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Food Science and Human Nutrition, Iowa State University, Ames, IA 50011-1123
* To whom correspondence should be addressed. E-mail: shendric{at}iastate.edu.
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ABSTRACT |
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 TOP ABSTRACT IntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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Introduction |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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The hamster model has also provided contrasting results in studies of the effects of isoflavones. Song et al. (6) fed male and female 6–8-wk-old Golden Syrian hamsters various diets with or without isoflavones. Plasma total cholesterol and non-HDL cholesterol (non-HDL-C) concentrations were significantly less in hamsters fed soy protein with or without isoflavones or fed 1.3 mmol daidzein/kg diet in a casein-based diet (approximately the concentration of total isoflavones in a 25% soy protein diet) compared with concentrations in those fed a high-fat, casein control diet, illustrating similar efficacy for cholesterol lowering by soy protein and daidzein). The cholesterol-lowering effects of isoflavones increased with increasing isoflavone doses in 6-mo-old ovariectomized Golden Syrian hamsters (7). In another study, plasma isoflavones showed a positive correlation with plasma LDL + VLDL-cholesterol concentration in female hamsters. Male hamsters fed isoflavones as part of a casein/lactalbumin diet did not have lower cholesterol concentrations than those fed the control diet, but isoflavones within a soy diet increased the cholesterol-lowering effect (8). The addition of lactalbumin to casein seemed to alter the effects of isoflavones on blood lipids.
Inter-individual variability in apparent absorption of isoflavones may account for the observed negligible effects of isoflavones in meta-analyses of soy protein feeding trials (1). In a randomized crossover design, 7 women fed various doses of soy milk showed distinct patterns of urinary isoflavone excretion; 2 women had greater excretion of isoflavones than did the other 5 women (9). Human fecal isoflavone disappearance phenotypes were observed in vitro (10). The phenotypes were stable in 12 of 15 subjects reexamined after 10 mo, and fecal isoflavone disappearance was strongly inversely correlated with plasma isoflavones in 8 men fed a single isoflavone dose (10). Gut microbes seemed played an important role in the metabolism of isoflavones. Zheng et al. (11) identified fecal isoflavone disappearance phenotypes among 35 Chinese and 33 Caucasian women. The high urinary isoflavone excretion phenotype affected urinary excretion of genistein when accompanied by relatively rapid gut transit time. Low fecal isoflavone degradation rate in 12 women fed soy for 7 d was related to 
50% greater apparent absorption of isoflavones compared with 13 women with high fecal isoflavone degradation rates (12).
Golden Syrian hamsters may be good models to study the role of isoflavone bioavailability in the cholesterol-lowering efficacy of isoflavones, because greater isoflavone bioavailability was associated with increased cholesterol lowering by these compounds when isoflavones were fed in a casein-based diet (13). Inter-individual variability in isoflavone bioavailability, which we think is an important issue in understanding the health effects of these compounds in humans, seemed to be greater among female than among male hamsters (13), making female hamsters a particularly useful model for humans. The objectives of this study were to identify in a hamster model urinary isoflavone excretion phenotypes using statistical methods and to test the hypothesis that a high excreter isoflavone bioavailability phenotype was associated with cholesterol-lowering efficacy of dietary soy protein.
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Materials and Methods |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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In the experimental period, hamsters were fed a hyperlipidemic casein-based control diet high in saturated fatty acids (14) or a treatment diet with soy protein (Supro 670, Protein Technologies International) substituted for casein. The isoflavone concentration of soy protein was analyzed by Waters HPLC linear gradient with UV detection (15). The soy protein contained 405 µg of daidzein, 647 µg of genistein, and 72 µg of glycitein/g. The saponin fractions of soy protein were analyzed by HPLC (16); the soy diet contained 1.2 mg saponin/g. We prepared and stored experimental diets at 4°C and measured food intake weekly and over 2–3 consecutive d in metabolic cages.
Animals and sample collection
Fifty 8-wk-old female Golden Syrian hamsters, 118–128 g, were obtained from Charles River Breeding Laboratories. Hamsters were fed AIN 93 mol/L diet for 2 mo (17), then assigned to 2 groups to achieve similar mean body wt/group: 10 hamsters fed casein diet and 40 hamsters fed soy protein diet. The hamsters had free access to food and drinking water in a temperature-controlled room (23°C) with a 12-h light/dark cycle during the 4-wk experimental period. Clinical observation and body wt measurements were conducted 1x/wk. Urinary and fecal samples were collected over 24 h at the end of wk 1 and 4 when hamsters were put in metabolic cages individually for 2–3 d. At the end of the feeding period, diets were withdrawn from hamsters 14–16 h before they were killed by exposure to CO2. We collected blood samples by cardiac puncture in EDTA tubes and centrifuged at 5000 x g;10 min, 4°C. Plasma samples were then frozen at –20°C until analysis. The animal experimental protocol was approved by the Iowa State University Animal Use Committee.
Plasma lipid analysis
Plasma total cholesterol, HDL cholesterol, and triglyceride concentrations were measured with Infinity Cholesterol and Triglycerides Reagent (Thermo DMA) and HDL Cholesterol Reagent (PTA/MgCl2) Diagnostics kits (Sigma Diagnostics). Non-HDL-C was calculated by subtracting HDL cholesterol (HDL-C) from total cholesterol and represented LDL + intermediate density lipoprotein (IDL) + VLDL cholesterol.
Chemicals
Daidzein (4',7-dihydroxyisoflavone), genistein (4',5,7-trihydroxyisoflavone), glycitein (4',7-dihydroxy-6-methoxyisoflavone), and THB (2,4,4'-trihydroxybeozoin) were synthesized (18). Milli-Q (Millipore) HPLC grade water was used. We purchased other HPLC solvents from Fisher Scientific.
Analytical methods
Fecal and urinary sample preparation and HPLC analysis. Isoflavone extraction from urine and fecal samples was performed as described previously (19). Fecal saponin metabolite analysis in wk 4 samples was as described (16,20).
Statistical analysis. We conducted pairwise correlation plots analysis and hierarchical clustering test to classify isoflavone excretion phenotypes. One-way ANOVA followed by t tests determined the differences in urinary and fecal isoflavones between 2 isoflavone excretion phenotypes at wk 1 and 4, differences in excretion of daidzein, genistein, and glycitein within excretion phenotype, differences in the plasma lipid levels between the soy protein-fed and control groups, between isoflavone excretion phenotypes, and between isoflavone excretion phenotypes and the controls. We used McNemar's test (21) to verify high and low isoflavone phenotype stability. Intraclass Correlation Analysis (22) was done to verify individual phenotype stability. Pearson correlation analysis determined the correlations between urinary and fecal isoflavone excretion and between isoflavone excretion and plasma lipids. All results were reported as mean ± SEM. All differences were considered significant at P < 0.05. Statistical analysis was performed with R software (23) and SAS (SAS Institute, version 8.1; 1998).
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Results |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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Urinary isoflavone excretion phenotypes were classified using pairwise correlation plots. The plot of the ranked values showed a gap in urinary glycitein excretion between individuals at wk 1 (Fig. 1). From these rankings, hamster 2 had the maximum excretion of all isoflavones. Hamster 8 remained among the least in urinary excretion of all isoflavones. The highlighted data for these 2 individuals indicates the consistency of apparent absorption for all isoflavones and the extreme difference among individuals. The number of observations in the 2 categories were great enough for inference; thus, the statistical analysis showed a natural cut-off for high and low urinary glycitein excreters (Fig.1).
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According to total urinary isoflavone excretion at wk 1 (Figs. 2 and 3), high and low urinary excretion phenotypes were identified by a hierarchical cluster test. Individuals of the high excretion phenotype (cluster 3) excreted more urinary daidzein, genistein, glycitein, and total isoflavone (P < 0.05) than did those of the low urinary excretion phenotype (cluster 2) at wk 1 and 4 (Table 3).
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Stability of urinary excretion phenotypes over 4 wk. McNemar's test showed the stability of the urinary excretion phenotypes between wk 1 and 4 (Tables 2 and 3). Only 4 of 39 individuals switched their urinary glycitein excretion phenotypes between wk 1 and 4. Twenty-six hamsters remained low excreters and 1 hamster became a high excreter. The low urinary glycitein excretion phenotype was more stable than the high glycitein urinary excretion phenotype. Three of 11 hamsters of the high glycitein excreter phenotype became low excreters. By the other classification method, the low excretion phenotype (cluster 2) was more stable (2 individuals switched) than the high excretion phenotype (cluster 3) in which 3 individuals switched.
Individual glycitein excretion phenotype stability from wk 1 to 4 (Fig. 4) was investigated using Intraclass Correlation Analysis, defined as 
), which compared the variability between hamsters to the total variability in the data. The 95% CI estimate for this ratio was [0, 0.89]. This interval was wide due to high variability, or low precision, in the estimate of this ratio. However, the variability of the values within a hamster can be seen as the length of the lines connecting the 2 measurements. In general, there was less change from wk 1 to 4 when a hamster had a relatively low original glycitein excretion phenotype.
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Relation between plasma lipids and isoflavone or saponin metabolite excretion. Strong relations existed between urinary and fecal isoflavone excretion within individuals at wk 1 and 4 according to correlation analysis (r = 0.79–0.93, P < 0.05). The plasma total cholesterol level was positively correlated with non-HDL-C and the ratios of non-HDL-C to HDL-C (r = 0.87 and 0.79, P < 0.05). Non-HDL-C was positively correlated with non-HDL-C/HDL-C (r = 0.97, P < 0.05). There was no correlation of HDL-C or triglyceride with total or non-HDL-C.
Total and non-HDL-C were negatively correlated with urinary daidzein, glycitein, and total isoflavone excretion (r = –0.45 to –0.58, P < 0.05). HDL-C and triglycerides were not significantly correlated with urinary or fecal isoflavone excretion. Total and non-HDL-C were not significantly correlated with fecal saponin metabolite excretion.
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Discussion |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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50% greater urinary isoflavone excretion (apparent absorption) than did 12 women of a high fecal isoflavone disappearance phenotype and slow GTT after consuming soy isoflavones for 7 d. Hamsters showed in vitro cecal isoflavone degradation rate phenotypic clusters (24) that could account for the urinary isoflavone excretion phenotypes observed in the present study, suggesting that the isoflavone bioavailability phenotypes observed in both humans (11,12) and hamsters (13) are due to gut microbial degradation of isoflavones that differs greatly and relatively stably among individuals. In hamsters, urinary isoflavone excretion was greater than fecal isoflavone excretion, presumably due to gut microbial degradation of these compounds, as observed in humans (9,25; Tables 2 and 3). Glycitein showed significantly greater excretion than daidzein or genistein in this study (Tables 2 and 3), indicating its greater bioavailability than the other isoflavones. Feeding 1.18 or 1.77 mmol total isoflavones/kg diet to 1-y-old male (n = 10) and female (n = 9) hamsters for 10 d produced similar 24-h urinary isoflavone excretion differences among the isoflavones (13). Daidzein was a more bioavailable isoflavone than genistein in women fed single doses of soymilk, as reflected in urinary excretion (9,25). Urinary glycitein excretion was similar to that of daidzein and greater than genistein in 7 women and 7 men fed single doses of soymilk (19). Not all human studies have shown greater bioavailability for daidzein than for genistein. Women who were high excreters of isoflavones did not differ in daidzein and genistein excretion, whereas low excreters showed greater daidzein than genistein excretion (9). We also found that the low glycitein urinary excretion phenotype was more stable and the high glycitein urinary excretion phenotype was less stable in hamsters (Fig. 4). In women, a high fecal daidzein isoflavone disappearance phenotype was not as stable as a low daidzein disappearance phenotype, but both high and low genistein disappearance phenotypes were relatively stable (11). Thus, in a broad sense, hamsters may usefully model human isoflavone bioavailability and its stable phenotypic variations to better study the health effects of these compounds.
In the current study, either glycitein or total isoflavone bioavailability as reflected in urinary excretion clusters determined at wk 1 or 4 predicted cholesterol-lowering efficacy of soy protein (Table 3). Hamsters fed 1.3 mmol daidzein/kg diet for 10 wk had less plasma cholesterol compared with casein-fed controls (20). In part because of the apparent similarity in daidzein and glycitein bioavailability in humans (19), the present results are likely to be relevant to humans for cholesterol-lowering efficacy of soy proteins containing isoflavones. To our knowledge, no studies of isoflavone effect on blood lipids have compared isoflavone bioavailability indices with cholesterol status; however, Urban et al. (25) showed that serum isoflavones increased as serum cholesterol significantly decreased after 34 elderly men consumed isolated soy protein high in isoflavone concentration for 6 wk in a randomized crossover design that compared the high isoflavone soy protein to a soy protein that had most of the isoflavones removed. These subjects showed serum isoflavone concentrations after eating the soy protein high in isoflavones that varied among subjects >10-fold for genistein and more than 30-fold for daidzein. This study, along with several others (2–5), supported the role of isoflavones in cholesterol-lowering but did not attempt to relate individual isoflavone status to blood lipid status. Based on our hamster model and the wide range of human variability in isoflavone status after soy intake (25), controlling for the isoflavone bioavailability phenotypes of human subjects may optimize conditions to observe isoflavone effects.
Several studies have found that isoflavones did not improve the blood lipid profile, including a recent meta-analysis (1). For example, dietary intake of isoflavone-rich (80.4 mg isoflavone aglucones/d; n = 24) or isoflavone-poor soy protein (4.4 mg isoflavones/d; n = 24) for 6 mo did not improve circulating lipid and lipoprotein concentrations in perimenopausal women or in subjects who were mildly hypercholesterolemic (n = 30) (26). Hamsters fed isoflavones (0.02 g/100 g diet) for 5 wk did not differ from the casein control in total cholesterol, triglycerides, or HDL-C (27). These studies did not attempt to control for inter-individual variability in isoflavone bioavailability.
Soyasaponins, associated with soy protein in addition to isoflavones, were also assessed for cholesterol-lowering effect. A diet containing 2.4 g group B soyasaponins/kg (saponin concentration similar to that in 25% soy protein diet) caused less plasma total cholesterol and non-HDL-C (by 20 and 33%) in 10 11–12-wk-old female Golden Syrian hamsters compared with those fed casein (20). High producers of a putative fecal saponin metabolite had less total cholesterol/HDL-C compared with low producers. In the present study, high and low saponin metabolite producers did not differ in plasma lipid status (data not shown), and the dietary saponin concentration (1.1 g/kg) was about one-half that shown to be effective in lessening cholesterol in a previous study (20). Thus, isoflavones and not saponins were the main modifiers of plasma lipids, but both components vary among soy protein sources and may contribute to lessening of plasma cholesterol by soy protein.
The cholesterol-lessening effect of soy protein in hamsters largely depended on the extent of apparent absorption of isoflavones, which varies according to distinct phenotypes. These urinary isoflavone excretion phenotypes may be defined according to glycitein or total isoflavone excretion over a given period. These isoflavone absorption phenotypes are probably determined by gut microbial ecology, but isoflavone degrading microbial species and other factors determining this relatively stable variability in isoflavone absorption remain to be identified. Studies examining the effects of isoflavones in humans and animal models might be improved by considering inter-individual variability in isoflavone absorption, as it seems to be a controllable factor.
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FOOTNOTES |
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Manuscript received 12 May 2006. Initial review completed 13 June 2006. Revision accepted 14 August 2006.
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11. Zheng Y, Hu J, Murphy PA, Alekel DL, Franke WD, Hendrich S. Rapid gut transit time and slow fecal isoflavone disappearance phenotype are associated with greater genistein bioavailability in women. J Nutr. 2003;133:3110–6.
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