcelli C, Grand jean H, Muller
C, Cormier C, Breart G, Meunier PJ, Delmas PD. Markers of bone
resorption predict hip fracture in elderly women: the EPIDOS
Prospective Study. J Bone Miner Res 1996;11:1531~8.
77. Greene JG. A factor analytic study of climacteric symptoms. J
Psychosom Res 1976;20:425~30.
78. Gustafsson JA. Therapeutic potential of selective estrogen receptor
modulators. Curr Opin Chern Bioi 1998;2:508~ 11.
79. Hahn RG. Compliance considerations with estrogen replacement:
Withdrawal bleeding and other factors. Am J Obstet Gynecol
1989;161 :1854~1858.
186. 80. Hartman PE, Shankel DM. Antimutagens and anticarcinogens: A
survey of putative interceptor molecules. Environ Mol Mutagenesis
1990; 15:145-82.
81. Hertog MGL, Kromhout D, Aravanis C, Blackburn H, Buzina R, Fidanza
F, Giampaoli S, Jansen A, Menotti A, Nedeljkovic S, et al. Flavonoid
intake and long-term risk of coronary heart disease and cancer in the
seven countries study. Arch Intern Med 1995; 155:381-386.
82. Hirano T, Oka K, Akiba M. Antiproliferative activity of synthetic and
naturally occurring flavonoids on tumour cells of the human breast
carcinoma cell line, ZR-75-1. Res Commun Chern Pathol Pharmacol
1989;64:69-78.
83. Hirano T, Fukuoka K, Oka K, Naito T, Hosaka K, Mitsuhashi H,
Matsumoto Y. Antiproliferative activity of mammalian lignan derivatives
against the human breast carcinoma cell line, ZR-75-1 . Cancer Invest
1990;8:595-602.
84.Hodgson JM, Puddey IB, Beilin LJ, Mori TA, Croft KD.
Supplementation with isoflavonoid phytoestrogens does not alter
187. serum lipid concentrations: a randomized controlled trial in humans. J
Nutr 1998;128:728-32.
85. Honore EK, Williams JK, Anthony MS, Clarkson TB. Soy isoflavones
enhance coronary vascular reactivity in atherosclerotic female
macaques. Fertil Steril 1996;67: 148-154.
86. Howes JB, Eden JA, Howes L. Acute and chronic pharmacokinetics of
daidzein and genistein in man. [abs] St George Hospital Scientific
Symposium, Sydney, 1997.
87. Howanitz JH, Howanitz PJ. Ch 13; Radioimmunoassay and related
techniques. In; Henry JB (ed), Clinical Diagnosis and Management by
Laboratory Methods; Vol 1, 16 edition. Philadelphia, WB Saunders Co.
1979;385-386.
88. Hughes CL Jr. Effects of phytoestrogens on GnRH-induced luteinizing
hormone secretion in ovariectomized rats. Rep rod Toxicol, 1987-
88;1 :179-81.
89. Hughes Jr, CL. Phytochemical mimicry of reproductive hormones and
modulation of herbivore fertility by phytoestrogens. Environ Health Pers
1988;78:171-175.
188. 90. Hughes CL Jr, Kaldas RS, Weisinger AS, McCants CE, Basham KB.
Acute and subacute effects of naturally occurring estrogens on
luteinizing hormone secretion in the ovariectomized rat: Part 1. Reprod
Toxicol, 1991;5:127-32.
91. Hulley S, Grady D, Bush T, Furberg C, Herrington D, Riggs B,
Vittinghoff E. Randomized trial of estrogen plus progestin for secondary
prevention of coronary heart disease in postmenopausal women. Heart
and Estrogen/progestin Replacement Study (HERS) Research Group.
JAMA. 1998;280:605-13.
92. Humfrey CD. Phytoestrogens and human health effects: weighing up
the current evidence. Nat Toxins 1998;6:51-9.
93. Hunter WM, Greenwood FC. Preparation of iodine 1131 labelled human
growth hormone of high specific activity. Nature 1962;194:495-496.
94. Husband AJ, Howes JB, Knight DC, Eden JA. The correlation between
phenolic estrogen levels and menopause symptoms in women. [abs]
EEC, COST-916 Phytoestrogens Meeting, Amsterdam, April, 1998.
189. 95.1shida H, Uesugi T, Hirai K, Toda T, Nukaya H, Yokotsuka K, Tsuji K.
Preventive effects of the plant isoflavones, daidzin and genistin, on
bone loss in ovariectomized rats fed a calcium-deficient diet. Bioi
Pharm Bull 1998;21 :62-6.
96.1smael NN. A study of menopause in Malaysia. Maturitas 1994; 19:
205-9.
97.Jha HC, Von Recklinghausen G, Zilliken F. Inhibition of in vitro
microsomal lipid peroxidation by isoflavonoids. Biochem Pharmacal
1985;34:1367-9.
98. Jordan VC, Koch R, Bain RR. Prolactin synthesis by cultured rat
pituitary cells: An assay to study estrogens, antiestrogens and their
metabolites in vitro. In: McLachlan JA, ed. Estrogens in the
environment Vol II. New York: Elsevier, 1985:221-37.
99.lrvine C, Fitzpatrick M, Robertson I, Woodhams D. The potential
adverse effects of soybean phytoestrogens in infant feeding[letter].
New Z Med J 1995;108:208-209.
100. Jenkins OJ, Kendall CW, Vidgen E, Agarwal S, Rao AV, Rosenberg
RS, Diamandis EP, Novokmet R, Mehling CC, Perera T, Griffin LC,
190. Cunnane SC. Health aspects of partially defatted flaxseed, including
effects on serum lipids, oxidative measures, and ex vivo androgen and
progestin activity: a controlled crossover trial. Am J Clin Nutr
1999;69:395-402.
101. Kaldas R, Hughes Jnr CL. Reproductive and general metabolic
effects of phytoestrogens in mammals. Reprod Toxicol 1989;3:81-9.
102. Kalu ON, Masoro EJ, Yu BP, Hardin RR, Hollis BW. Modulation of
age-related hyperparathyroidism and senile bone mass in Fischer rats
by soy protein and food restriction. Endocrinology 1988;122:1847-54.
103. Kanis JA, Passmore R. Calcium supplementation of the diet: not
justified by present evidence. BMJ 1989;298:205-8.
104. Kardinaal AF, Morton MS, Bruggemann-Rotgans IE, van Beresteijn
EC. Phyto-estrogen excretion and rate of bone loss in postmenopausal
women. Eur J Clin Nutr 1998 Nov;52(11 ):850-5.
105. Karpanou EA, Vyssoulis GP, Papakyriakou SA, Toutouza MG,
Toutouzas PK. Effects of menopause on aortic root function
hypertensive women. JAm Coli Cardiol1996;28:1562-1566.
191. 106. Kelly GE, Joannou GE, Reeder AY, Nelson C, Waring MA. The
variable metabolic response to dietary isoflavones in humans. Proc
Soc Exp Bioi Med 1995;208:40-43.
107. Kelly RP, Hayward CS, Avolio AP, o•Rourke MF. Non-invasive
determination of age-related changes in the human arterial pulse.
Circulation 1989;80:1652-1659.
108. Kelly RP, Gibbs HH, Morgan JJ, Daley JE, Mang K, Avolio AP,
o·Rourke MF. Nitroglycerine has more favourable effects on left
ventricular afterload than apparent from measurement of pressure in a
peripheral artery. European HeartJ 1990;11:138-144.
109. Kim CJ, Jang HC, Cho DH, Min YK. Effects of hormone
replacement therapy on lipoprotein (a) levels and lipids in
postmenopausal women. Arerioscler Thromb 1994;14:275-81.
11 0. King RA, Bursill DB. Plasma and urinary kinetics of the isoflavones
daidzein and genistein after a single soy meal in humans. Am J Clin
Nutr 1998;67:867-72.
192 111. Knuckles BE, de Fremery D, Kohler GO. Coumestrol content of of
fractions obtained from wet processing of alfalfa. J Agric Food Chern
1976;24:1177-1180.
112. Koss, LG. Cytological Evaluation of the Endocrine Status of the
Woman. In: Diagnostic cytology and its histopathological bases, Vol 1.
Philadelphia, JB Lippincott Company. 1979;208.
113. Kuiper GGJM, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson
JA. Cloning of a novel estrogen receptor expressed in rat prostate and
ovary. Proc Natl Acad Sci USA 1996;93:5925-5930.
114. Kuiper GG, Carlsson B, Grandien K, Enmark E, Haggblad J,
Nilsson S, Gustafsson JA. Comparison of ligand binding specificity and
transcript tissue distribution of estrogen receptor a and b.
Endocrinology 1997;138:863-70.
115. Kuiper GG, Lemmen JG, Carlsson B, Gorton JC, Safe SH, van der
Saag PT, van der Burg B, Gustafsson JA. Interaction of estrogenic
chemicals and phytoestrogens with estrogen receptor beta.
Endocrinology 1998; 139:4252-4263.
193. 116. Kurzer MS, Xu X. Dietary phytoestrogens. Annu Rev Nutr
1997;17:353-381.
117. Lamartiniere CA, Moore J, Holland M, Barnes S. Neonatal genistein
chemoprevents mammary cancer. Proc Soc Exp Bioi Med
1995;208: 120-3.
118. Lampe JW, Martini MC, Kurzer MS, Adlercreutz H, Slavin JL.
Urinary lignan and isoflavonoid excretion in premenopausal women
consuming flaxseed powder. Am J Clin Nutr 1994;60:122-128.
119. Laogun AA, Gosling RG. In vivo arterial compliance in man. Clin
Phys Physiol Meas 1982;3:201-212.
120. Leavitt WW, Wright PA. Effects of legumes on reproduction in mice.
J Rep rod Fert 1963;6: 115-123.
121. Leopold AS, Erwin M, Oh J, Browning B. Phytoestrogens: adverse
effects on reproduction in Californian quail. Science 1976; 191 :98-99.
122. Levy JR, Faber KA, Ayyash L, Hughes Jnr CL. The effect of
prenatal exposure to the phytoestrogen genistein on sexual
differentiation in rats. Proc Soc Exp Bioi Med 1995;208:60-6.
194. 123. Linassier C, Pierre M, Le Pecq JB, Pierre J. Mechanisms of action
in NIH-3T3 cells of genistein, an inhibitor of EGF receptor tyrosine
kinase activity. Biochem Pharmacol1990;39:187-93.
124. Lip GYH, Beevers M, Churchill D, Beevers DG. Hormone
replacement therapy and blood pressure in hypertensive women. J
Human Hypert 1994;8:491-494.
125. Loewe S, Lange F, Spohr E. Uber weiliche sexual hormones
(thelytropine). Biochem Zeitschr 1927;180:1-26.
126. Leopold AS, Erwin M, Oh J, Browning B. Phytoestrogens: adverse
effects on reproduction in Californian quail. Science 1976;191:98-100.
127. Lu LJ, Lin SN, Grady JJ, Nagamani M, Anderson KE. Altered
kinetics and extent of urinary daidzein and genistein excretion in
women during chronic soy exposure. Nutr Cancer 1996;26:289-302.
128. Lu LJ, Anderson KE, Grady JJ, Nagamani M. Effects of soy
consumption for one month on steroid hormones in premenopausal
women: implications for breast cancer risk reduction. Cancer Epidemiol
Biomarkers Prev 1996;5:63-70. (b)
195. 129. Lundh T J, Pettersson H, Kiessling KH. Liquid chromatographic
determination of the estrogens daidzein, formononetin, coumestrol, and
equol in bovine blood plasma and urine. J Assoc Off Anal Chern. 1988
Sep-Oct;71 (5):938-41.
130. Maclennan AH, Wilson DH, Taylor AW. Prevalence and costs of
alternative medicine in Australia. Lancet 1996;347:569-73.
131. MacRae WD, Hudson JB, Towers GHN. The antiviral action of
lignans. Planta Med 1989;55:531-5.
132. Markiewicz L, Garey J, Adlercreutz H, Gurpide E. In vitro bioassays
of non-steroidal phytoestrogens. J Steroid Biochem Melee Bioi
1993;45:399-405.
133. Martin PM, Horwitz KB, Ryan DS, McGuire WL. Phytoestrogen
interaction with estrogen receptors in human breast cancer cells.
Endocrinology 1978; 103:1860-7.
134. McCarthy T. The prevalence of symptoms in menopausal women in
the Far East: Singapore segment. Maturitas 1994; 19:199-204.
196. 135. Melton LJ. Hip fractures: a worldwide problem today and tomorrow.
Bone 1993;14:S1-S8.
136. Menotti A, Keys A, Blackburn H, Aravanis C, Dontas A, Fidanza F,
Giampaoli S, Karvonen M, Kromhout D, Nedeljkovic S, et al. Twenty
year stroke mortality and prediction in twelve cohorts of the Seven
Countries Study. lnt J Epidemiol1990;19:309-315.
137. Messina M, Messina V. Increasing use of soyfoods and their role in
cancer prevention. JAm Diet Assoc 1991 ;91 :836-40.
138. Messina MJ, Persky V, Setchell KDR, Barnes S. Soy intake and
cancer risk: A review of the in vitro and in vivo data. Nutr Cancer
1994;21 :113-31.
139. Messina M. lsoflavone intakes by Japanese were overestimated.
Am J Clin Nutr 1995;62:645.
140. Miksicek RJ. Estrogenic flavonoids: structural requirements for
biological activity. Proc Soc Exp Bioi Med 1995;208:44-50.
141. Morton MS. Determination of lignans and isoflavonoids in human
female plasma following dietary supplementation. J Endoc
1994; 142:251-259.
197. 142. Murgo JP, Westerhof N, Giolma JP, Altobelli S. Aortic impedance in
normal man: relationship to pressure waveforms. Circulation
1980;62:1 05-116.
143. Murkies AL, Lombard C, Strauss BJG, Wilcox G, Burger HG,
Morton MS. Dietary flour supplementation decreases post-menopausal
hot flushes: Effect of soy and wheat. Maturitas 1995;21 :189-195.
144. Nagata C, Takatsuka N, lnaba S, Kawakami N, Shimizu H. Effect of
soymilk consumption on serum estrogen concentrations in
premenopausal Japanese women. J Natl Cancer lnst 1998;90:1830-5.
145. Nairn M, Gestetner B, Zilkah S, Birk Y, Bondi A. Soybean
isoflavones. Characterization, determination, and antifungal activity. J
Agric Food Chern 1974;22:806-10.
146. National Research Council. Evidence on dietary components and
chronic diseases. In: Commission on Life Sciences, Food and Nutrition
Board, Committee on Diet and Health, eds. Diet and Health:
implications for reducing chronic disease risk. Washington DC:
National Academy Press. 1989;139-528.
198. 147. Nestel PJ, Yamashita T, Sasahara T, Pomeroy S, Dart A,
Komesaroff P, Owen A, Abbey M. Soy isoflavones improve systemic
arterial compliance but not plasma lipids in menopausal and
perimenopausal women. Arterioscler Thromb Vase Bioi 1997;17:3392-
8.
148. Nestel PJ, Pomeroy S, Kay S, Komesaroff P, Behrsing J, Cameron
JD, West L. lsoflavones from red clover improve systemic arterial
compliance but not plasma lipids in menopausal women. J Clin
Endocrinol Metab 1999;84:895-898.
149. Nilsson S, Mellbin T, Hofvander Y, Sundelin C, Valentin J, Nygren
KG. Long-term followup of children breastfed by mothers using oral
contraceptives. Contraception 1986;34:443-57.
150. Nishimura J, Huang JS, Duel TF. Platelet-derived growth factor
stimulates tyrosine-specific protein kinase activity in Swiss mouse 3T3
cells membranes. Proc Natl Acad Sci USA 1982;79:4303-6.
151. Oelsner G, Barnea ER, Mullen MV. Simultaneous measurements of
clomiphene citrate in plasma and follicular fluid in women undergoing
IVF & ET. Program, American Fertility Society, Abstract 39,1986.
199. 152. Okura A, Arakawa H, Oka H, Yoshinari T, Monden Y. Effect of
genistein on topoisomerase activity and on the growth of [val 12]Ha
ras-transformed NIH 3T3 cells. Biochim Biophys Res Commun
1988;157:183-9.
153. Packer AI, Hsu YC, Besmer P, Bachvarova RF. The ligand of the c
kit receptor promotes oocyte growth. Dev Bioi, 1994;161:194-205.
154. Petersen TG, Barnes S. Genistein inhibition of the growth of human
breast cancer cells: independence from estrogen receptors and the
multi-drug resistance gene. Biochem Biophys Res Commun
1991;179:661-7.
155. Petruzelli LM, Ganguly S, Smith CJ, Cobb MH, Rubin CS, Rosen
OM. Insulin activates a tyrosine-specific protein kinase in extracts of
3T3-L 1 adipocytes and human placenta. Proc Natl Acad Sci USA
1982;79:6792-6.
156. Phipps WR, Martini MC, Lampe JW, Slavin JL, Kurzer MS. Effect of
flax seed ingestion on the menstrual cycle. J Clin Endocrinol Metab
1993;77:1215-9.
200. 157. Potter SM. Soy protein and serum lipids. Curr Opin Lipidol
1996;4:260-4.
158. Potter SM, Baum JA, Teng H, Stillman RJ, Shay NF, Erdman JW
Jr. Soy protein and isoflavones: their effects on blood lipids and bone
density in postmenopausal women. Am J Clin Nutr 1998;68:1375S-
1379S.
159. Price KR, Fenwick GR. Naturally occurring estrogens in foods - a
review. Food Add Contam 1985;2:73-106.
160. Quella SK, Loprinzi CL, Barton Dl Knost JA, Sloan JA, LaVasseur
Bl, Swan D, Krupp KR, Miller KD, Novotny PJ. Evaluation of soy
phytoestrogens for the treatment of hot flashes in breast cancer
survivors: A North Central Cancer Treatment Group Trial. J Clin Oncol
2000; 18:1 068-7 4.
161. Radziszewski B. Zintersmchung uber Ydrobenzamid, amarin, und
lophin. Chern Ber 1877;1 0:70.
162. Reinli K, Block G. Phytoestrogen content of foods- a compendium
of literature values. Nutr Cancer 1996;26:123-148.
201. 163. Rekers H. Mastering the menopause. In: Burger H, Boulet M. eds.
A portrait of the menopause. Peak Ridge, New Jersey: The Parthenon
Publishing Group, 1991:23-43.
164. Report of a WHO Study Group. Assessment of fracture risk and its
application to screening for postmenopausal osteoporosis. Geneva,
Switzerland: WHO Technical Report Series 843. 1994:11-3.
165. Richards JS, Jahnsen T, Hedin L, Lifka J, Ratoosh S, Durica JM,
Goldring NB. Ovarian follicular development: from physiology to
molecular biology. Recent Prog Horm Res 1987;43:231-76.
166. Rubin JB, Shia MA, Pilch PF. Stimulation of tyrosine-specific
phosphorylation in vitro by insulin-like growth factor I. Nature
1983;305:438-40.
167. Saba PS, Roman MJ, Pini R, Spitzer M, Ganau A, Devereux RB.
Relation of arterial pressure waveform to left ventricular and carotid
anatomy in normotensive subjects. JAm Coli Cardiol1993;22:1873-80.
168. Sacks FM, Kass EH. Low blood pressure in vegetarians: effects of
specific foods and nutrients. Am J Clin Nutr 1988;48:S795-S800.
202 169. Saloniemi H, Wahala K, Nykanen-Kurki P, Kallela K, Saastamoinen
I. Phytoestrogen content and estrogenic effect of legume fodder. Proc
Soc Exp Bioi Med 1995;208:13-17.
170. Sarrel PM. Hormone replacement therapy in the menopause. lnt J
Fertil1997;42:78-84.
171. Sato M, Glasebrook AL, Bryant HU. Raloxifene: a selective
estrogen receptor modulator. J Bone Miner Metab 1994; 12:89-820.
172. Scambia G, Mango D, Signorile PG, Anselmi Angeli RA, Palena C,
Gallo D, Bombardelli E, Morazzoni P, Riva A, Mancuso S. Clinical
effects of a standardized soy extract in postmenopausal women: a pilot
study. Menopause 2000;7:1 05-11.
173. Schoental R. Precocious sexual development in Puerto Rico and
estrogenic mycotoxins. Lancet. 1983;1 (8323):537.
174. Schroeder RR, Vogelhut PO, Carrico RJ, Bogulaski RC, Buckler
RT. Competitive binding assay for biotin monitored by
chemiluminescence. Anal Chern 1976;48: 1933-7.
203. 175. Schwid HA, Taylor LA, Smith NT. Computer model analysis of the
radial artery pressure wave. J Clin Monitor 1987;3:220-228.
176. Seely S. The possible connection between phytoestrogens, milk
and coronary heart disease. Med Hypoth 1982;8:349-354.
177. Setchell KOR, Lawson AM, Mitchell FL, Adlercreutz H, Kirk ON,
Axelson M. Lignans in man and in animal species. Nature
1980;287:740-7 42.
178. Setchell KO, Lawson AM, Conway E, Taylor NF, Kirk ON, Cooley G,
Farrant RO, Wynn S, Axelson M. The definitive identification of the
lignans trans-2,3-bis(3-hydroxybenzyl)-gamma-butyrolactone and 2,3-
bis(3-hydroxybenzyl)butane-1 ,4-diol in human and animal urine.
Biochem J 1981 ;197:447-58. (a)
179. Setchell KOR, Lawson AM, Borriello SP, Harkness R, Gordon H,
Morgan OM, Kirk ON, Adlercreatz H, Anderson LC, Axelson M. Lignan
formation in man-microbial involvement and possible role in cancer.
Lancet 1981 ;ii:4-7. (b)
204. 180. Setchell KDR, Lawson AM, Borriello SP, Adlercreutz H, Axelson M.
Formation of lignans by intestinal microflora. In Malt RA, Williamson
RCN Eds. Colonic Carcinogenesis Lancaster, UK:MTP, 1982,93-97.
181. Setchell KDR, Gosselin SJ, Welsh MB, et al. Dietary estrogens- a
probable cause of infertility and liver disease in captive cheetah.
Gastroenterol 1987;93:225-223. (a}
182. Setchell KD, Welsh MB, Lim CK. High-performance liquid
chromatographic analysis of phytoestrogens in soy protein
preparations with ultraviolet, electrochemical and thermospray mass
spectrometric detection. J Chromatogr 1987;386:315. (b)
183. Setchell KDR. Adlercreutz H. Mammalian Lignans and Phyto
estrogens. Recent studies on their Formation, Metabolism and
Biological Role in Health and Disease. In: Rowland IR, ed. Role of the
Gut Flora in Toxicity and Cancer, New York: Academic Press Limited,
1988:315-345.
184. Sharma RD. lsoflavone content of Bengalgram (Cicer arietinum) at
various stages of germination. J Plant Foods 1981 ;3:259-264.
205. 185. Shewmon DA, Stock JL, Rosen CJ, Heiniluoma KM, Hogue MM,
Morrison A, Doyle EM, Ukena T, Weale V, Baker S. Tamoxifen and
estrogen lower circulating lipoprotein (a) concentrations in healthy
postmenopausal women. Arterioscler Thromb 1994;14:1586-93.
186. Shutt 01, Cox Rl. Steroid and phytoestrogen binding to sheep
uterine receptors in vitro. J Endocrinol 1972;52:299-31 0.
187. Simkus GJ, Fitchett DH. Radial arterial pressure measurements
may be a poor guide to the beneficial effects of nitroprusside on left
ventricular systolic pressure in congestive heart failure. Am J Cardiel
1990;66:323-326.
188. Smith DA, Banks SW. Formation and biological properties of
isoflavonoid phytoalexins. Prog Clin Bioi Res 1986;213:113-124.
189. Speroff L. Hormonal contraception. ln:Adashi EY, Rock JA,
Rosenwaks Z, eds. Reproductive Endocrinology, Sugery, and
Technology. Philadelphia: Lippincott-Raven Publishers, 1996, 1683-
1708.
190. Spinozzi F, Pagliacci MC, Migliorati G, Moraca R, Grignani F,
Riccardi C, Nicoletti I. The natural tyrosine kinase inhibitor genistein
206. produces cell cycle arrest and apoptosis in Jurkat T -leukaemia cells.
Leuk Res 1994;18:431-9.
191 . Stampfer MJ, Colditz GA. Estrogen replacement therapy and
coronary heart disease: a quantitative assessment of the
epidemiological evidence. Prev Med 1991 ;20:47 -63.
192. Strahler BL,Trotter JR. Firefly luminescence in the study of energy
transfer mechanisms. Arch Biochem Biophy. 1952;40:28-41.
193. Sturdee 0, Brincat M. The Hot Flush. In: Studd JWW, Whitehead Ml
eds. The Menopause. Oxford: Blackwell Scientific Publications.
1988;24-42.
194. Takeya Y, Popper JS, Shimizu Y, Kato H, Rhoads GG, Kagan A.
195. Epidemiologic studies of coronary heart disease and stroke in
Japanese men living in Japan, Hawaii and California: Incidence of
stroke in Japan and Hawaii. Stroke 1984; 15:15-23.
196. Stuting HH, Krull IS. Complete on-line determination of biopolymer
molecular weight via high-performance liquid chromatography coupled
to low-angle laser light scattering, ultraviolet, and differential refractive
index detection. Anal Chern 1990;62:2107-14.
207. 197. Takazawa K. A clinical study of the second component of left
ventricular systolic pressure. J Tokyo Med College 1987;45:256-270.
198. Takeya Y, Popper JS, Shimizu Y, Kato H, Rhoads GG, Kagan A.
Epidemiologic studies of coronary heart disease and stroke in
Japanese men living in Japan, Hawaii and California: Incidence of
stroke in Japan and Hawaii. Stroke 1984; 15:15-23.
199. Tang GWK. The climacteric of Chinese factory workers. Maturitas
1994;19:177-82.
200. Thomson BIOAdvantage™, Columns For Peptides, Proteins and
Biomolecules. http://www.hplc.com/Advantage, 2000}
201. Thompson LU, Robb P, Serraino M, Cheung F. Mammalian lignan
production from various foods. Nutr Cancer 1991 ;16:43-52.
202. United States of America Department of Agriculture. Agricultural
Research Service; Phytochemical database. http://www.ars
grin.gov/-ngrlsb/
208. 203. Upmalis DH, Lobo R, Bradley L, Warren M, Cone FL, Lamia CA.
Vasomotor symptom relief by soy isoflavone extract tablets in
postmenopausal women: a multicenter, double-blind, randomized,
placebo-controlled study. Menopause 2000;7:236-42.
204. Ushiro H, Cohen S. Identification of phosphotyrosine as a product
of epidermal growth factor-activated protein kinase in A-431 cell
membranes. J Bioi Chern 1980;255:8363-5.
205. Valente M, Bufalino L, Castiglione GN, o•Angelo R, Mancuso A,
Galoppi P, Zichella L. Effects of 1-year treatment with lpriflavone on
bone in postmenopausal women with low bone mass. Calcif Tissue lnt
1994;54:377-80.
206. Wagner JD, Cefalu WT, Anthony MS, Litwak KN, Zhang L, Clarkson
TB. Dietary soy protein and estrogen replacement therapy improve
cardiovascular risk factors and decrease aortic cholesteryl ester
content in ovariectomized cynomolgus monkeys. Metabolism
1997;46:698-705.
207. Wang H, Murphy PA. lsoflavone content in commercial soybean
foods. J Agric Food Chern 1994;42:1666-1673.
209. 208. Wang LY, Zhao AP, Chai XS. Effects of puerarin on cat vascular
smooth muscle in vitro. ACTA Pharmacal Sin 1994;15:180-182.
209. Wei H, Wei L, Frenkel K, Bowen R, Barnes S. Inhibition of tumour
promoter-induced hydrogen peroxide formation in vitro and in vivo by
genistein. Nutr Cancer 1993;20:1-12.
210. Wei H, Bowen R, Cai Q, Barnes S, Wang Y. Antioxidant and
antipromotional effects of the soybean isoflavone genistein. Proc Soc
Exp Bioi Med 1995;208:124-30.
211. Wen D, Peles E, Cupples R, Suggs SV, Bacus SS, Luo Y, Trail G,
Hu S, Silbiger SM, Levy RB, et al. Neu differentiation factor: A
transmembrane glycoprotein containing an EGF domain and an
immunoglobulin homology unit. Cell 1992;69:559-72.
212. Wilcox G, Wahlqvist ML, Burger HG, Medley G. Estrogenic effects
of plant foods in postmenopausal women. Brit Med J 1990;301 :905-6.
213. Williams JK, Clarkson TB. Dietary soy isoflavones inhibit in-vivo
constrictor responses of coronary arteries to collagen-induced platelet
activation. Coron Artery Dis 1998;9:759-64.
210. 214. Whitten PL, Lewis C, Russell E, Naftolin F. Potential adverse
effects of phytoestrogens. J Nutr 1995;125:771 S-6S.
215. Woodhead JS, Campbell AK, McCapra F, Beheshti T,.Weeks I.
Acridinium esters as high specific-activity labels in immunoassays. Clin
Chern 1983; 29/8: 1474-79.
216. Wu ES, Loch JT 3d, Toder BH, Borrelli AR, Gawlak D, Radov LA,
Gensmantel NP. Flavones. 3. Synthesis, biological activities, and
conformational analysis of isoflavone derivatives and related
compounds. J Med Cham 1992;35:3519-25.
217. Wyman JG, Van Etten HD. Antibacterial activity of selected
isoflavonoids. Phytopathology 1978;68:583-589.
218. Xu X, Duncan AM, Merz BE, Kurzer MS Effects of soy isoflavones
on estrogen and phytoestrogen metabolism in premenopausal women.
Cancer Epidemiol Biomarkers Prev 1998;7:1101-8.
219. Yalow RS, Berson SA. Immunoassay of endogenous plasma insulin
in man. J Clin Invest 1960;39: 1157-75.
211. 220. Yalow RS, Berson SA. Problems of validation of of considerations.
In; Odell WD, Daughaday WH (eds). Competitive Protein Binding
Assays. Philidelphia, JB Lippincott Co, 1971.
221. Yamashita T, Sasahara T, Pomeroy SE, Collier G, Nestel PJ.
Arterial compliance, blood pressure, plasma leptin, and plasma lipids in
women are improved with weight reduction equally with a meat-based
diet and a plant-based diet. Metabolism 1998;47:1308-1314.
222. Zava DT, Dollbaum CM, Bien M. Estrogen and progestin bioactivity
of foods, herbs, and spices. Proc Soc Exp Bioi Med 1998;217:369-378.
212. Ll~