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Soy isoflavone metabolism in cats compared with other species: Urinary metabolite concentrations and glucuronidation by liver microsomes
Abstract
1. Soybean is a common source of protein in many pet foods. Slow glucuronidation of soy-derived isoflavones in cats has been hypothesized to result in accumulation with adverse health consequences. Here, we evaluated species’ differences in soy isoflavone glucuronidation using urine samples from cats and dogs fed a soy-based diet and liver microsomes from cats compared with microsomes from 12 other species.
2. Significant concentrations of conjugated (but not unconjugated) genistein, daidzein and glycitein, and the gut microbiome metabolites, dihydrogenistein and dihydrodaidzein, were found in cat and dog urine samples. Substantial amounts of conjugated equol were also found in cat urine but not in dog urine.
3. β-Glucuronidase treatment showed that all these compounds were significantly glucuronidated in dog urine while only daidzein (11%) and glycitein (37%) showed any glucuronidation in cat urine suggesting that alternate metabolic pathways including sulfation predominate in cats.
4. Glucuronidation rates of genistein, daidzein and equol by cat livers were consistently ranked within the lowest 3 out of 13 species’ livers evaluated. Ferret and mongoose livers were also ranked in the lowest four species.
5. Our results demonstrate that glucuronidation is a minor pathway for soy isoflavone metabolism in cats compared with most other species.
... Although these compounds have low affinity for estrogen receptors compared to estradiol (α-ER and β-ER), they can bind to these receptors and induce estrogenic and antiestrogenic effects in mammals [3][4][5]. Additionally, some of their metabolites, such as equol, exhibit a high affinity for estrogen receptors [6,7]. These compounds are also associated with increased energy expenditure due to their ability to stimulate the coactivator PGC-1β, which enhances the expression of genes involved in fatty acid oxidation and mitochondrial activity in muscle cells [8]. ...
... Commercial diets for cats naturally contain these molecules (up to 154 mg genistein and 147 mg daidzein per kilogram of dry matter in dry food) due to the inclusion of soybean-derived ingredients such as isolated protein, concentrated protein, and soybean oil in diet formulations [6,9,10]. Furthermore, these compounds and their metabolites have been detected in the bloodstream of felines, confirming their absorption [6,11,12]. ...
... Commercial diets for cats naturally contain these molecules (up to 154 mg genistein and 147 mg daidzein per kilogram of dry matter in dry food) due to the inclusion of soybean-derived ingredients such as isolated protein, concentrated protein, and soybean oil in diet formulations [6,9,10]. Furthermore, these compounds and their metabolites have been detected in the bloodstream of felines, confirming their absorption [6,11,12]. ...
Isoflavones are composed of phytoestrogens (genistein and daidzein), which can be metabolized by cats. These compounds can promote the maintenance of lean body mass and control food intake. These effects are desirable in neutered animals, as they are predisposed to obesity. The objective of this study was to evaluate the effects of dietary supplementation of 1.0% isoflavone on the metabolizable energy intake, serum concentrations of satiety-related hormones and peptides, and body composition of neutered cats. Sixteen neutered adult cats were blocked by gender and divided into two groups (n = 8): the control group (CG) received a commercial diet, while the isoflavone group (IG) received the same diet supplemented by 1% of isoflavone for 99 days. Computed tomography was performed on the first and last experimental days to assess the animals’ body composition. Satiety challenges were conducted on days 19 and 44. In the last day of the study, blood samples were collected to determine the concentration of insulin, ghrelin, leptin, peptide YY, and GLP-1. A statistical analysis was conducted using R software 3.5.2, considering both the interaction and individual effects of group and time (p < 0.05). The average intake of genistein in the IG was 0.75 ± 0.10 mg/kg body weight, and daidzein intake was 51.73 ± 7.05 mg/kg. No significant individual or interaction effects were observed for any of the analyzed variables. Therefore, the inclusion of 1.0% isoflavone in the diet did not affect the energy requirements, satiety responses, or body composition of neutered adult cats.
... Previous metabolic studies on humans and experimental animals reported that daidzein is predominantly conjugated at the 7-and/or 4′-positions in the liver and intestines, and glucuronides are its major metabolites ( Fig. 1) formed by UDP-glucuronosyltransferase (UGT) (Bayer et al. 2001;Thomas et al. 2001;Hosoda et al. 2011;Redmon et al. 2016). Glucuronides undergo enterohepatic recycling due to β-glucuronidase and the subsequent intestinal reabsorption of daidzein, and extensive first-pass metabolism has been suggested as a reason for the poor bioavailability (< 5%) of daidzein (Barnes et al. 2011;Franke et al. 2014;Soukup et al. 2016;Zaheer and Humayoun Akhtar 2017). ...
... Thus, the biological activity of daidzein is considered to be closely associated with glucuronidation in the body. Although glucuronidation activities toward daidzein in the liver and intestinal microsomes of humans and experimental animals, such as monkeys and rodents, have already been examined (Chen et al. 2005;Pritchett et al. 2008;Redmon et al. 2016), there is currently little information on species differences in the regioselective and tissue-dependent glucuronidation of daidzein. In the present study, the hepatic and intestinal glucuronidation of daidzein at the 7-and 4′-positions by microsomal fractions of humans, monkeys, rats, and mice was investigated to elucidate the relationship between the metabolism and bioactivities of daidzein. ...
... Consequently, the activity ratio of 7-/4′-glucuronidation in the liver microsomes of humans, monkeys, rats, and mice varied by approximately tenfold (monkeys ≫ rats ≥ mice > humans), suggesting that the regioselectivity of daidzein glucuronidation by the liver microsomes markedly differs among species. Redmon et al. (2016) measured the glucuronidation activities of daidzein in the liver microsomes of 13 species including humans, monkeys, rats, and mice at a substrate concentration of 35 µM. 7-Glucuronidation activity in the liver microsomes was the highest in monkeys, at a level that was > 20-fold higher than those of humans, rats, and mice. ...
Daidzein, one of the major soy isoflavones, has a number of beneficial bioactivities for human health. It is mainly metabolized into 7- and/or 4′-glucuronides by UDP-glucuronosyltransferase (UGT) enzymes in mammals, including humans. The present study was conducted to examine the regioselective glucuronidation of daidzein at the 7- and 4′-hydroxyl groups in the liver and intestinal microsomes of humans, monkeys, rats, and mice. Daidzein glucuronidation activities at substrate concentrations of 1.0–200 µM were assessed, and Eadie–Hofstee plots were constructed. The kinetics for 7- and 4′-glucuronidation in the liver microsomes fit the Michaelis–Menten model, except for an atypical model for 7-glucuronidation in rats and a biphasic model for 4′-glucuronidation in monkeys. These kinetics in the intestinal microsomes followed the Michaelis–Menten model, except for a biphasic model for 7-glucuronidation in mice. The CLint values for 7-glucuronidation were in the order of monkeys (49) ≫ rats (5.3) > humans (1.0) > mice (0.7) for liver microsomes, and rats (2.4) ≥ monkeys (2.2) > humans (1.0) ≥ mice (0.8) for intestinal microsomes. On the other hand, the CLint values for 4′-glucuronidation were in the order of monkeys (4.0) > mice (1.0) ≈ humans (1.0) > rats (0.4) for liver microsomes, and humans (1.0) ≫ monkeys (0.08) ≥ mice (0.07) > rats (0.05) for intestinal microsomes. These results demonstrated that the metabolic abilities of UGT enzymes toward daidzein in the liver and intestines markedly differed among humans, monkeys, rats, and mice, and suggest that species and regioselective differences are closely associated with the bioactivities of soy isoflavones.
... To date, there is limited information on the conjugation profile of DAI (Doerge et al. 2000;Shelnutt et al. 2002;Zhang et al. 2003;Hosoda et al. 2011), and those relating to the EQU are even less (Koh & Mitchell 2011;Schwen et al. 2012;Legette et al. 2014;Redmon et al. 2016). This was due to non-availability of reference standards and, until recently, to sensitive and specific analytical techniques. ...
... On the contrary, Clarke et al. (2002) identified in human urine, by authentic standard, EQU-7-glucuronide and EQU-4 0 -glucuronide and the main peak was related to the 7-glucuronide. Redmon et al. (2016) reported that in dog, cat and human the Table 1. Plasma kinetic indexes in 12 healthy volunteers for daidzein and its metabolites after ingestion of about 100 mg daidzein in soy milk. ...
... (Figure 3, peak C) was not considered EQU-sulfate. Regarding plasma, EQU-7-glucuronide was the only metabolite found, and EQU pharmacokinetics studies in human and animals such as pig, rat, dog, horse and monkey show similar metabolism (Redmon et al. 2016). ...
The main aim of the study was to establish in vivo a correlation between equol (EQU) production and a number of intestinal bacteria able to perform the transformation. Thus, healthy female volunteers were selected for their ability to convert slowly (n = 6, 10(5)-10(9) cells/g wet feces) or quickly (n = 6, 10(10)-10(12) cells/g wet feces) daidzein (DAI) in EQU. After oral administration of 100 mg DAI in soymilk, plasma (0-99 h) and urine (0-96 h) samples were collected. DAI and its metabolites were determined by LC-MS/MS and EQU -conjugates by UPLC-High Resolution-MS. Only for EQU a direct correlation was found between the number of transforming microorganisms and parameters such as tmax and t1/2 (p = 0.027). Peak serum concentration time, Cmax, AUC0-72 h and t1/2 for total EQU (n = 12) were 36 ± 10 h, 89 ± 78 nM, 2.4 ± 1.7 (μmol × h/L) and 15.6 ± 3.3 h, respectively. In plasma and urine EQU was found mainly as 7-O-glucuronide.
... They belong to a class of phytoestrogens, which are plant-derived compounds with estrogen-like activity. Redmon et al. (2016) investigated the metabolism of soy isoflavones in cats compared to other species. The study found that cats showed significantly lower glucuronidation rates of soy isoflavones, such as genistein, daidzein, and equol, in liver microsomes compared to dogs and other species. ...
... Additionally, cats excreted higher levels of equol in urine compared to dogs, possibly due to differences in the intestinal microbiome. These findings highlight species-specific differences in soy isoflavone metabolism and the potential impact on health [26]. ...
Plant extracts, derived from various natural sources, encompass primary and secondary metabolites, which include plant polysaccharides, polyphenols, alkaloids, flavonoids, glycosides, terpenes, and volatile oils. These compounds exhibit a range of biological activities such as antioxidant, anti-inflammatory, and antimicrobial functions. Currently, polyphenols and other bioactive compounds are being incorporated into the diets of farm animals, fish, and pets to promote health benefits. Despite this, the application and potential of plant extracts in canine and feline nutrition have not been comprehensively explored. Many aspects of the mechanisms underlying the action of these plant metabolites remain to be analyzed and elucidated. Furthermore, leveraging natural plant extracts for the treatment of clinical conditions in dogs and cats is a crucial component of clinical nutrition. Consequently, this review aims to highlight the impact of plant extracts on overall health, gastrointestinal health, immune health, cardiovascular health, redox balance, and pathology in dogs and cats.
... It was found that one-fifth of D1 will enter the urine through circulation and glucuronides were the main metabolites. Redmon (Redmon et al., 2016) et al. investigated metabolites in urine samples from cats and dogs and liver microsomes from cats compared with microsomes from 12 other species. The results showed that sulfation was probably the major metabolic pathway, while glucuronidation was a minor pathway for D1 metabolism in cats. ...
... distributed in the liver but is mainly distributed in the marginal zone in the kidney (Figure 7). The results reported by Redmon (Redmon et al., 2016) showed that glucuronidation was the main metabolic pathway of D1 in mouse liver microsomes. Therefore, it was inferred that glucuronidation of its derivative DD2 occurred primarily in the liver. ...
Daidzein (D1) has been proved to be of great benefit to human health. More and more attention was paid to the metabolic process of D1. Most studies focused on the metabolites of D1 and analogs were determined through the excretion of animals and humans by traditional HPLC-MS, while their in situ distribution and metabolism in organs in vivo has not been reported. In our group, novel daidzein sulfonate derivatives were synthesized and confirmed to have excellent pharmaceutical properties. They exhibited good anti-inflammatory, inhibitory activities on human vascular smooth muscle cell proliferation and other bioactivities. Compared with traditional analytical methods, matrix-assisted laser desorption ionization time-of-flight mass spectrometry imaging (MALDI-TOF MSI) can directly analyze the distribution of compounds in tissues and organs. In this study, we investigate the in situ distribution and metabolism of D1 and its derivatives (DD2, DD3) in the organs of mice based on MALDI-TOF MSI for the first time. Trace prototype compounds were detected in the plasma 4 h after the intravenous injection of D1, DD2, and DD3. Seven phase I metabolites and seven phase II metabolites were detected. D1 sulfates were found in the plasma and in organs except the heart. The presence of D1 and DD3 monosulfates in the brain indicated that they could penetrate the blood–brain barrier. DD2 and DD3 could be hydrolyzed into D1 and their metabolic pathways were similar to those of D1. In addition, a ligand-receptor docking of D1 and DD2 with mitogen-activated protein kinase 8 (JNK1) was performed because of their significant anti-inflammatory activities through the JNK signaling pathway. It showed that the binding energy of DD2 with JNK1 was obviously lower than that of D1 which was consistent with their anti-inflammatory activities. It provided a theoretical basis for further validation of their anti-inflammatory mechanism at the protein level. In summary, the research will provide beneficial guidance for further pharmacological, toxicological studies and the clinical-use research of these compounds.
... We also observed strong contraction of UGT1As, especially the UGT1A6 pseudogene, UGT1A7-12 loss, and complete loss of UGT2Bs in all Felidae. These findings are in accordance with in vitro studies of limited capacity of UGTs for a wide range of chemicals in cats [55][56][57]. Similar features of in vitro limited glucuronidation were also observed in pinnipeds [17,18]. ...
UDP-glucuronosyltransferases (UGTs) are one of the most important enzymes for xenobiotic metabolism or detoxification. Through duplication and loss of genes, mammals evolved the species-specific variety of UGT isoforms. Among mammals, Carnivora is one of the orders that includes various carnivorous species, yet there is huge variation of food habitat. Recently, lower activity of UGT1A and 2B were shown in Felidae and pinnipeds, suggesting evolutional loss of these isoforms. However, comprehensive analysis for genetic or evolutional features are still missing. This study was conducted to reveal evolutional history of UGTs in Carnivoran species. We found specific gene expansion of UGT1As in Canidae, brown bear and black bear. We also found similar genetic duplication in UGT2Bs in Canidae, and some Mustelidae and Ursidae. In addition, we discovered contraction or complete loss of UGT1A7–12 in phocids, some otariids, felids, and some Mustelids. These studies indicate that even closely related species have completely different evolution of UGTs and further imply the difficulty of extrapolation of the pharmacokinetics and toxicokinetic result of experimental animals into wildlife carnivorans.
... Diets formulated for cats are known to vary greatly in protein sources and they can be classified into either vegetable or animal origin. Soybean and soybean-derived products are the main vegetable-based protein sources in cat diets, especially in vegetarian diets; however, they also have low palatability that limits inclusion in diets for cats (Redmon et al. 2016). One common industry practice is to use other foods or additives such as porcine liver or polyphosphates to increase the overall palatability of the cat diets (Zentek and Schulz 2004). ...
The taste of food is an essential characteristic for cats and has been shown to affect food selection. However, understanding of food selection by cats using taste characteristics is far from complete. Therefore, the aim of the present review was to summarize the current knowledge on food preference and the role of taste on this selection in domestic cats. Appetite regulation is one of the determinants of palatability in cats and involves a highly complex interplay between hypothalamus, adipose tissue, and digestive tract. However, knowledge on this interplay is scarce in cats. When evaluating different foods for cats, behavioural responses such as facial expressions involving the movements and positions of ears, tongue, and head can provide increased insight into the effectiveness of formulating a more palatable diet. This paper also reviews food additives currently used in industry for enhancing the palatability of cat foods. In summary, a better understanding of the factors that affect the food preference in cats is essential to produce high-quality foods because cats will not eat a food with a flavour they dislike even though it is complete and nutritionally balanced.
... The ability of 17β-E 2 to regulate GPx transcription may contribute to increase of the expression or activity of the GPx in females 38 , and this, in part, can contribute to reduce lipid hydrogen peroxides, and decrease LPO. However, our results suggest that the phytoestrogens provided by the diet could prevent the net loss of serum 17β-E 2 , and thus contribute to restore the activity of this enzyme 39 . However, more studies are needed to elucidate this mechanism. ...
There are few studies evaluating the oxidant-antioxidant status after oophorosalpingohysterectomy (OSH) in female dogs. Here we determined the effect of OSH on antioxidant enzymes in serum, and quantified morphological changes in subcutaneous adipocytes. Lateral OSH was performed in 12 female dogs. The concentration of 17β-estradiol (17β-E2), the activities of extracellular superoxide dismutase (SOD-ec), glutathione peroxidase (GPx), glutathione-S-transferase (GST) and glutathione reductase (GR) were determined. Glutathione (GSH), glutathione disulfide (GSSG), lipid peroxidation (LPO), total antioxidant capacity (TAC), carbonylation and vitamin C were measured in serum. Subcutaneous adipose tissue was obtained to determine morphological changes and cell number, under basal conditions and six months after OSH. The SOD-ec, GPx and GST activities increased significantly (p ≤ 0.05), LPO, carbonylation and GSSG also increased. GSH and vitamin C decreased (p = 0.03). 17β-E2 tended to decrease six months after OSH. Hypertrophy of subcutaneous adipocytes was observed after OSH from the first month and was accentuated after six months (p = 0.001). The results suggest that 17β-E2 decreases after OSH and alters the antioxidant enzyme activities in serum thus, redox balance is altered. These changes are associated with an increase in body weight and hypertrophy of subcutaneous adipose tissue.
... E-7-G was also described as the major metabolite produced by human pooled liver microsomes, while E-4′-G was not detected. 30 Our data also ...
Equol is a product formed during the biotransformation of the naturally occurring isoflavone daidzein by intestinal bacteria. The role of equol in the prevention of several hormone‐dependent diseases such as prostate cancer and osteoporosis as well as vasomotor symptoms has been extensively investigated. Equol primarily occurs in the form of major metabolites such as glucuronides and sulfates, while intact equol has been detected at only ca. 1% in human plasma. However, to date, conjugated metabolites have been evaluated by measuring the free equol obtained after selective enzymatic hydrolysis. Thus, the precise types of conjugates circulating in vivo and the position(s) of the conjugation sites on the equol skeleton have yet to be clarified. Our study describes the identification of polar equol metabolites in the plasma of 2 equol‐producers obtained at 8 hours after consuming 50 g of kinako (approximately 37 mg of daidzein). The structural identification of these conjugated metabolites in plasma was performed by comparison to the LC‐ESI‐MSn and 1H‐NMR spectral data of the corresponding chemically synthesized compounds. The results of the LC‐ESI‐MS/MS analysis indicated that the main conjugated metabolite in plasma was (S)‐equol‐7‐glucuronide‐4′‐sulfate along with lower amounts of 7‐ and 4′‐monoglucuronides as well as 7‐ and 4′‐monosulfates.
... The aglycons released can be absorbed or transformed. Equol, a gut microbiome metabolite of daidzein, was found in cat blood plasma (4) and urine (5). About 30% of orally administered, pure genistein was absorbed by cats (6) and 5% excreted in urine (7). ...
... There are number of issues involving soyfoods that have given rise to quite lively discussions in the peer-reviewed literature. One of those somewhat ironically, given the large populations of Asian countries that have historically consumed soy, is the possible adverse impact of soy on fertility On the other hand, breeding problems have been noted in some animal species in response to isoflavone ingestion [371][372][373][374] although it was subsequently shown that these issues arose because of either excessive intake [375] or because of differences in isoflavone metabolism between animals and humans [69,81,376,377]. ...
Soyfoods have long been recognized as sources of high-quality protein and healthful fat, but over the past 25 years these foods have been rigorously investigated for their role in chronic disease prevention and treatment. There is evidence, for example, that they reduce risk of coronary heart disease and breast and prostate cancer. In addition, soy alleviates hot flashes and may favorably affect renal function, alleviate depressive symptoms and improve skin health. Much of the focus on soyfoods is because they are uniquely-rich sources of isoflavones. Isoflavones are classified as both phytoestrogens and selective estrogen receptor modulators. Despite the many proposed benefits, the presence of isoflavones has led to concerns that soy may exert untoward effects in some individuals. However, these concerns are based primarily on animal studies, whereas the human research supports the safety and benefits of soyfoods. In support of safety is the recent conclusion of the European Food Safety Authority that isoflavones do not adversely affect the breast, thyroid or uterus of postmenopausal women. This review covers each of the major research areas involving soy focusing primarily on the clinical and epidemiologic research. Background information on Asian soy intake, isoflavones, and nutrient content is also provided.
... Diverses études menées chez l'animal ont suggéré dans le passé que la consommation d'isoflavones pourrait avoir un impact défavorable sur la fertilité (Lundh, 1990 ;Setchell, 1987). Il a été montré ultérieurement que ce problème était lié à une consommation excessive (Urpi, 2008) et qu'il existe des différences très marquées entre les animaux et les humains quant au métabolisme des isoflavones (Redmon, 2015 ;Rowland, 2000Rowland, , 2003Setchell, 2002). À l'âge adulte, chez la femme, la consommation d'aliments au soja augmente légèrement la durée du cycle menstruel, mais n'empêche pas l'ovulation (Hooper, 2009). ...
Aujourd’hui, 4 Français sur 10 consomment des aliments au soja. Les consommateurs les ont
intégrés dans leur alimentation, sans bouleverser leurs habitudes alimentaires, parce
qu’ils les considèrent naturels et sains. Les non consommateurs sont en attente de plus
d’informations (origine géographique du soja et qualités nutritionnelles). La consommation
est assez récente et devrait progresser dans les prochaines années. Les aliments élaborés
à partir de la graine de soja fournissent des protéines de très bonne qualité
nutritionnelle, des acides gras insaturés, des fibres, des vitamines et des minéraux. La
graine de soja contient des isoflavones, dont une grande partie est éliminée lors de
l’élaboration des aliments au soja (qui en contiennent 1 à 3 mg/g de protéine). La
consommation actuelle d’aliments au soja est largement compatible avec l’apport maximal de
1mg/kg/j recommandé par l’Afssa en 2005. Le développement infantile avec les préparations
à base de protéines de soja est comparable à celui observé avec celles à base de protéines
de lait de vache ou avec le lait maternel, sur tous les paramètres étudiés (croissance,
santé osseuse et fonctions métaboliques, reproductives, endocrines, immunitaires et
neurologiques). Les aliments au soja semblent exercer un effet protecteur vis-à-vis du
risque de cancer du sein (probablement lié aux isoflavones), surtout si la consommation
est débutée avant l’adolescence. Chez les femmes ayant des antécédents de cancer du sein,
l’alimentation peut intégrer les aliments au soja. Introduire des aliments à base de soja
dans l’alimentation contribue aussi à maintenir la santé cardiovasculaire, du fait de leur
effet favorable sur le taux de LDL-cholestérol, sur la fonction endothéliale, et
potentiellement sur la tension artérielle.
Objective:
The purpose of this study was to investigate whether soy isoflavone supplementation is effective in preventing periodontal destruction exacerbated by estrogen deficiency (ED) and its potential mechanism.
Background:
The progression of periodontitis is affected by host factors, such as smoking, diabetes mellitus, and steroid use. Bone loss in periodontitis can be aggravated by ED.
Methods:
A rat model of experimental periodontitis (EP) with ED was established by silk ligature and inoculation with Porphyromonas gingivalis, and some EP rats were subjected to bilateral ovariectomy (OVX). The treatment groups received an intravenous injection of 17-β-estradiol (E2 B) or soy isoflavones (SI) by gavage. The rats were euthanized, and the maxillary jaws, gingiva, and serum were harvested. Tight junction protein and interleukin (IL)-17 expression, reactive oxygen species (ROS) level, and periodontal destruction were assessed. In addition, we determined whether grainyhead-like 2 (GRHL2) is required for enhancing the epithelial barrier by SI in an in vitro P. gingivalis infection model.
Results:
Estrogen deficiency impaired the expression of genes encoding tight junction proteins in the gingiva, increased IL-17 level, and accelerated alveolar bone resorption. SI treatment alleviated tight junction protein expression, decreased IL-17 and ROS levels, and prevented the absorption of alveolar bone. Furthermore, GRHL2 expression was significantly induced by SI in human oral keratinocytes-1 (HOK-1) cells; GRHL2 knockdown impaired the expression of OCLN and ZO-1 induced by SI treatment.
Conclusion:
Soy isoflavones alleviates periodontitis in OVX rats, as observed by the increased expression of tight junction proteins, and reduced IL-17 level and alveolar bone loss. The in vitro studies suggested that the enhancement of oral epithelial barrier by SI treatment was partially dependent on GRHL2.
Two valine carbamate prodrugs of daidzein were designed to improve its bioavailability. To compare the pharmacokinetic behavior of these prodrugs with different protected phenolic hydroxyl groups of daidzein, a rapid and sensitive method for simultaneous quantification of daidzein, its valine carbamate prodrug, and daidzein‐7‐O‐glucuronide in rat plasma was developed and validated in this study. The samples were processed using a fast one‐step protein precipitation method with methanol added to 50 μL of plasma and were analyzed by ultra‐high performance liquid chromatography with tandem mass spectrometry. To improve the selectivity, peak shape, and peak elution, several key factors, especially stationary phase and the composition of the mobile phase, were tested, and the analysis was performed using the Kinetex® C18 column (100 × 2.1 mm, 2.6 μm) within only 2.6 min under optimal conditions. The established method exhibited good linearity over the concentration range of 2.0‐1000 ng/mL for daidzein, and 8.0‐4000 ng/mL for the prodrug and daidzein‐7‐O‐glucuronide. The accuracy of the quality control samples was between 95.5% and 110.2% with satisfactory intra‐ and inter‐day precision (RSD values < 10.85%), respectively. This sensitive, rapid, low‐cost and high‐throughput method was successfully applied to compare the pharmacokinetic behavior of different daidzein carbamate prodrugs. This article is protected by copyright. All rights reserved
S-equol, an active metabolite of the soy isoflavone daidzein, is mainly metabolized into glucuronide(s) by UDP-glucuronosyltransferase (UGT) enzymes in mammals. In the present study, S-equol glucuronidation was examined in the liver and intestinal microsomes of humans, monkeys, dogs, rats, and mice using a kinetic analysis. CLint values for 7- and 4′-glucuronidation by liver microsomes were higher than those by intestinal microsomes in all species. CLint values for total glucuronidation (sum of 7- and 4′-glucuronidation) were rats (7.6) > monkeys (5.8) > mice (4.9) > dogs (2.8) > humans (1.0) for liver microsomes, and rats (9.6) > mice (2.8) > dogs (1.3) ≥ monkeys (1.2) > humans (1.0) for intestinal microsomes, respectively. Regarding regioselective glucuronidation by liver and intestinal microsomes, CLint values were 7-glucuronidation > 4′-glucuronidation for humans, monkeys, dogs, and mice, and 4′-glucuronidation > 7-glucuronidation for rats. These results suggest that the metabolic abilities of UGT enzymes toward S-equol in the liver and intestines markedly differ among humans, monkeys, dogs, rats, and mice.
Glucuronidation is a well-recognized phase II metabolic pathway for a variety of chemicals including drugs and endogenous substances. Although it is usually the secondary metabolic pathway for a compound preceded by phase I hydroxylation, glucuronidation alone could serve as the dominant metabolic pathway compounds, including some with high aqueous solubility. Glucuronidation involves the metabolism of parent compound by UDP-glucuronosyltransferases (UGTs) into hydrophilic and negatively charged glucuronides that cannot exit the cell without the aid of efflux transporters. Therefore, elimination of parent compound via glucuronidation in a metabolic active cell is controlled by two driving forces; the formation of glucuronides by UGT enzymes and the (polarized) excretion of these glucuronides by efflux transporters located on the cell surfaces in various drug disposition organs.
Contrary to the common assumption that the glucuronides reaching the systemic circulation were destined for urinary excretion, recent evidences suggest that hepatocytes are capable of highly efficient biliary clearance of the gut-generated glucuronides. Furthermore, the biliary- and enteric- eliminated glucuronides participate into recycling schemes involving intestinal microbes, which often prolong their local and systemic exposure, albeit at low systemic concentrations. Taken together, these recent research advances indicate that though UGT determines the rate and extent of glucuronide generation, the efflux and uptake transporters determine the distribution of these glucuronides into blood and then to various organs for elimination. Recycling schemes impact the apparent plasma half-life of parent compounds and their glucuronides that reach intestinal lumen, in addition to prolonging their gut and colon exposure.
Some felid diets contain isoflavones but the metabolic capacity of cats towards isoflavones is relatively unknown, despite the understanding that isoflavones have divergent biological potential according to their metabolite end-products. The objective of this study was to determine the plasma metabolites detectable in domestic cats after exposure to 2 different dietary forms of isoflavones, either as a soy extract tablet (n = 6) or as part of a dietary matrix (n = 4). Serial blood samples were collected after isoflavone exposure to identify the plasma metabolites of each cat. Genistein was detected in its unconjugated form or as a monosulfate. Daidzein was detected as both a mono- and di-sulfate, as well as in its unconjugated form. Other daidzein metabolites detected included equol mono- and di-sulfate, dihydrodaidzein, and O-desmethylangolensin. No β-glucuronide metabolites of either isoflavone were detected. Equol was produced in markedly fewer cats after ingestion of a soy extract tablet as a single oral bolus compared to cats consuming an isoflavone-containing diet. The detectable metabolites of the isoflavones, genistein, and daidzein in domestic cat plasma after dietary ingestion may have been described in the present study for the first time. The metabolic capacity for isoflavones by domestic cats appears to be efficient, with only minimal proportions of the ingested amount detected in their unconjugated forms. This has implications for the potential of isoflavones to exert physiological activity in the domestic cat when consumed at concentrations representative of dietary intake.
Whether soy food consumption may protect against coronary heart disease (CHD) remains controversial. No previous study has used biomarkers of soy intake in assessing the relationship between soy consumption and CHD. Biomarkers that reflect both intake and metabolism may be more informative than self-reports of dietary intake.
We examined associations of urinary isoflavonoids, a biomarker of soy or soy isoflavone intake, with risk of CHD in a case-control study nested within two prospective cohort studies of Chinese adults in Shanghai. Cases were defined as subjects with no history of CHD at baseline who developed incident CHD during follow-up. Control subjects were randomly selected from those who remained free of CHD and matched to cases by sex, age, date and time of sample collection and antibiotic use. Baseline urinary isoflavonoids (daidzein, genistein, glycitein, equol, O-desmethylangolensin, dihydrodaidzein and dihydrogenistein) were compared between cases (n = 377) and control subjects (n = 753). Conditional logistic regression was used to evaluate the associations.
Total urinary isoflavonoids were not associated with CHD in either women or men. However, urinary equol excretion showed a significant inverse association with CHD in women. The adjusted odds ratios (95% confidence intervals) for CHD across increasing quartiles of equol levels in women were 1 (reference), 0.61 (0.32, 1.15), 0.51 (0.26, 0.98) and 0.46 (0.24, 0.89) (P = 0.02 for trend).
Our study suggests for the first time that equol, a bioactive metabolite of soy isoflavone daidzein, may be inversely associated with risk of CHD in women.
The domestic cat (Felis catus) shows remarkable sensitivity to the adverse effects of phenolic drugs, including acetaminophen and aspirin, as well as structurally-related toxicants found in the diet and environment. This idiosyncrasy results from pseudogenization of the gene encoding UDP-glucuronosyltransferase (UGT) 1A6, the major species-conserved phenol detoxification enzyme. Here, we established the phylogenetic timing of disruptive UGT1A6 mutations and explored the hypothesis that gene inactivation in cats was enabled by minimal exposure to plant-derived toxicants. Fixation of the UGT1A6 pseudogene was estimated to have occurred between 35 and 11 million years ago with all extant Felidae having dysfunctional UGT1A6. Out of 22 additional taxa sampled, representative of most Carnivora families, only brown hyena (Parahyaena brunnea) and northern elephant seal (Mirounga angustirostris) showed inactivating UGT1A6 mutations. A comprehensive literature review of the natural diet of the sampled taxa indicated that all species with defective UGT1A6 were hypercarnivores (>70% dietary animal matter). Furthermore those species with UGT1A6 defects showed evidence for reduced amino acid constraint (increased dN/dS ratios approaching the neutral selection value of 1.0) as compared with species with intact UGT1A6. In contrast, there was no evidence for reduced amino acid constraint for these same species within UGT1A1, the gene encoding the enzyme responsible for detoxification of endogenously generated bilirubin. Our results provide the first evidence suggesting that diet may have played a permissive role in the devolution of a mammalian drug metabolizing enzyme. Further work is needed to establish whether these preliminary findings can be generalized to all Carnivora.
The pathologic changes associated with hyperthyroidism (adenomatous hyperplasia, adenoma of the thyroid gland) have been well characterized in cats, but the pathogenesis of these changes remains unclear. In this research, we undertook a case-control study to search for potential risk factors for this disease. Owners of 379 hyperthyroid and 351 control cats were questioned about their cats' exposure to potential risk factors including breed, demographic factors, medical history, indoor environment, chemicals applied to the cat and environment, and diet. The association between these hypothesized risk factors and outcome of disease was evaluated by conditional logistic regression. Two genetically related cat breeds (ie, Siamese and Himalayan) were found to have diminished risk of developing hyperthyroidism. Cats that used litter had higher risk of developing hyperthyroidism than those that did not. Use of topical ectoparasite preparations was associated with increased risk of developing hyperthyroidism. Compared with cats that did not eat canned food, those that ate commercially prepared canned food had an approximate 2-fold increase in risk of disease. When these 4 variables (breed, use of cat litter, consumption of canned cat food, and use of topical ectoparasite preparations) from the univariate analysis were selected for further study as candidate risk factors and analyzed by multivariate conditional logistic regression, a persistent protective effect of breed (ie, Siamese or Himalayan) was found. In addition, results suggested a 2- to 3-fold increase in risk of developing hyperthyroidism among cats eating a diet composed mostly of canned cat food and a 3-fold increase in risk among those using cat litter. In contrast, the use of commercial flea products did not retain a strong association. The results of this study indicate that further research into dietary and other potentially important environmental factors (eg, cat litter) is warranted.
Equol [7-hydroxy-3-(4'-hydroxyphenyl)-chroman] is a nonsteroidal estrogen of the isoflavone class. It is exclusively a product of intestinal bacterial metabolism of dietary isoflavones and it possesses estrogenic activity, having affinity for both estrogen receptors, ERalpha and ERbeta. Equol is superior to all other isoflavones in its antioxidant activity. It is the end product of the biotransformation of the phytoestrogen daidzein, one of the two main isoflavones found in abundance in soybeans and most soy foods. Once formed, it is relatively stable; however, equol is not produced in all healthy adults in response to dietary challenge with soy or daidzein. Several recent dietary intervention studies examining the health effects of soy isoflavones allude to the potential importance of equol by establishing that maximal clinical responses to soy protein diets are observed in people who are good "equol-producers." It is now apparent that there are two distinct subpopulations of people and that "bacterio-typing" individuals for their ability to make equol may hold the clue to the effectiveness of soy protein diets in the treatment or prevention of hormone-dependent conditions. In reviewing the history of equol, its biological properties, factors influencing its formation and clinical data, we propose a new paradigm. The clinical effectiveness of soy protein in cardiovascular, bone and menopausal health may be a function of the ability to biotransform soy isoflavones to the more potent estrogenic isoflavone, equol. The failure to distinguish those subjects who are "equol-producers" from "nonequol producers" in previous clinical studies could plausibly explain the variance in reported data on the health benefits of soy.
To determine whether the increasing prevalence of feline hyperthyroidism is the result of aging of the cat population and whether consumption of canned foods at various times throughout life is associated with increased risk of hyperthyroidism.
Retrospective and case-control studies.
Medical records of 169,576 cats, including 3,570 cats with hyperthyroidism, evaluated at 9 veterinary school hospitals during a 20-year period, and 109 cats with hyperthyroidism (cases) and 173 cats without hyperthyroidism (controls).
Age-adjusted hospital prevalence of hyperthyroidism was calculated by use of Veterinary Medical Database records. On the basis of owners' questionnaire responses, logistic regression was used to evaluate associations between consumption of canned food and development of hyperthyroidism.
Age-specific hospital prevalence of feline hyperthyroidism increased significantly from 1978 to 1997. Overall, consumption of pop-top canned (vs dry) food at various times throughout life and each additional year of age were associated with greater risk of developing hyperthyroidism. In female cats, increased risk was associated with consumption of food packaged in pop-top cans or in combinations of pop-top and non-pop-top cans. In male cats, increased risk was associated with consumption of food packaged in pop-top cans and age.
These findings suggest that the increasing prevalence of feline hyperthyroidism is not solely the result of aging of the cat population and that canned foods may play a role.
The indigenous intestinal microflora are involved in a variety of processes within the human body, and are important for maintaining host health. As such, interindividual differences in the ability to harbor certain intestinal bacteria might be associated with interindividual differences in health and/or disease susceptibility. In the last decade there has been considerable interest in phytoestrogen intakes in relation to human health. Daidzein, an isoflavone phytoestrogen found in soy, is metabolized to equol and O-desmethylangolensin (O-DMA) by intestinal bacteria. The specific bacterium/bacteria responsible for equol and O-DMA production in humans have yet to be identified definitively, but in vitro and animal studies have suggested that equol and O-DMA are more biologically active than their precursor daidzein. Interestingly, substantial interindividual differences in daidzein metabolism exist; following soy or daidzein consumption, approximately 30%-50% of the human population produce equol, and approximately 80%-90% produce O-DMA. Observational and intervention studies in humans have suggested that the ability to produce equol and O-DMA may be associated with reduced risk of certain diseases including breast and prostate cancers. However, relatively few studies have been conducted to date. In this review, we discuss the available evidence for a relationship between daidzeinmetabolizing phenotypes and human health, and suggest potential mechanisms for some of the reported relationships.
Although it is widely appreciated that cats respond differently to certain drugs compared with other companion animal species, the causes of these differences are poorly understood. This article evaluates published evidence for altered drug effects in cats, focusing on pharmacokinetic differences between cats, dogs, and humans, and the molecular mechanisms underlying these differences. More work is needed to better understand drug metabolism and disposition differences in cats, thereby enabling more rational prescribing of existing medications, and the development of safer drugs for this species.
The soybean has been implicated in diet-induced goiter by many studies. The extensive consumption of soy products in infant formulas and in vegetarian diets makes it essential to define the goitrogenic potential. In this report, it was observed that an acidic methanolic extract of soybeans contains compounds that inhibit thyroid peroxidase- (TPO) catalyzed reactions essential to thyroid hormone synthesis. Analysis of the soybean extract using HPLC, UV-VIS spectrophotometry, and LC-MS led to identification of the isoflavones genistein and daidzein as major components by direct comparison with authentic standard reference isoflavones. HPLC fractionation and enzymatic assay of the soybean extract showed that the components responsible for inhibition of TPO-catalyzed reactions coeluted with daidzein and genistein. In the presence of iodide ion, genistein and daidzein blocked TPO-catalyzed tyrosine iodination by acting as alternate substrates, yielding mono-, di-, and triiodoisoflavones. Genistein also inhibited thyroxine synthesis using iodinated casein or human goiter thyroglobulin as substrates for the coupling reaction. Incubation of either isoflavone with TPO in the presence of H2O2 caused irreversible inactivation of the enzyme; however, the presence of iodide ion in the incubations completely abolished the inactivation. The ic50 values for inhibition of TPO-catalyzed reactions by genistein and daidzein were ca. 1–10 μM, concentrations that approach the total isoflavone levels (ca. 1 μM) previously measured in plasma from humans consuming soy products. Because inhibition of thyroid hormone synthesis can induce goiter and thyroid neoplasia in rodents, delineation of anti-thyroid mechanisms for soy isoflavones may be important for extrapolating goitrogenic hazards identified in chronic rodent bioassays to humans consuming soy products.
We characterized the isoform specific glucuronidation of six isoflavones, genistein, daidzein, glycitein, formononetin, biochanin A and prunetin, using 12 expressed human UGTs and human intestinal and liver microsomes. The results indicated that these isoflavones are metabolized most rapidly at three different concentrations by one of these four UGT isoforms: UGT1A1, UGT1A8, UGT1A9 and UGT1A10. Furthermore, glycitein was usually metabolized the fastest whereas prunetin the slowest. Using the rates of metabolism by 12 UGT isoforms as a means to establish the metabolic "fingerprint", we found that each isoflavone had distinctive concentration-dependent patterns. Determination of kinetic parameters of glucuronidation using genistein and prunetin indicated that the distinct concentration-dependent metabolic patterns were the result of differences in K(m) and V(max) values. We then measured how well metabolic "fingerprinting" predicted metabolism of these isoflavones by human intestinal and liver microsomes. We found that the prediction was rather successful for five isoflavones in the liver microsomes, but not successful in the intestinal microsomes. We propose that a newly discovered UGT3A1 isoform capable of metabolizing phenols and estrogens might be responsible for the metabolism of isoflavones such as formononetin in humans. In conclusion, the first systematic study of metabolic "fingerprinting" of six common isoflavones showed that each isoflavone has UGT isoform-specific metabolic patterns that are concentration-dependent and predictive of metabolism of the isoflavones in liver microsomes.
To evaluate the effect of a soy-based diet on general health and adrenocortical and thyroid gland function in dogs. Animals-20 healthy privately owned adult dogs.
In a randomized controlled clinical trial, dogs were fed a soy-based diet with high (HID; n = 10) or low (LID; 10) isoflavones content. General health of dogs, clinicopathologic variables, and serum concentrations of adrenal gland and thyroid gland hormones were assessed before treatment was initiated and up to 1 year later. Differences between groups with respect to changes in the values of variables after treatment were assessed by means of a Student t test (2 time points) and repeated-measures ANOVA (3 time points).
No differences were detected between the 2 groups with respect to body condition and results of hematologic, serum biochemical, and urine analyses. Most serum concentrations of hormones did not change significantly after treatment, nor were they affected by diet. However, the mean change in serum concentration of total thyroxine was higher in the HID group (15.7 pmol/L) than that in the LID group (-1.9 pmol/L). The mean change in estradiol concentration after ACTH stimulation at 1 year after diets began was also higher in the HID group (19.0 pg/mL) than that in the LID group (-5.6 pg/mL).
Phytoestrogens may influence endocrine function in dogs. Feeding soy to dogs on a long-term basis may influence results of studies in which endocrine function is evaluated, although larger studies are needed to confirm this supposition.
Examination was made of the urinary and biliary excretion of metabolites of daidzin and daidzein, the major components of roots of Pueraria lobata Ohwi (Leguminosae) in rats. The urine of rats administered daidzin orally contained four major metabolites, daidzein 7,4'-di-O-sulfate (M-1), daidzein 7-O-beta-D-glucuronide (M-2), daidzein 4'-O-sulfate (M-3), daidzein (M-4), as determined from spectroscopic and chemical data. The urine of rats treated with daidzein contained M-2--M-4 in the above metabolites. Total cumulative amounts of the four metabolites excreted in the urine at 48 h following the oral administration of daidzin and daidzein were approximately 4.8% and 4.6% of the doses administered, respectively. The bile of rats administered daidzin orally contained M-1--M-4. Daidzein 7-O-beta-D-glucuronide 4'-O-sulfate (M-5), a major biliary metabolite, was identified by the high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) spectra. At least daidzin appeared to be hydrolyzed to aglycone after absorption in the body, and as a part of metabolites, M-1--M-4 having free hydroxyl, glucuronided or sulfated hydroxyls at the C-7 position, may then be excreted in the urine and bile.
We have discussed the etiopathology of feline toxic nodular goiter in the context of human nodular goiter pathogenesis. We have reviewed thyroid heterogeneity, growth regulation, functional and growth autonomy, nodule and tumor formation, and the evolution of toxic nodular goiter in the human being. By addressing toxic nodular goiter of the cat, the history, morphologic findings, xenotransplantation and cell culture studies, evidence for and against circulating thyroid stimulators and epizootiological studies of the feline disease have been summarized. Due to its structure, the thyroid gland offers some unique possibilities to study the mechanisms that are responsible for cellular heterogeneity, the emergence of autonomous nodular growth and function, and, ultimately, the development of tumors. The demonstration of naturally occurring clones of cells with high intrinsic proliferation potential within the follicular epithelium of the thyroid has fostered promising new concepts on the genesis of nodular growth of benign and possibly malignant endocrine tumors. Hyperthyroid cat goiters contain single or multiple, autonomously (i.e., TSH-independently) functioning and growing nodules. Neither hyperfunction nor growth of these nodules depends on extrathyroidal circulating stimulators. The basic lesion appears to be an excessive intrinsic growth capacity of some thyroid cells. The factors enhancing the transformation of a normal thyroid into a nodular hyperfunctioning goiter over many years are still unknown. Immunological, environmental, and nutritional factors are the focus of ongoing studies, but an infectious agent can not yet be excluded.
Biotransformation of the triazolobenzodiazepine alprazolam (ALP) was studied in vitro using hepatic microsomal preparations from human, monkey, mouse, and rat liver tissue. Two principal hydroxylated metabolites were identified: 4-hydroxy- and alpha-hydroxy-alprazolam (4-OH-ALP and alpha-OH-ALP). In all species, rates of 4-OH-ALP formation exceeded those of alpha-OH-ALP. In human liver microsomes, ratios of 4-OH-ALP/alpha-OH-ALP reaction velocities calculated at clinically relevant plasma concentrations of ALP ranged from 7 to 17, qualitatively consistent with, but numerically larger than, the ratio of the plasma levels of the two metabolites during clinical use of ALP in humans. Km values for both 4-OH-ALP (170-305 microM) and alpha-OH-ALP (63-441 microM) considerably exceeded the usual maximum plasma concentration observed in humans (200 ng/ml, 0.65 microM), consistent with the linear (dose-independent) pharmacokinetic characteristics of ALP observed in humans. Thus formation of 4-OH-ALP via hydroxylation is the major route of ALP metabolism. This pathway is probably mediated by the cytochrome P-450-3A subfamily. Factors that impair the activity of this cytochrome subtype are likely to impair clearance of ALP in vivo.
Examination was made of the urinary and biliary excretion of the metabolites of genistein and genistein, the major components of Glycine and Sophora genus in rats. The urine of rats administered genistein orally contained eight metabolites. Three of these metabolites, genistein 4'-O-sulfate (M-1), genistein 7-O-beta-D-glucuronide (M-3), genistein 4'-O-sulfate 7-O-beta-D-glucuronide (M-6), were identified from spectroscopic and chemical data. The bile of rats administered genistein orally contained M-2, M-3 and M-6. M-6, a major biliary metabolite, was isolated and identified from spectroscopic and chemical data. The urine or bile of rats treated with genistein, the glycoside of genistein, contained M-1-M-8 or M-2, M-3, M-6 in the above metabolites. These findings suggest that genistein is absorbed as genistein after hydrolysis in the gastrointestinal tract. The total cumulative amounts of the two metabolites and genistein excreted in the urine during 48h, or of M-6 excreted in the bile during 36h following the oral administration of genistein, were approximately 5.7% or 16.0% of the doses administered, respectively. The result show that M-1, M-3 and M-6, having a free hydroxyl, glucuronide- or sulfate-conjugated hydroxyls at the C-7 or C-4' position, are excreted in the urine and bile as parts of the metabolites of genistein.
Flavonoids are widely distributed in plant-derived foods and possess a variety of biological activities including antithyroid effects in experimental animals and humans. A structure-activity study of 13 commonly consumed flavonoids was conducted to evaluate inhibition of thyroid peroxidase (TPO), the enzyme that catalyzes thyroid hormone biosynthesis. Most flavonoids tested were potent inhibitors of TPO, with IC50 values ranging from 0.6 to 41 microM. Inhibition by the more potent compounds, fisetin, kaempferol, naringenin, and quercetin, which contain a resorcinol moiety, was consistent with mechanism-based inactivation of TPO as previously observed for resorcinol and derivatives. Other flavonoids inhibited TPO by different mechanisms, such as myricetin and naringin, showed noncompetitive inhibition of tyrosine iodination with respect to iodine ion and linear mixed-type inhibition with respect to hydrogen peroxide. In contrast, biochanin A was found to be an alternate substrate for iodination. The major product, 6,8-diiodo-biochanin A, was characterized by electrospray mass spectrometry and 1H-NMR. These inhibitory mechanisms for flavonoids are consistent with the antithyroid effects observed in experimental animals and, further, predict differences in hazards for antithyroid effects in humans consuming dietary flavonoids. In vivo, suicide substrate inhibition, which could be reversed only by de novo protein synthesis, would be long-lasting. However, the effects of reversible binding inhibitors and alternate substrates would be temporary due to attenuation by metabolism and excretion. The central role of hormonal regulation in growth and proliferation of thyroid tissue suggests that chronic consumption of flavonoids, especially suicide substrates, could play a role in the etiology of thyroid cancer.
The soybean has been implicated in diet-induced goiter by many studies. The extensive consumption of soy products in infant formulas and in vegetarian diets makes it essential to define the goitrogenic potential. In this report, it was observed that an acidic methanolic extract of soybeans contains compounds that inhibit thyroid peroxidase- (TPO) catalyzed reactions essential to thyroid hormone synthesis. Analysis of the soybean extract using HPLC, UV-VIS spectrophotometry, and LC-MS led to identification of the isoflavones genistein and daidzein as major components by direct comparison with authentic standard reference isoflavones. HPLC fractionation and enzymatic assay of the soybean extract showed that the components responsible for inhibition of TPO-catalyzed reactions coeluted with daidzein and genistein. In the presence of iodide ion, genistein and daidzein blocked TPO-catalyzed tyrosine iodination by acting as alternate substrates, yielding mono-, di-, and triiodoisoflavones. Genistein also inhibited thyroxine synthesis using iodinated casein or human goiter thyroglobulin as substrates for the coupling reaction. Incubation of either isoflavone with TPO in the presence of H2O2 caused irreversible inactivation of the enzyme; however, the presence of iodide ion in the incubations completely abolished the inactivation. The IC50 values for inhibition of TPO-catalyzed reactions by genistein and daidzein were ca. 1-10 microM, concentrations that approach the total isoflavone levels (ca. 1 microM) previously measured in plasma from humans consuming soy products. Because inhibition of thyroid hormone synthesis can induce goiter and thyroid neoplasia in rodents, delineation of anti-thyroid mechanisms for soy isoflavones may be important for extrapolating goitrogenic hazards identified in chronic rodent bioassays to humans consuming soy products.
Most methods for detecting isoflavones in biological samples do not measure the concentration of sulfate conjugates. An LC-MS method is reported here to estimate urinary concentrations of genistein and daidzein, their sulfate and glucuronide conjugates and other major metabolites. Human and rat urine samples were extracted with diethyl ether, or pre-digested with sulfatase and/or beta-glucuronidase followed by extraction. The isoflavones were separated using gradient LC methods and detected by negative single ion monitoring on an MS system using a heated nebulizer atmospheric pressure chemical ionization interface. CVs for inter- and intra-assay variability were generally < 20 and 10%, respectively. Preliminary studies using these procedures demonstrate 52+/-4 and 26+/-4% of genistein in rat urine was found as the aglycone and sulfate conjugates, respectively, compared to 0.36 and 4%, respectively, in human urine. This method is suitable for the study of isoflavone sulfate conjugates in biological fluids.
Soybean products containing isoflavones are widely consumed in Western and Asian diets for putative health benefits, but adverse effects are also possible. The conjugated forms of isoflavones present in a soy nutritional supplement (predominately acetyl glucosides) and in blood from two human volunteers after consuming the supplement (7- and 4'-glucuronides and sulfates) were identified using liquid chromatography coupled with electrospray/tandem mass spectrometry. Circulating conjugates of genistein and daidzein were quantified using selective enzymatic hydrolysis and deuterated internal standards for liquid chromatography-electrospray/mass spectrometry. The levels of isoflavone glucuronides were much greater than the corresponding sulfates or aglycones. The substrate activities of genistein and daidzein were evaluated with recombinant human UDP glucuronosyl transferase (UGT) and sulfotransferase (SULT) by using enzyme kinetics. The SULTs 1A1*2, 1E, and 2A1 catalyzed formation of a single genistein sulfate; however, SULTs 1A2*1 and 1A3 had no observed activity. None of the SULTs showed activity with daidzein. Although several UGTs (1A1, 1A4, 1A6, 1A7, 1A9, and 1A10) catalyzed 7- and 4'-glucuronidation of genistein or daidzein, the UGT 1A10 isoform, which is found in human colon but not liver, was found to be specific for genistein. Glucuronidation of only genistein was observed in human colon microsomes, although nearly equal activity was observed for daidzein in human liver and kidney microsomes. These findings suggest a prominent role for glucuronidation of genistein in the intestine concomitant with absorption, although hepatic glucuronidation of absorbed genistein and daidzein aglycones is also likely.
The domestic cat has a significantly lower capacity to glucuronidate planar phenolic xenobiotics compared with most other mammalian species. The aim of this study was to determine the mechanistic basis for this anomaly. Current knowledge of the substrate specificity of UDP-glucuronosyltransferase (UGT) isoforms indicates that the cat may either lack or poorly express UGT1A6. Initially, a novel cloning technique was used to identify UGT1A genes expressed in cat liver. Only two unique UGT1A isoforms could be discriminated. The first (28%, of clones) was most homologous to UGT1A1 (the bilirubin-UGT), while the second (72% of clones) showed homology to several isoforms, but could not be unambiguously identified, and was designated cat UGT1A02. Southern blot analysis confirmed the presence of a single UGT1A6-homologous region in the cat genome. Subsequent cloning and sequencing of the entire UGT1A6 exon 1 coding region revealed five deleterious genetic mutations. Identical mutations were found by sequencing of UGT1A6 exon 1 from five other unrelated cats. Four of these five genetic lesions were also identified in the UGT1A6 exon 1 region of a margay (Leopardus wiedii). Finally, RT-PCR of liver mRNA from four different cats confirmed the presence of UGT1A1 and UGT1A02, but not UGT1A6. In conclusion, UGT1A6 is a pseudogene in the domestic cat and in at least one other phylogenetically related species. Furthermore, cats appear to have a less diverse pattern of UGT1A isoform expression compared with other species. Such differences most likely reflect the highly carnivorous diet of Feliform species and resultant minimal exposure to phytoalexins.
To identify dietary and environmental risk factors for hyperthyroidism in cats.
Case-control study.
100 cats with hyperthyroidism and 163 control cats.
Medical records were examined, and owners completed a mailed questionnaire. Data collected included information regarding demographic variables, environmental exposures, and diet, including preferred flavors of canned cat food.
Case cats were significantly less likely to have been born recently than control cats. Housing; exposure to fertilizers, herbicides, or plant pesticides; regular use of flea products; and presence of a smoker in the home were not significantly associated with an increased risk of disease, but cats that preferred fish or liver and giblets flavors of canned cat food had an increased risk.
Results suggest that cats that prefer to eat certain flavors of canned cat food may have a significantly increased risk of hyperthyroidism.
To determine the absolute and relative soy isoflavone content in commercial cat foods.
14 dry, 6 semimoist, and 22 moist commercial cat foods.
Soy isoflavone content of each food was determined by use of acid-methanol hydrolysis and high-pressure liquid chromatography with ultraviolet absorbance detection. Isoflavones were identified and quantified by reference to authentic standards.
Genistein and daidzein were the major soy isoflavones identified in 24 of 42 foods, with concentrations ranging from 1 to 163 microg/g of food. Foods labeled as containing soybean solids (16/42) had isoflavone concentrations > 11 microg/g. More dry (13/14) and semimoist (6/6) foods contained isoflavones than moist foods (5/22). Isoflavone content and food cost were negatively correlated for dry and semimoist foods but not for moist foods. Total amount of isoflavone consumed by cats fed these soy-containing foods as a sole maintenance diet was estimated to be between 0.6 and 4.5 mg/kg of body weight/d, which is comparable to concentrations in humans that result in a measurable although modest effect on serum concentrations of steroid and thyroid hormones.
Genistein and daidzein are common constituents of commercial cat foods. Predictors of isoflavone content included ingredient labeling, food type, and food cost. Soy isoflavones in some commercial cat foods were detected in amounts predicted to have a biological effect.
The principal objective of this study was to determine whether ferrets glucuronidate acetaminophen more slowly compared with other species, and if so investigate the molecular basis for the difference. Acetaminophen-UDP-glucuronosyltransferase (UGT) activities were measured using hepatic microsomes from eight ferrets, four humans, four cats, four dogs, rat, mouse, cow, horse, monkey, pig and rabbit. Gender differences between male and female ferret livers were explored using enzyme kinetic analysis. Immunoblotting of microsomal proteins was also performed using UGT-specific antibodies. Finally, the exon 1 region of UGT1A6, a major acetaminophen-UGT, was sequenced. Glucuronidation of acetaminophen was relatively slow in ferret livers compared with livers from all other species except cat. Gender differences were also apparent, with intrinsic clearance (Vmax/Km) values significantly higher in male compared with female ferret livers. Furthermore, Vmax values correlated with densitometric measurements of two protein bands identified with a UGT1A subfamily-specific antibody. No deleterious mutations were identified in the exon 1 or flanking regions of the ferret UGT1A6 gene. In conclusion, like cats, ferret livers glucuronidate acetaminophen relatively slowly. However, unlike cats, in which UGT1A6 is encoded by a pseudogene and dysfunctional, there are no defects in the ferret UGT1A6 gene which could account for the low activity.
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.
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.
Six men and 6 women ( +/- 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 beta-glucuronidase (EC 3.2.1.31) and sulfatase (EC 3.1.6.1) digestion and liquid chromatography-mass spectrometry.
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(1/2 lambdaz)) was 3.1, 5.7, 3.2, and 8.4 h, respectively.
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.
To identify and determine the concentrations of phytoestrogens in commercial dog foods.
24 commercial dog foods, including 12 moist or dry extruded commercial dog foods that contained soybeans or soybean fractions and 12 foods without any soybean-related ingredients listed on the label.
Foods were analyzed for phytoestrogen content, including 4 isoflavones (genistein, glycitein, daidzein, and biochanin A), 1 coumestan (coumestrol), and 2 lignans (secoisolariciresinol and matairesinol) by use of acid-methanol hydrolysis and high-pressure liquid chromatography with UV-absorbance detection. Phytoestrogens were identified and quantified by reference to authentic standards.
Isoflavones, coumestans, and lignans were undetectable in diets that did not list soybean-related ingredients on the label. Only 1 of the 12 diets that included soybean or soybean fractions had undetectable concentrations of phytoestrogens and that product contained soy fiber. The major phytoestrogens were the isoflavones daidzein (24 to 615 microg/g of dry matter) and genistein (4 to 238 microg/g of dry matter).
Soybean and soybean fractions are commonly used ingredients in commercial dog foods. Dietary intake of phytoestrogens may have both beneficial and deleterious health effects. Our results indicated that certain commercial dog foods contain phytoestrogens in amounts that could have biological effects when ingested long-term.
To compare effects of short-term administration of a soy diet with those of a soy-free diet on serum thyroid hormone concentrations in healthy adult cats.
18 healthy adult cats.
Cats were randomly assigned to receive either a soy or soy-free diet for 3 months each in a crossover design. Assays included CBC, serum biochemical profile, thyroid hormone analysis, and measurement of urinary isoflavone concentrations.
Genistein, a major soy isoflavone, was identified in the urine of 10 of 18 cats prior to dietary intervention. Compared with the soy-free diet, cats that received the soy diet had significantly higher total thyroxine (T4) and free T4 (fT4) concentrations, but unchanged total triiodothyronine (T3) concentrations. The T3/fT4 ratio was also significantly lower in cats that received the soy diet. Although the magnitudes of the increases were small (8% for T4 and 14% for fT4), these changes resulted in an increased proportion of cats (from 1/18 to 4/18) that had fT4 values greater than the upper limit of the laboratory reference range. There was no significant effect of diet on any other measured parameter.
Short-term administration of dietary soy has a measurable although modest effect on thyroid hormone homeostasis in cats. Increase in T4 concentration relative to T3 concentration may result from inhibition of 5'-iodothyronine deiodinase or enhanced T3 clearance. Soy is a common dietary component that increases serum T4 concentration in cats.
The majority of UDP-glucuronosyltransferases (UGT), like other drug-metabolizing enzymes, display broad and often overlapping substrate specificities, complicating evaluation of the function of individual UGT isoforms within human tissues. Despite this, there have been recent advances in identifying UGT-selective probes--UGT substrates that are primarily glucuronidated by a single isoform. Such probes can be used to (1) facilitate identification of UGT isoforms mediating a particular glucuronidation activity in human liver through activity correlation analysis; (2) evaluate the role of particular UGTs in drug-drug interactions through either enzyme induction or inhibition; and (3) elucidate the functional significance of genetic polymorphisms associated with the gene encoding the UGT of interest. UGT-selective probes currently being used in our laboratory for the evaluation of glucuronidation activities in human liver tissues include estradiol (3OH-glucuronidation; UGT1A1), trifluoperazine (UGT1A4) serotonin (UGT1A6), propofol (UGT1A9), 3'-azidothymidine (UGT2B7), and S-oxazepam (UGT2B15). In vitro incubation protocols and the HPLC analysis methods used to determine each of these activities are described in detail. Future work is needed to elucidate more highly selective probes than those in current usage, as well as probes for the extrahepatic UGT isoforms.
Soy isoflavones have received considerable attention. Individuals with isoflavones-rich diets have significantly lower occurrences of cardiovascular disease, osteoporosis, and some cancers. The clinical effectiveness of soy isoflavones may be a function of the ability to biotransform soy isoflavones to the more potent estrogenic metabolite, equol, which may enhance the actions of soy isoflavones, owing to its greater affinity for estrogen receptors, unique antiandrogenic properties, and superior antioxidant activity. However, not all individuals consuming daidzein produce equol. Only approximately one-third to one-half of the population is able to metabolize daidzein to equol. This high variability in equol production is presumably attributable to interindividual differences in the composition of the intestinal microflora, which may play an important role in the mechanisms of action of isoflavones. But, the specific bacterial species in the colon involved in the production of equol are yet to be discovered. Therefore, future researches are aimed at identifying the specific bacterial species and strains that are capable of converting daidzein to equol or increasing equol production.
The soyabean isoflavones genistein and daidzein, which may protect against some cancers, cardiovascular disease and bone mineral loss, undergo substantial Phase 2 metabolism, predominantly glucuronidation. We observed a correlation between rates of metabolism of marker substrates of specific UGTs and rates of glucuronidation of genistein and daidzein in vitro by a panel of human liver microsomes, demonstrating that UGT1A1 and UGT1A9, but not UGT1A4, make a major contribution to the metabolism of these isoflavones by human liver. These findings were substantiated by observations that recombinant human UGT1A1 and UGT1A9, but not UGT1A4, catalysed the production of the major glucuronides of both genistein and daidzein in vitro. Recombinant human UGT1A8 also metabolised both genistein and daidzein, whereas UGT1A6 was specific to genistein and UGTs 2B7 and 2B15 were inactive, or only marginally active, with either isoflavone as substrate. The intestinal isoform UGT1A10 metabolised either both isoflavones or genistein only, depending on the commercial supplier of the recombinant enzyme, possibly as a result of a difference in amino acid sequence, which we were unable to confirm. Daidzein (16 microM) increased cell death in the MCF-7 human breast cancer cell line and this effect was reversed by glucuronidation. In view of a well-characterised functional polymorphism in UGT1A1, these observations may have implications for inter-individual variability in the potential health-beneficial effects of isoflavone consumption.