Background: The soy isoflavones genistein and daidzein are found in blood and tissues as aglycones, glucuronides, and sulfates. Isoflavone conjugates may serve as sources of aglycones at specific target tissues and may have bioactivity. Yet, very little is known about the plasma pharmacokinetics of isoflavone conjugates after soy ingestion. Objective: The objective of this study was to determine the plasma pharmacokinetics of glucuronide and sulfate conjugates of genistein and daidzein in humans after the consumption of a drink made with soy-protein isolate. Design: Six men and 6 women (x̄ ± SD age: 40.8 ± 3 y) consumed a soy-protein-isolate drink. The pharmacokinetics of isoflavone glucuronide and sulfate conjugates were studied with the use of β-glucuronidase (EC 3.2.1.31) and sulfatase (EC 3.1.6.1) digestion and liquid chromatography–mass spectrometry. Results: Glucuronides of genistein and daidzein made up a significantly lower percentage (P < 0.05) of the total isoflavone concentration in plasma (48% and 33%, respectively) than in urine. The percentages of sulfates of genistein and daidzein in plasma (8% and 26%, respectively) were 2- to 6-fold those in urine (P < 0.05). Approximately 30% of the total genistein or daidzein was comprised of mixed conjugates (one glucuronide and one sulfate). For daidzein sulfate, genistein sulfate, daidzein glucuronide, and genistein glucuronide, the time to peak (t_max) was 4.5, 4.5, 4.5, and 6.0 h, respectively, and the apparent half-life (t_1/2λz) was 3.1, 5.7, 3.2, and 8.4 h, respectively. Conclusions: These data suggest that there are significant differences in the pharmacokinetics of sulfate and glucuronide conjugates of isoflavones. This may have important implications for the meal frequency and maintenance of target tissue bioactivity required to elicit potential health benefits.

Various physiologic effects of soy food consumption have been attributed to the estrogenic actions of isoflavones. The order of estrogen receptor binding potency of soy-derived isoflavone aglycones is equol > genistein > daidzein, and their conjugates are less potent. Because the metabolic profile may be an important determinant of bioactivity after soy intake, we studied the serum and urine isoflavone concentrations in 3 animal models and compared them with isoflavone profiles in women. Female Sprague-Dawley rats, Hampshire/Duroc Cross pigs, cynomolgus monkeys, and women were fed diets containing soy protein isolate. Isoflavones and their metabolites were measured by LC-MS or electrochemical detection. Equol represented ∼77 and 52% (molar ratio) of summed serum isoflavones (isoflavones plus metabolites) in rats and cynomolgus monkeys, respectively. Equol was undetectable in pig serum and human plasma, but daidzein and genistein contributed >88% of summed circulating isoflavones. Monkey and rat urine contained high levels of aglycones (>85% and >32%, respectively), whereas pigs and women excreted isoflavone mainly in the form of glucuronides (>80%), with <10% as aglycones. Isoflavones in human plasma were predominantly glucuronides (75%) with 24% as sulfates and <1% as aglycones; in monkey serum, however, 64% of isoflavones were sulfates, 30% glucuronides, and 6% aglycones. Equol was also a major serum metabolite of 6-mo-old rhesus monkeys (80% of summed isoflavones). Thus, there were significant interspecies differences in isoflavone metabolism, and the overall metabolic profile of pigs was closer to that of women than that of rats or monkeys.

We previously reported that lifetime consumption of soy proteins or whey proteins reduced the incidence of azoxymethane (AOM)-induced colon tumors in rats. To obtain insights into these effects, global gene expression profiles of colons from rats with lifetime ingestion of casein (CAS, control diet), soy protein isolate (SPI), and whey protein hydrolysate (WPH) diets were determined. Male Sprague Dawley rats, fed one of the three purified diets, were studied at 40 weeks after AOM injection and when tumors had developed in some animals of each group. Total RNA, purified from non-tumor tissue within the proximal half of each colon, was used to prepare biotinylated probes, which were hybridized to Affymetrix RG_U34A rat microarrays containing probes sets for 8799 rat genes. Microarray data were analyzed using DMT (Affymetrix), SAM (Stanford) and pair-wise comparisons. Differentially expressed genes (SPI and/or WPH vs. CAS) were found. We identified 31 induced and 49 repressed genes in the proximal colons of the SPI-fed group and 44 induced and 119 repressed genes in the proximal colons of the WPH-fed group, relative to CAS. Hierarchical clustering identified the co-induction or co-repression of multiple genes by SPI and WPH. The differential expression of I-FABP (2.92-, 3.97-fold down-regulated in SPI and WPH fed rats; P = 0.023, P = 0.01, respectively), cyclin D1 (1.61-, 2.42-fold down-regulated in SPI and WPH fed rats; P = 0.033, P = 0.001, respectively), and the c-neu proto-oncogene (2.46-, 4.10-fold down-regulated in SPI and WPH fed rats; P < 0.001, P < 0.001, respectively) mRNAs were confirmed by real-time quantitative RT-PCR. SPI and WPH affected colonic neuro-endocrine gene expression: peptide YY (PYY) and glucagon mRNAs were down-regulated in WPH fed rats, whereas somatostatin mRNA and corresponding circulating protein levels, were enhanced by SPI and WPH. The identification of transcripts co- or differentially-regulated by SPI and WPH diets suggests common as well as unique anti-tumorigenesis mechanisms of action which may involve growth factor, neuroendocrine and immune system genes. SPI and WPH induction of somatostatin, a known anti-proliferative agent for colon cancer cells, would inhibit tumorigenesis.

The gut microbiota of breast-fed and formula-fed infants differ significantly, as do the risks for allergies, gut dysfunction, and upper respiratory tract infections. The connections between breast milk, various formulas, and the profiles of gut bacteria to these childhood illnesses, as well as the mechanisms underlying the effects, are not well understood. We investigated distal colon microbiota by 16S RNA amplicon sequencing, morphology by histomorphometry, immune response by cytokine expression, and tryptophan metabolism in a pig model in which piglets were sow-fed, or fed soy or dairy milk-based formula from postnatal day (PND) 2 to 21. Formula feeding significantly (p < 0.05) altered the colon microbiota relative to the sow feeding. A significant reduction in microbial diversity was noted with formula groups in comparison to sow-fed. Streptococcus, Blautia, Citrobacter, Butrycimonas, Parabacteroides, Lactococcus genera were increased with formula feeding relative to sow feeding. In addition, relative to sow feeding, Anaerotruncus, Akkermansia, Enterococcus, Acinetobacter, Christensenella, and Holdemania were increased in milk-fed piglets, and Biliophila, Ruminococcus, Clostridium were increased in soy-fed piglets. No significant gut morphological changes were noted. However, higher cytokine mRNA expression (BMP4, CCL11, CCL21) was observed in the distal colon of formula groups. Formula feeding reduced enterochromaffin cell number and serotonin, but increased tryptamine levels relative to sow feeding. Our data confirm that formula diet alters the colon microbiota and appears to shift tryptophan metabolism from serotonin to tryptamine, which may lead to greater histamine levels and risk of allergies in infants.

Infants fed soy formula are the segment of the U. S. population that consumes the most soy. Before birth and after weaning, most Americans are not exposed to appreciable levels of soyfoods other than foods that have small amounts of processed soy components. The opposite scenario occurs in Asia, because Asians are more likely to consume relatively high levels of soyfoods throughout life, except between birth and weaning, when breastfeeding or milk-based formula are common. Soy formula is made with soy protein isolate containing isoflavones (SPI+) and supports normal growth and development in term infants. Recent data suggest that there are no long-term adverse effects of early exposure to soy formula through young adulthood. It is as yet unknown whether soy formula consumption by infants will result in health problems or benefits upon aging, but multigenerational animal studies with diets made with SPI+ have not revealed any problems. Soy isoflavones can function as estrogen agonists, antagonists or selective estrogen receptor modulators, depending on the conditions, and much research has focused on health effects of purified isoflavones. Results from several studies suggest that the effects of diets made with SPI+ differ significantly from those of diets to which purified soy isoflavones are added. Furthermore, it seems that soy protein processed to contain lower levels of isoflavones also provides significant health benefits. Further research is needed to confirm the results of the few studies that have been conducted and new studies are needed to investigate the more subtle effects that could occur during development or that could surface later in life.

Soy formula (SF) has been fed to millions of infants worldwide. It has been shown to promote growth and development as well as milk-based formula (MF). Controversy has developed over the adequacy and safety of SF. Most concerns are based on in vivo and in vitro data that raise the possibility of estrogenic effects of isoflavones contained in SF. There are few studies of children who were fed SF, and thus insufficient data are available to judge if SF feeding results in clinically significant developmental effects and if there are any long-term health consequences (adverse or beneficial). However, the Arkansas Children's Nutrition Center is conducting a prospective longitudinal study comparing growth, development, and health of breastfed children with formula-fed (SF and MF) children from birth through age 6 y. After 5 y of study, children in all 3 groups (n > 300) are growing and developing within normal limits, and there are no indications of adverse effects in the soy-fed children. Neonatal pig studies comparing SF, MF, and breast milk (BM) have shown diet-specific gene expression profiles in various target tissues. Therefore, although SF differed significantly from BM, MF also differed from BM, and SF differed from MF. Nonetheless, these animals grew and developed normally, and SF piglets had several health benefits (eg, increased bone quality) and no observable adverse effects. Thus, to date, our results suggest that SF supports normal growth and may have advantages in promoting bone development.