Fermentation of calcium-fortified soya milk does not appear to enhance acute calcium absorption in osteopenic post-menopausal women

Ageing women may choose to drink soya milk to reduce menopausal symptoms. As fermentation enriches soya milk with isoflavone aglycones, its beneficial qualities may improve. To reduce osteoporotic risk, however, soya milk must be Ca enriched, and it is not known how fermentation affects Ca bioavailability. A randomised crossover pilot study was undertaken to compare the Ca absorption of fortified soya milk with that of fermented and fortified soya milk in twelve Australian osteopenic post-menopausal women. The fortified soya milk was inoculated with Lactobacillus acidophilus American Type Culture Collection (ATCC) 4962 and fermented for 24 h at 37°C. Ca absorption from soya milk samples was measured using a single isotope radiocalcium method. Participants had a mean age of 54·8 (sd 12·3) years, with mean BMI of 26·5 (sd 5·5) kg/m2 and subnormal to normal serum 25-hydroxyvitamin D (mean 62·5 (sd 19·1) nmol/l). Participants consumed 185 kBq of 45Ca in 44 mg of Ca carrier. The mean fractional Ca absorption (α) from soya milk and fermented soya milk was 0·64 (sd 0·23) and 0·71 (sd 0·29), respectively, a difference not of statistical significance (P = 0·122). Although fermentation of soya milk may provide other health benefits, fermentation had little effect on acute Ca absorption

Growth and body composition in pku children— a three-year prospective study comparing the effects of l-amino acid to glycomacropeptide protein substitutes

Protein quality and quantity are important factors in determining lean body (muscle) mass (LBM). In phenylketonuria (PKU), protein substitutes provide most of the nitrogen, either as amino acids (AA) or glycomacropeptide with supplementary amino acids (CGMP-AA). Body composition and growth are important indicators of long-term health. In a 3-year prospective study comparing the impact of AA and CGMP-AA on body composition and growth in PKU, 48 children were recruited. N = 19 (median age 11.1 years, range 5–15 years) took AA only, n = 16 (median age 7.3 years, range 5–15 years) took a combination of CGMP-AA and AA, (CGMP50) and 13 children (median age 9.2 years, range 5–16 years) took CGMP-AA only (CGMP100). A dual energy X-ray absorptiometry (DXA) scan at enrolment and 36 months measured LBM, % body fat (%BF) and fat mass (FM). Height was measured at enrolment, 12, 24 and 36 months. No correlation or statistically significant differences (after adjusting for age, gender, puberty and phenylalanine blood concentrations) were found between the three groups for LBM, %BF, FM and height. The change in height z scores, (AA 0, CGMP50 +0.4 and CGMP100 +0.7) showed a trend that children in the CGMP100 group were taller, had improved LBM with decreased FM and % BF but this was not statistically significant. There appeared to be no advantage of CGMP-AA compared to AA on body composition after 3-years of follow-up. Although statistically significant differences were not reached, a trend towards improved body composition was observed with CGMP-AA when it provided the entire protein substitute requirement.publishersversionpublishe

A Three-Year Longitudinal Study Comparing Bone Mass, Density, and Geometry Measured by DXA, pQCT, and Bone Turnover Markers in Children with PKU Taking L-Amino Acid or Glycomacropeptide Protein Substitutes

From MDPI via Jisc Publications RouterHistory: accepted 2021-06-09, pub-electronic 2021-06-17Publication status: PublishedFunder: Vitaflo International Ltd; Grant(s): 001In patients with phenylketonuria (PKU), treated by diet therapy only, evidence suggests that areal bone mineral density (BMDa) is within the normal clinical reference range but is below the population norm. Aims: To study longitudinal bone density, mass, and geometry over 36 months in children with PKU taking either amino acid (L-AA) or casein glycomacropeptide substitutes (CGMP-AA) as their main protein source. Methodology: A total of 48 subjects completed the study, 19 subjects in the L-AA group (median age 11.1, range 5–16 years) and 29 subjects in the CGMP-AA group (median age 8.3, range 5–16years). The CGMP-AA was further divided into two groups, CGMP100 (median age 9.2, range 5–16years) (n = 13), children taking CGMP-AA only and CGMP50 (median age 7.3, range 5–15years) (n = 16), children taking a combination of CGMP-AA and L-AA. Dual X-ray absorptiometry (DXA) was measured at enrolment and 36 months, peripheral quantitative computer tomography (pQCT) at 36 months only, and serum blood and urine bone turnover markers (BTM) and blood bone biochemistry at enrolment, 6, 12, and 36 months. Results: No statistically significant differences were found between the three groups for DXA outcome parameters, i.e., BMDa (L2–L4 BMDa g/cm2), bone mineral apparent density (L2–L4 BMAD g/cm3) and total body less head BMDa (TBLH g/cm2). All blood biochemistry markers were within the reference ranges, and BTM showed active bone turnover with a trend for BTM to decrease with increasing age. Conclusions: Bone density was clinically normal, although the median z scores were below the population mean. BTM showed active bone turnover and blood biochemistry was within the reference ranges. There appeared to be no advantage to bone density, mass, or geometry from taking a macropeptide-based protein substitute as compared with L-AAs

A three-year longitudinal study comparing bone mass, density, and geometry measured by DXA, pQCT, and bone turnover markers in children with PKU taking L-amino acid or glycomacropeptide protein substitutes

In patients with phenylketonuria (PKU), treated by diet therapy only, evidence suggests that areal bone mineral density (BMDa) is within the normal clinical reference range but is below the population norm. Aims: To study longitudinal bone density, mass, and geometry over 36 months in children with PKU taking either amino acid (L-AA) or casein glycomacropeptide substitutes (CGMP-AA) as their main protein source. Methodology: A total of 48 subjects completed the study, 19 subjects in the L-AA group (median age 11.1, range 5–6 years) and 29 subjects in the CGMP-AA group (median age 8.3, range 5–16years). The CGMP-AA was further divided into two groups, CGMP100 (median age 9.2, range 5–16years) (n = 13), children taking CGMP-AA only and CGMP50 (median age 7.3, range 5–15years) (n = 16), children taking a combination of CGMP-AA and L-AA. Dual X-ray absorptiometry (DXA) was measured at enrolment and 36 months, peripheral quantitative computer tomography (pQCT) at 36 months only, and serum blood and urine bone turnover markers (BTM) and blood bone biochemistry at enrolment, 6, 12, and 36 months. Results: No statistically significant differences were found between the three groups for DXA outcome parameters, i.e., BMDa (L2–L4 BMDa g/cm 2 ), bone mineral apparent density (L2–L4 BMAD g/cm 3 ) and total body less head BMDa (TBLH g/cm 2 ). All blood biochemistry markers were within the reference ranges, and BTM showed active bone turnover with a trend for BTM to decrease with increasing age. Conclusions: Bone density was clinically normal, although the median z scores were below the population mean. BTM showed active bone turnover and blood biochemistry was within the reference ranges. There appeared to be no advantage to bone density, mass, or geometry from taking a macropeptide-based protein substitute as compared with L-AAs

Neurocognitive outcome and mental health in children with tyrosinemia type 1 and phenylketonuria:A comparison between two genetic disorders affecting the same metabolic pathway

Tyrosinemia type 1 (TT1) and phenylketonuria (PKU) are both inborn errors of phenylalanine-tyrosine metabolism. Neurocognitive and behavioral outcomes have always featured in PKU research but received less attention in TT1 research. This study aimed to investigate and compare neurocognitive, behavioral, and social outcomes of treated TT1 and PKU patients. We included 33 TT1 patients (mean age 11.24 years; 16 male), 31 PKU patients (mean age 10.84; 14 male), and 58 age- and gender-matched healthy controls (mean age 10.82 years; 29 male). IQ (Wechsler-subtests), executive functioning (the Behavioral Rating Inventory of Executive Functioning), mental health (the Achenbach-scales), and social functioning (the Social Skills Rating System) were assessed. Results of TT1 patients, PKU patients, and healthy controls were compared using Kruskal-Wallis tests with post-hoc Mann-Whitney U tests. TT1 patients showed a lower IQ and poorer executive functioning, mental health, and social functioning compared to healthy controls and PKU patients. PKU patients did not differ from healthy controls regarding these outcome measures. Relatively poor outcomes for TT1 patients were particularly evident for verbal IQ, BRIEF dimensions "working memory", "plan and organize" and "monitor", ASEBA dimensions "social problems" and "attention problems", and for the SSRS "assertiveness" scale (all p value

Polygenic scores and onset of major mood or psychotic disorders among offspring of affected parents

Objective: Family history is an established risk factor for mental illness. The authors sought to investigate whether polygenic scores (PGSs) can complement family history to improve identification of risk for major mood and psychotic disorders. Methods: Eight cohorts were combined to create a sample of 1,884 participants ages 2–36 years, including 1,339 offspring of parents with mood or psychotic disorders, who were prospectively assessed with diagnostic interviews over an average of 5.1 years. PGSs were constructed for depression, bipolar disorder, anxiety, attention deficit hyperactivity disorder (ADHD), schizophrenia, neuroticism, subjective well-being, p factor, and height (as a negative control). Cox regression was used to test associations between PGSs, family history of major mental illness, and onsets of major mood and psychotic disorders. Results: There were 435 onsets of major mood and psychotic disorders across follow-up. PGSs for neuroticism (hazard ratio=1.23, 95% CI=1.12–1.36), schizophrenia (hazard ratio=1.15, 95% CI=1.04–1.26), depression (hazard ratio=1.11, 95% CI=1.01–1.22), ADHD (hazard ratio=1.10, 95% CI=1.00–1.21), subjective well-being (hazard ratio=0.90, 95% CI=0.82–0.99), and p factor (hazard ratio=1.14, 95% CI=1.04–1.26) were associated with onsets. After controlling for family history, neuroticism PGS remained significantly positively associated (hazard ratio=1.19, 95% CI=1.08–1.31) and subjective well-being PGS remained significantly negatively associated (hazard ratio=0.89, 95% CI=0.81–0.98) with onsets. Conclusions: Neuroticism and subjective well-being PGSs capture risk of major mood and psychotic disorders that is independent of family history, whereas PGSs for psychiatric illness provide limited predictive power when family history is known. Neuroticism and subjective well-being PGSs may complement family history in the early identification of persons at elevated risk

Phytochemicals in human health and disease

As I began this body of work, there were no published experimental studies regarding the clinical or biological effects of isoflavones or lignans in humans. Phytochemicals were typically described as ‘plant toxicants’ or ‘non-nutrients’, with a reputation for adverse rather than beneficial effects and generally pertaining to livestock. ‘Phytoestrogens’ (or ‘phyto-oestrogens’) was an obscure term in the medical literature. Following my initial publication in 1990, there was an extraordinary rise in the number of publications in the phyoestrogen field alone, such that by 1996 there were more than 600 publications reported on Medline. The significance of equol, a product of gastrointestinal microbial fermentation of the isoflavones daidzein and formononetin, had long been well established in numerous animal species, particularly sheep. However, though anticipated in our early human studies, it was a minor part of this literature until recently. Overlooking the key role of gut flora in the bioavailability and biotransformation of isoflavones, and the factors permissive to this process, has led to confusion and controversy in the literature regarding inconsistent biological effects in humans. Attempting to address this issue are a series of publications in this thesis arising from a decade of collaboration with colleagues at Victoria University working in the area of food science and biological science. Following demonstration that, in vitro, certain probiotic bifido-bacteria strains could hydrolyse isoflavone glucosides and biotransform daidzein into equol, potentially yielding more bioavailable and bioactive forms, preliminary studies in-vivo suggested more consistent urinary isoflavone recovery with co-ingestion of live bifido-bacteria and soy isoflavones in the form of fermented soy milk. Also suggested was a possible change in equol-producing status in this setting. If confirmed these findings could have clinical implications. Phytoestrogens are, strictly speaking, phytochemicals with oestrogenic activity, variably defined. However, not all the diverse biological properties of these compounds are mediated through oestrogen-like actions. Furthermore some plant components appear to have effects on the reproductive system, in the absence, as yet, of a defined hormonal mechanism, as with Lepidium Meyenii (Maca). The observed effects, on anxiety and depression scores, are likely phytochemical properties independent of oestrogenic activity. Having demonstrated biological activities of certain phytochemicals in humans, as found naturally occurring in foods, and further defined the role of the gut flora, the field has now moved from the perspective of hormone-dependent diet-disease relationships to a greater understanding of the potential to modify disease expression in rare, and as well as more common, conditions. It is on this basis that I am submitting this thesis as an original contribution to the advancement of scientific knowledge. Oestrogenic activity in plants was first described in 1926 (Dohrn, et al. 1926). The subsequent wealth of data regarding such effects in numerous animal species (Price and Fenwick 1985), along with then recent publications reporting the presence of isoflavone phytoestrogens including equol, the microflora metabolite of daidzein, in human urine (Setchell, Borriello, et al. 1984), began a line of enquiry by the author from mid-1986, regarding possible biological effects in humans. Preparing for a third year undergraduate essay, I had been inspired by the concept, raised by Seely, and others, from the University of Manchester, of naturally occurring compounds in plants [phytochemicals], including those with oestrogenic activity [phytoestrogens], as potentially having effects on human health (Seely, et al. 1985). From this, I developed further hypotheses regarding the role of phytochemicals in human health, and the beginning of my research career. The hypotheses that underpin this thesis included the following:- Hypothesis 1: Phytochemicals may be biologically active in humans with potentially beneficial as well as adverse effects on health Hypothesis 2 Phytochemicals may be modified in their properties by intestinal and/or probiotic microflora Hypothesis 3 Individual phytochemicals may have multiple mechanisms of action and individual foods may contain multiple phytochemicals. Therefore observed effects of phytochemical-rich food supplementation may be complex and not predictable by a single mechanism. Postmenopausal women, potentially most sensitive to such effects, supplemented with phytoestrogen-rich foods based on recent literature, were studied and an oestrogenic effect on vaginal cytology was reported in the BMJ (Wilcox et al 1990/Chapter 1). This was the first report of oestrogenic effects of isoflavone and lignan phytoestrogens (then described as plant oestrogens) in humans. Following this publication we were approached by Tenovus Institute of Cancer Research in Cardiff, leading to collaboration with Morton and others; our findings confirmed retrospectively the presence of these compounds in the plasma of the study subjects during the intervention period (Morton et al 1994/Chapter 2). In late 1991 I was also approached by Murkies who was seeking to explore the potential effect of isoflavone-rich foods in modifying symptoms of hot flushes in postmenopausal women. I had a major role in presenting the background literature, designing the clinical study protocol, data interpretation and critical input into the manuscript as well as bringing together other key members of the research team (Murkies et al 1995/Chapter 3). Six weeks’ supplementation with soy flour, rich in isoflavones, reduced hot flushes, compared with the refined wheat flour placebo lacking significant isoflavones or lignans as confirmed on biochemical analysis. Interestingly the effects on hormonal cytology were not seen in this study. This raised the question regarding possible differences between subjects recruited for the respective studies. In my original clinical study healthy volunteers from the wider community were recruited whereas symptomatic post-menopausal women were recruited in this subsequent study. A later, longer-term study in asymptomatic post-menopausal women confirmed a positive effect on vaginal cytology (Chiechi, et al. 2003). One potential explanation was the possibility of a difference in gut microflora, and consequently the capacity to biotransform daidzein into the more potent metabolite equol, between the two groups of women. In the original study, 4 out of 12 women (33%) tested were equol producers (Chapter 2) compared with the later study with Murkies, where only 5 of a total 58 (8.6%) women were equol producers (Chapter 3). In a review of the effects of soy on menopausal symptoms, presented as an invited lecture at the 8th International Congress on the Menopause in 1996, I summarized the variable findings of the studies reported to that date (Wilcox 1997/Chapter 4). A further review written jointly with Murkies, with additional critical input by Davis, summarized the literature regarding phytoestrogens in a broader clinical context (Murkies et al 1998/Chapter 5). The pivotal role of equol, anticipated from animal data and early pre-clinical studies, was confirmed in subsequent reports by others. (Setchell, Brown and Lydeking-Olsen, The clinical importance of the metabolite equol-a clue to the effectiveness of soy and its isoflavones. 2002) In 1997, I was approached by Stojanovska, leading to subsequent collaboration together with Shah, an academic and applied food scientist with a strong track record in dairy fermentation. In 1999, Shah and I obtained Australian Research Council (in partnership with Sanitarium Health Foods) for 2001-2004: ARC-Spirt Grant (AUD $79,116, combined Scholarship & cash contribution) to develop in-vitro fermentation of soy milk with live bifidobacteria with the aim of enhancing the bioavailability of ingested isoflavones (Tsangalis et al 2004/Chapter 6). In vitro hydrolysis of isoflavone glucosides and biotransformation of daidzein to equol in fermented soy-milk containing live bifido-bacteria was associated with greater urinary isoflavone recovery in postmenopausal women (Tsangalis et al 2005/Chapter 7) and a trend to increased prevalence of equol production after 2 weeks’ supplementation (Tsangalis et al 2007/Chapter 8). These findings have formed the basis of a clinical study (to be reported) comparing the effects of fermented soy milk, non-fermented soymilk and casein over a 12 week period involving 40 postmenopausal women in a parallel arm study. The study has been designed to look at markers of oestrogen action, as well as androgen profiles and SHBG. The latter may be both influenced by oestrogenic activity as well as hepatic effects of systemic insulin resistance; Jayagopal et al (Jayagopal, et al. 2002) reported a reduction in insulin resistance in 32 postmenopausal women with type 2 diabetes supplemented with 30g daily soy protein (Jayagopal, et al. 2002), though this may have been confounded by differences in protein intake. Although early population studies (Adlercreutz H 1987) reported an association between dietary phytoestrogen intake, particularly dietary lignans, and SHBG levels, the majority of early human intervention studies (Chapter 4) failed to confirm this. One study that did (Brzezinski A 1997)used a combination of flaxseed (linseed) and soy as a phytoestrogen supplement. However Pino et al (Pino AM 2000) subsequently reported a positive effect of soy supplementation in raising SHBG in postmenopausal women that was essentially dependent on the baseline SHBG level: this effect was only observed where the baseline SHBG level was less than 55 nmol/L. Reduced SHBG is commonly seen in states of insulin resistance, as associated with central adiposity, as well with as androgen excess. In this context, the topic of insulin and insulin resistance, including the hepatic effects of insulin action on androgen profiles and SHBG, along with reported effects of phytochemicals, was broadly reviewed in a further publication (Wilcox G 2005/Appendix 1; references 28, 71-73). Furthermore there may be greater sensitivity to the hepatic effects of ingested phytoestrogens, and other phytochemicals, given the vastly greater concentration of ingested phytoestrogens in the portal circulation, reported by Axelson & Setchell in rodent studies in 1981 (Axelson and Setchell 1981). This discussion underscores the potentially complex mechanisms of actions of phytochemicals particularly when consumed as whole foods. In response to public enquiry, and in collaboration with Industry and colleagues at Victoria University, studies comparing the absorption of calcium from commercial soy milk, with that from cows’ milk, were undertaken. As well as researching the methodology, designing and oversighting the clinical aspects of these studies, I proposed, in addition, investigating the effect of fermentation of soy milk on calcium absorption. This was because of the acute effects of fermentation on the hydrolysis of phytate (a known inhibitor of calcium bioavailability) as well as the potentially positive longer-term role of isoflavones on bone health. There was no significant difference in acute calcium absorption between the commercial soy protein isolate based soy milk, which was demonstrated to be low in phytate, and cows’ milk (Tang AL et al APJCN 2010/Chapter 9), nor was there a significant effect of fermentation in enhancing calcium absorption acutely with this preparation (Tang et al BJN 2011/Chapter10) despite improved calcium solubility and reduced pH (Tang AL et al, J Food Sci 2007/Chapter 11). However, demonstration of phytase activity in lactobacillus species (Tang AL et al JFS 2010/Chapter 12), has potential to improve the bioavailability of calcium, as well as zinc and iron, in whole soy bean preparations and other wholegrain products, where the phytochemical phytate is known to be an inhibitory factor (Schlemmer, et al. 2009). Oestrogenic activity and/or oestrogenically active compounds have been identified in over half the plants consumed by humans. Many of these plants have had traditional uses relating to their reputed effects on endocrine and/or reproductive systems (Farnsworth, et al. 1975). Lepidium Meyenii (Maca), an edible plant in the radish family growing at very high altitude in the Andes, has traditional uses as a general tonic, aphrodisiac and fertility agent (Gonzales 2012). Based on anecdotal experience, and in collaboration with colleagues at Victoria University, I designed a randomized double-blind cross-over trial of Maca, including biochemical analyses, which was conducted in postmenopausal women. Whilst the extract used failed to show any oestrogenic or androgenic activity in vitro, a significant effect on psychological symptoms on the Greene Scale was noted, particularly with respect to symptoms associated with depression and anxiety. Phytochemicals such as flavonoids with monoamine oxidase activity have been isolated in Maca, as one possible mechanism (Brooks et al 2008/Chapter 13) Most studies looking at oestrogenic effects of isoflavones in postmenopausal women have failed to show significant effects on psychological symptoms. It is known that with genistein, for example, only ten percent of the free compound crosses the blood brain barrier (Tsai 2005). The finding of such beneficial effects of Maca on psychological symptoms is therefore of significance. Much work in this field has concentrated on the role of these compounds in normal physiology across the lifecycle and in chronic common disease states, such as osteoporosis, breast cancer and cardiovascular risk factors (Chapter 5). However there are emerging developments with respect to less common conditions. Biotransformation of diet-derived compounds by intestinal microflora, exemplified by the conversion of daidzein to equol and consequent systemic endocrine effects, is a model for the treatment of diseases such as inherited metabolic disorders where, conceptually, the microbiome may supplement the human genome in the presence of genetic metabolic enzyme deficiency. This is discussed in the final Chapter (Wilcox /Chapter 14) Chapter 1: Oestrogenic effects of plant foods in postmenopausal women. British Medical Journal 1990;301:905-906 Chapter 2: Determination of plasma lignans and isoflavonoids in human female plasma following dietary supplementation Journal of Endocrinology 1994;142:251-259 Chapter 3: Dietary flour supplementation decreases post-menopausal hot flushes: effects of soy or wheat Maturitas 1995;21(3):189-195 Chapter 4: The effect of soy on menopausal symptoms. Progress in the Management of the Menopause: Parthenon: New York 1997:268-276 Chapter 5: Clinical Review 92: Phytoestrogens. Journal of Clinical Endocrinology & Metabolism 1998;83(2):297-303 Chapter 6: Development of an isoflavone aglycone enriched soymilk using soy germ, soy protein isolate and bifidobacteria. Food Research International 2004;37:301-312 Chapter 7: Bioavailability of isoflavone phytoestrogens in postmenopausal women consuming soymilk fermented with probiotic bifidobacteria British Journal of Nutrition 2005 Jun;93(6):867-77. Chapter 8: Urinary excretion of equol by postmenopausal women consuming soymilk fermented by bifidobacteria European Journal of Clinical Nutrition 2007 Mar;61(3):438-41. Chapter 9: Calcium absorption in Australian osteopenic post-menopausal women: an acute comparative study of fortified soymilk to cows' milk. Asia Pacific Journal of Clinical Nutrition. 2010;19(2):243-9. Chapter 10: Fermentation of calcium fortified soymilk does not appear to enhance acute calcium absorption in osteopenic post-menopausal women. British Journal of Nutrition 2011 Jan;105(2):282-6. Chapter 11: Fermentation of calcium fortified soymilk with Lactobacilli: effects on calcium solubility, isoflavone conversion and production of organic acids. Journal of Food Science 2007 Nov;72(9):M431-6. Chapter 12: Phytase Activity from Lactobacillus spp. In Calcium Fortified Soymilk Journal of Food Science 2010 Aug 1;75(6):M373-6. Chapter 13: Beneficial effects of Lepidium Meyenii (Maca) on psychological symptoms and measures of sexual dysfunction in post-menopausal women are not related to estrogen or androgen content Menopause 2008 Nov-Dec;15(6):1157-62. Chapter 14: Probiotics in Human Health and Disease: A New Avenue of Understanding between Diet, Disease and Metabolic Disorders Journal of Probiotics and Health 2013, 1:4 Appendix 1: Insulin and insulin resistance. Clinical Biochemist Reviews 2005;26:19-39   Conclusions and Synthesis: The papers included in this thesis give support to the hypotheses outlined in the introductory chapter. Namely that 1. Phytochemicals may be biologically active in humans with potentially beneficial as well as adverse effects on health 2. Phytochemicals may be modified in their properties by intestinal and/or probiotic microflora 3. Individual phytochemicals may have multiple mechanisms of action and individual foods may contain multiple phytochemicals. Therefore observed effects of phytochemical-rich food supplementation may be complex and not predictable by a single mechanism. The initial studies seeking to delineate the significance of phytoestrogens in human health, using postmenopausal women as a model, have set the scene as a ‘proof of concept’ for the significance of phytochemicals in the broader context of human health and nutrition. This is supported by the respective sequential expansion of the literature containing the key words ‘phytoestrogen or plant oestrogen’ and ‘phytochemical’. Biotransformation of dietary components by intestinal flora and/or probiotic bacteria, elegantly demonstrated with respect to isoflavones and lignans, is now part of an emerging field in human nutrition and metabolism, where intestinal flora are likely to be a key mediator of diet-disease relationships. The final paper (Chapter 14) brings together a number of concepts and future possible directions for this field, particularly as it may relate to rare metabolic disorders. Other recent publications by others have explored further the potential for phytochemicals, consumed as intact plant foods, to influence the breadth of human health and disease states. Phytochemicals are now considered to be a significant contributor to cross cultural diet-disease relationships such as those reported for the Mediterranean Diet (Widmer RJ 2015), and for Asian diets for example. Differences in intestinal flora between different populations have been attributed to divergent risks for chronic disease, with potential for therapeutic interventions by means of probiotic organisms (Flint HJ 2012). Equol-producing status is suggested as, both, a mediator, as well as a correlate, of more favourable health outcomes (Majee 2011), an intriguing development given its early notoriety in ovine ‘Clover Disease’. The variable and at times conflicting findings in the literature regarding phytoestrogens in menopausal women likely reflect the complex interactions with baseline hormonal status, background diet, gut flora, antibiotic exposure and effects of the entero-hepatic circulation, as well as nature of supplement used and effects of other phytochemicals present when food sources rather than isolated compounds are used. Currently I am working as consultant in metabolic medicine, in a large specialist centre for the care of adults with inherited metabolic disorders, in Manchester, United Kingdom. Interestingly phytochemicals, including flavonoids such as the isoflavone genistein, are currently under investigation as potential therapeutic agents in a group of rare inherited metabolic disorders known as the mucopolysaccaridoses (MPS) including the devastating neurologically manifest disorder MPS III, otherwise known as San Filippo disease. Promising results of pharmacological dosing with genistein in a rodent model of the condition have led to the commencement of clinical trials of genistein either as the pure aglycone (Kim, et al. 2013) or ion the form of soy extract, in humans, specifically children and young adults, with the condition (Wegrzyn G 2010). Pharmacological dosing is being used to compensate for the 1:10 transfer across the blood brain barrier. The contribution of the studies on the bioavailability, metabolism and effects of isoflavone phytoestrogens in humans, as included in this body of work, may help inform future clinical trials in these patients, as well as others. This thesis has therefore, contributed to the understanding and treatment of a wide spectrum of human diseases as well as to human health.   Bibliography: Adlercreutz H, Höckerstedt K, Bannwart C, Bloigu S, Hämäläinen 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 27, no. 4-6 (1987): 1135-44. Axelson, M, and KD. Setchell. “The excretion of lignans in rats -- evidence for an intestinal bacterial source for this new group of compounds.” FEBS Lett, 26 Jan 1981: 337-42. Brzezinski A, Adlercreutz H, Shaoul R, Rosier A, Shmueli A, Tanos V, SchenkerJ G. “Short-term Effects of Phytoestrogen-rich Diet on Postmenopausal Women.” Menopause 4, no. 2 (1997): 89–94. Chiechi, LM1, G Putignano, V Guerra, MP Schiavelli, AM Cisternino, and C. Carriero. “The effect of a soy rich diet on the vaginal epithelium in postmenopause: a randomized double blind trial.” Maturitas, 20 Aug 2003: 241-6. Dohrn, M, W Faure, H Poll, and W. Blutevogel. “Tokokinine, Stoffe mit Sexualhormonartiger Wirkung aus Planzenellen.” Med Klin, 1

Phytochemicals in human health and disease

As I began this body of work, there were no published experimental studies regarding the clinical or biological effects of isoflavones or lignans in humans. Phytochemicals were typically described as ‘plant toxicants’ or ‘non-nutrients’, with a reputation for adverse rather than beneficial effects and generally pertaining to livestock. ‘Phytoestrogens’ (or ‘phyto-oestrogens’) was an obscure term in the medical literature. Following my initial publication in 1990, there was an extraordinary rise in the number of publications in the phyoestrogen field alone, such that by 1996 there were more than 600 publications reported on Medline. The significance of equol, a product of gastrointestinal microbial fermentation of the isoflavones daidzein and formononetin, had long been well established in numerous animal species, particularly sheep. However, though anticipated in our early human studies, it was a minor part of this literature until recently. Overlooking the key role of gut flora in the bioavailability and biotransformation of isoflavones, and the factors permissive to this process, has led to confusion and controversy in the literature regarding inconsistent biological effects in humans. Attempting to address this issue are a series of publications in this thesis arising from a decade of collaboration with colleagues at Victoria University working in the area of food science and biological science. Following demonstration that, in vitro, certain probiotic bifido-bacteria strains could hydrolyse isoflavone glucosides and biotransform daidzein into equol, potentially yielding more bioavailable and bioactive forms, preliminary studies in-vivo suggested more consistent urinary isoflavone recovery with co-ingestion of live bifido-bacteria and soy isoflavones in the form of fermented soy milk. Also suggested was a possible change in equol-producing status in this setting. If confirmed these findings could have clinical implications. Phytoestrogens are, strictly speaking, phytochemicals with oestrogenic activity, variably defined. However, not all the diverse biological properties of these compounds are mediated through oestrogen-like actions. Furthermore some plant components appear to have effects on the reproductive system, in the absence, as yet, of a defined hormonal mechanism, as with Lepidium Meyenii (Maca). The observed effects, on anxiety and depression scores, are likely phytochemical properties independent of oestrogenic activity. Having demonstrated biological activities of certain phytochemicals in humans, as found naturally occurring in foods, and further defined the role of the gut flora, the field has now moved from the perspective of hormone-dependent diet-disease relationships to a greater understanding of the potential to modify disease expression in rare, and as well as more common, conditions. It is on this basis that I am submitting this thesis as an original contribution to the advancement of scientific knowledge. Oestrogenic activity in plants was first described in 1926 (Dohrn, et al. 1926). The subsequent wealth of data regarding such effects in numerous animal species (Price and Fenwick 1985), along with then recent publications reporting the presence of isoflavone phytoestrogens including equol, the microflora metabolite of daidzein, in human urine (Setchell, Borriello, et al. 1984), began a line of enquiry by the author from mid-1986, regarding possible biological effects in humans. Preparing for a third year undergraduate essay, I had been inspired by the concept, raised by Seely, and others, from the University of Manchester, of naturally occurring compounds in plants [phytochemicals], including those with oestrogenic activity [phytoestrogens], as potentially having effects on human health (Seely, et al. 1985). From this, I developed further hypotheses regarding the role of phytochemicals in human health, and the beginning of my research career. The hypotheses that underpin this thesis included the following:- Hypothesis 1: Phytochemicals may be biologically active in humans with potentially beneficial as well as adverse effects on health Hypothesis 2 Phytochemicals may be modified in their properties by intestinal and/or probiotic microflora Hypothesis 3 Individual phytochemicals may have multiple mechanisms of action and individual foods may contain multiple phytochemicals. Therefore observed effects of phytochemical-rich food supplementation may be complex and not predictable by a single mechanism. Postmenopausal women, potentially most sensitive to such effects, supplemented with phytoestrogen-rich foods based on recent literature, were studied and an oestrogenic effect on vaginal cytology was reported in the BMJ (Wilcox et al 1990/Chapter 1). This was the first report of oestrogenic effects of isoflavone and lignan phytoestrogens (then described as plant oestrogens) in humans. Following this publication we were approached by Tenovus Institute of Cancer Research in Cardiff, leading to collaboration with Morton and others; our findings confirmed retrospectively the presence of these compounds in the plasma of the study subjects during the intervention period (Morton et al 1994/Chapter 2). In late 1991 I was also approached by Murkies who was seeking to explore the potential effect of isoflavone-rich foods in modifying symptoms of hot flushes in postmenopausal women. I had a major role in presenting the background literature, designing the clinical study protocol, data interpretation and critical input into the manuscript as well as bringing together other key members of the research team (Murkies et al 1995/Chapter 3). Six weeks’ supplementation with soy flour, rich in isoflavones, reduced hot flushes, compared with the refined wheat flour placebo lacking significant isoflavones or lignans as confirmed on biochemical analysis. Interestingly the effects on hormonal cytology were not seen in this study. This raised the question regarding possible differences between subjects recruited for the respective studies. In my original clinical study healthy volunteers from the wider community were recruited whereas symptomatic post-menopausal women were recruited in this subsequent study. A later, longer-term study in asymptomatic post-menopausal women confirmed a positive effect on vaginal cytology (Chiechi, et al. 2003). One potential explanation was the possibility of a difference in gut microflora, and consequently the capacity to biotransform daidzein into the more potent metabolite equol, between the two groups of women. In the original study, 4 out of 12 women (33%) tested were equol producers (Chapter 2) compared with the later study with Murkies, where only 5 of a total 58 (8.6%) women were equol producers (Chapter 3). In a review of the effects of soy on menopausal symptoms, presented as an invited lecture at the 8th International Congress on the Menopause in 1996, I summarized the variable findings of the studies reported to that date (Wilcox 1997/Chapter 4). A further review written jointly with Murkies, with additional critical input by Davis, summarized the literature regarding phytoestrogens in a broader clinical context (Murkies et al 1998/Chapter 5). The pivotal role of equol, anticipated from animal data and early pre-clinical studies, was confirmed in subsequent reports by others. (Setchell, Brown and Lydeking-Olsen, The clinical importance of the metabolite equol-a clue to the effectiveness of soy and its isoflavones. 2002) In 1997, I was approached by Stojanovska, leading to subsequent collaboration together with Shah, an academic and applied food scientist with a strong track record in dairy fermentation. In 1999, Shah and I obtained Australian Research Council (in partnership with Sanitarium Health Foods) for 2001-2004: ARC-Spirt Grant (AUD $79,116, combined Scholarship & cash contribution) to develop in-vitro fermentation of soy milk with live bifidobacteria with the aim of enhancing the bioavailability of ingested isoflavones (Tsangalis et al 2004/Chapter 6). In vitro hydrolysis of isoflavone glucosides and biotransformation of daidzein to equol in fermented soy-milk containing live bifido-bacteria was associated with greater urinary isoflavone recovery in postmenopausal women (Tsangalis et al 2005/Chapter 7) and a trend to increased prevalence of equol production after 2 weeks’ supplementation (Tsangalis et al 2007/Chapter 8). These findings have formed the basis of a clinical study (to be reported) comparing the effects of fermented soy milk, non-fermented soymilk and casein over a 12 week period involving 40 postmenopausal women in a parallel arm study. The study has been designed to look at markers of oestrogen action, as well as androgen profiles and SHBG. The latter may be both influenced by oestrogenic activity as well as hepatic effects of systemic insulin resistance; Jayagopal et al (Jayagopal, et al. 2002) reported a reduction in insulin resistance in 32 postmenopausal women with type 2 diabetes supplemented with 30g daily soy protein (Jayagopal, et al. 2002), though this may have been confounded by differences in protein intake. Although early population studies (Adlercreutz H 1987) reported an association between dietary phytoestrogen intake, particularly dietary lignans, and SHBG levels, the majority of early human intervention studies (Chapter 4) failed to confirm this. One study that did (Brzezinski A 1997)used a combination of flaxseed (linseed) and soy as a phytoestrogen supplement. However Pino et al (Pino AM 2000) subsequently reported a positive effect of soy supplementation in raising SHBG in postmenopausal women that was essentially dependent on the baseline SHBG level: this effect was only observed where the baseline SHBG level was less than 55 nmol/L. Reduced SHBG is commonly seen in states of insulin resistance, as associated with central adiposity, as well with as androgen excess. (...