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© 2006 American Society for Nutrition J. Nutr. 136:2975-2979, December 2006

Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions

Differential Biliary Excretion of Genistein Metabolites Following Intraduodenal and Intravenous Infusion of Genistin in Female Rats1

Jeevan K. Prasain2,5,*, Jun Xu2, Marion Kirk3, Michelle Smith Johnson2, Jeff Sfakianos2,6 and Stephen Barnes2,3,4,5

2 Department of Pharmacology and Toxicology, 3 Comprehensive Cancer Center Mass Spectrometry Shared Facility, 4 Center for Nutrient-Gene Interaction in Cancer Prevention, and 5 Purdue University-University of Alabama at Birmingham Botanicals Center for Age-Related Disease, Birmingham, AL 35294

* To whom correspondence should be addressed. E-mail: jeevan.prasain{at}ccc.uab.edu.


   ABSTRACT
TOP
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
The purpose of this study was to determine whether bioflavonoid glucoside O-conjugates are absorbed from the intestine in the intact form or as their aglycones following hydrolysis by intestinal ß-glucosidases. In this study, the intestinal absorption of genistin, the ß-glucoside of the isoflavone genistein, was examined in anesthetized, adult female rats fitted with indwelling biliary cannulas. To first establish whether genistein, once absorbed, was converted into unique metabolites, genistin was infused into the femoral or portal veins and bile samples quantitatively collected. Analysis of bile samples by HPLC-mass spectrometry revealed that almost full recovery of the genistein component occurred in the form of unreacted genistin (~20%) and genistein 7ß-O-glucuronide (~80%). However, when genistin was infused into the upper small intestine, only genistein 7ß-O-glucuronide and the aglycone genistein appeared in the bile. There was no evidence for any biliary secretion of the unreacted genistin, thereby excluding its uptake in the intact form from the small intestine in this animal model.


   Introduction
TOP
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
Bioflavonoids have been the target of many ongoing studies investigating their role in the prevention of atherosclerosis, e.g. the flavonoid quercetin (1); cancer, e.g. members of the tea catechin family and isoflavones (2,3); of osteoporosis, e.g. isoflavones (4); and diseases involving inflammatory components (5). Although the bioflavonoids are usually delivered in food matrices, they are nonetheless xenobiotics. They are therefore just like synthetic therapeutic agents, subject to uncertainties regarding intestinal absorption and metabolism before entering the peripheral blood compartment and reaching target organs (6).

The soy isoflavone genistein (4',5,7-trihydroxyisoflavone) is an important dietary phytochemical component and the subject of much enquiry for possible health-related benefits as well as toxicities. It is found in soybeans as 2 distinct ß-glucosides, those with and those without a 6"-O-malonyl ester group (7–9). Methods used to prepare the soy foods, soy milk and tofu, largely eliminate the malonyl group, leaving the ß-glucoside as the principal chemical form (8). In fermented soy foods, the isoflavones are mostly converted to their aglycones (10).

The bioavailability of genistin and genistein is a crucial aspect of their potential biological actions. Using the blood concentrations of genistein as the criterion for uptake, several investigators have reported bioavailabilities of 5–12% of the ingested dose (11). However, this does not take into account other forms of genistein that are formed after initial uptake. We have previously shown in the adult female rat that genistein is quickly absorbed from the small intestine and rapidly appears in the bile as its 7ß-O-glucuronide and that if all forms of genistein are taken into account, the intestinal absorption of genistein is at least 70% (12). When genistein was infused into the portal vein, close to 100% of the dose was recovered in bile within 60 min. However, only at the highest infusion rates was unconjugated genistein found in significant quantities in the peripheral blood, the principal form being its 7ß-O-glucuronide (12). Total urinary output over the period of the experiment was no greater than 1–2%. These data indicate that genistein and its metabolites are principally and quantitatively excreted into bile following genistein's absorption from the small intestine.

Using everted small intestinal sacs, we also demonstrated that during intestinal transport, genistein is converted to its 7ß-O-glucuronide, which accumulates on the serosal side of the everted sac (12). In work using the colonic cancer CaCo-2 cell line as a model of intestinal transport, it has been shown that in addition to bidirectional flux of genistein, its 7ß-O-glucuronide exhibited basolateral to apical efflux, i.e. the ß-glucuronide can be excreted into the lumen of the intestine (13). This result has also been reported by Andlauer et al. (14) using an isolated perfused intestinal preparation.

Less information about the absorption of genistin is available. Measuring all chemical forms, it is apparent in rats that the areas under the blood concentration-time curves are essentially the same for genistein and genistin (15), although the time to the peak concentration was much shorter for genistein. In humans, isoflavone ß-glucosides are purportedly more bioavailable than their aglycone counterparts (16). In addition, isoflavone ß-O-glucosides are not detected in human blood or urine (17), indirectly leading to the conclusion that these conjugated forms of isoflavones are not absorbed intact.

In preliminary experiments (J. Sfakianos and S. Barnes, unpublished data) using everted small intestinal sac preparations, we found that genistin was rapidly converted to new products or absorbed. However, at that time, the metabolite was not identified. Given the magnitude of the efflux of genistein 7ß-O-glucuronide in the CaCo-2 cells (13) and perfused intestinal preparations (14), it was essential to determine whether this occurred in intact animals, because such efflux would substantially reduce the absorption of genistein and its metabolites. Our strategy for detecting direct absorption of intact genistin was first to infuse it into a peripheral blood site (femoral vein) or close to the liver (portal vein). We wanted to determine whether genistin could be transported into bile and in what form(s). The presence in bile of a unique metabolite of genistin would then allow quantitative assessment of the extent to which genistin underwent absorption from the intestine when administered to the intestinal compartment.


   Materials and Methods
TOP
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 LITERATURE CITED
 
    Materials. Genistin, the ß-glucoside of genistein, was isolated from soy molasses, an aqueous alcohol extract of soy flour (18). Genistein was prepared from genistin by hydrolysis in methanol-HCl (18). These isoflavones, following recrystallization, were 98% pure (by reversed-phase HPLC). Melting points and molecular weights were determined by atmospheric pressure chemical ionization mass spectrometry, and 1H NMR spectra were consistent with genistin and genistein, as previously described (19). Genistein 7ß-O-glucuronide was prepared by infusing genistein into the portal vein of anesthetized adult rats fitted with indwelling biliary catheters. The collected bile was chromatographed over a Sephadex LH-20 column, treated with cholylglycine hydrolase to hydrolyze bile acid conjugates, and rechromatographed over the Sephadex LH-20 column, as previously described (20).

ß-Glucuronidase and sulfatase were purchased from Sigma Chemical. Acetonitrile and trifluoroacetic acid were sequencing grades obtained from Fisher Chemical and Pierce Chemical, respectively. All other chemicals were of the highest grades obtainable. Sep-Pak C18 cartridges were purchased from Waters.

    Animals. Female Sprague-Dawley rats (n = 6, body wt = 225–275 g) were purchased from Harlan Sprague-Dawley) and were fed an isoflavone- and soy-free AIN-76A diet (casein 20.0%, methionine 0.3%, carbohydrate 64.9%, and corn oil 5.0%, by weight, and vitamin and mineral mix) produced by Harland Teklad for 1 wk prior to use in the study (21). Rats were anesthetized with ketamine/xylocaine (0.1 mL/100 g body wt) and their body temperature was maintained at 38°C (monitored rectally) by placing them on a heating pad. Following a midline incision, the bile duct was exposed and cannulated with PE-10 tubing, tied, and secured with 5–0 silk (Ethicon).

    Femoral vein infusion. In the first experiment to determine the metabolites and routes of excretion of intravenously administered genistin, genistin (50 µmol/L) was infused (23.4 µL/min) into the exposed femoral vein at 1.17 nmol/min (505.4 ng/min) for 60 min in 154 mmol/L NaCl containing 10 mmol/L sodium taurocholate using a Harvard syringe pump. Bile was collected in 5-min intervals during the first 30 min and then at 10-min intervals over the next 2.5 h. Urine was collected from the bladder at the end of the experiment. Blood samples (0.5 mL) were taken from a cannula inserted in the carotid artery prior to the start of the infusion, after infusion for 1 h, 2 h, and at the end of the experiment. After allowing the blood samples to clot, serum samples were obtained by centrifugation.

    Portal vein infusion. Because infusion via the femoral vein causes genistin to enter the peripheral circulation prior to uptake and excretion by the liver, a method was developed to infuse genistin into the portal vein without obstructing portal vein blood flow. To do this, the portal vein was exposed and a 27-gauge stainless steel syringe needle, connected to PE10 tubing, was inserted. Super glue was used immediately to seal the puncture point. In this manner, genistin was infused into the portal vein while not blocking portal vein blood flow. Bile, blood, and urine samples were collected as described for the experiments using femoral vein infusion.

    Absorption from the small intestine. To examine the absorption of genistin from the small intestine, using anesthetized female rats fitted with biliary cannulas, genistin (in 10 mmol/L sodium taurocholate, 10 mmol/L glucose, and 154 mmol/L NaCl) was infused (70 µL/min, i.e. 1555 ng/min) over a 1-h period into the duodenum. After this, the genistin-free perfusate was infused for the remainder of the experimental period (4 h). Bile samples were collected every 5 min during the first 30-min period and then at 20-min intervals. Urine was collected from the bladder at the end of the experiment. Blood samples (0.25 mL) were taken prior to the start of the infusion and after infusion for 1, 2, 3, and 4 h.

    HPLC analysis of isoflavones in bile, serum, and urine samples. Bile, serum, and urine samples were prediluted with 1 mL (bile) or 10 volumes (serum and urine) of 50 mmol/L ammonium acetate buffer, pH 5.0, and the isoflavones extracted by passage over an activated Sep-Pak C18 cartridge equilibrated with 50 mmol/L ammonium acetate buffer, pH 5.0. The cartridge was washed with 3 x 1 mL 10 mmol/L ammonium acetate buffer, pH 5.0, and the isoflavones eluted with 2 x 2 mL methanol. The methanol was evaporated under a stream of nitrogen at room temperature and the residues reconstituted in 100 µL of 80% aqueous methanol. Aliquots (1–10 µL) were used for HPLC analysis.

To ascertain whether the individual peaks observed were glucuronides or sulfates, aliquots of the extracts were completely evaporated and reconstituted in 50 mmol/L Tris-HCl buffer, pH 7, containing 500 units of ß-glucuronidase or 0.5 units of sulfatase and incubated overnight at 37°C. The pH was lowered to 5.0 by the addition of 0.75 mL of 1.0 mol/L ammonium acetate, pH 5.0. The sample was diluted with 5 mL water and the isoflavones extracted by passage over an activated Sep-Pak C18 cartridge, as described above.

HPLC analysis was carried out on a Hewlett Packard model 1100 instrument with a 25-cm x 0.46-cm i.d. Brownlee Aquapore C8 reverse-phase column and using a linear 0–50% gradient (5%/min) of acetonitrile in either 0.1% trifluoroacetic acid or 10 mmol/L ammonium acetate at a flow rate of 1.5 mL/min. Isoflavones in the eluate were detected by their absorbance at 262 nm.

    LC-MS/MS analysis. Extracted samples of bile, sera, or urine were separated by reverse-phase HPLC on a 15-cm x 0.21-cm i.d. Brownlee Aquapore C8 column using either a linear 0–50% gradient (5%/min) of acetonitrile in 10 mmol/L ammonium acetate or isocratically with 30% acetonitrile in 10 mmol/L ammonium acetate at a flow rate of 0.2 mL/min. The column eluate was split 1:1 and 1 stream passed into the IonSpray interface of a PE-Sciex (Concord) API III triple quadrupole mass spectrometer operating in the negative ion mode, with an orifice potential of –60 V. In the MS-MS mode, product ion spectra were obtained by selecting parent ions in the first quadrupole, which were then collided with argon-10% nitrogen gas in the second quadrupole and analyzed in the third quadrupole. In the multiple reaction ion mode (MRM-MS), individual precursor and product ions for each metabolite were selected to obtain quantitative data. The MRM analysis was conducted by monitoring the precursor ion to product ion transitions from m/z 431/269 (genistin), 445/269 (genistin 7ß-O-glucuronide), and 269/133 (genistein). The operation of the mass spectrometer and analysis of data were carried out using 2 MacIntosh Quadra 950 computers interfaced with an Ethernet link. The calibration curve standards covered a wide range of concentrations of genistein, genistin, and genistein 7ß-O-glucuronide (0.1, 1, 5, 10, 25, and 50 µmol/L) and areas under each analyte peak were fitted using linear regression analysis.

    Statistics. Data are presented as means ± SD. Differences in the rates of biliary output of genistin and its metabolites were calculated by the Student's t test, with Tukey's correction for the multiple comparisons. Differences were considered significant at P < 0.05.


   Results
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 LITERATURE CITED
 
The infusion of genistin into the femoral and portal veins led to the rapid appearance of 2 new peaks with approximately equal areas when bile samples were analyzed by reverse-phase HPLC using their absorbance at 262 nm (data not shown). To establish the identity of these new peaks, they were collected and analyzed by negative ion electrospray ionization (ESI)-MS. The first peak had a m/z of 445, the expected molecular ion of genistein 7ß-O-glucuronide, whereas the other peak had a m/z of 431, consistent with it being unreacted genistin. Evident from LC-tandem MS analysis, interference from other nonisoflavone compounds occurs in bile (Fig. 1). Although 1 of the m/z 445 molecular ions gave rise to the expected m/z 269 product ion, the other major m/z 445 ion did not, indicating that it is not a genistin metabolite. Product ion spectra of the molecular ions confirmed the identities of each biliary metabolite; the principal product ion in each case was m/z 269 for genistein.


Figure 1
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  		Figure 1  LC-MS/MS of biliary metabolites of genistin. (A) Ion chromatogram of compounds with m/z 445. (B) Product ion spectrum of the first m/z 445 peak. (C) Product ion spectrum of the second m/z 445 peak.

 
To obtain quantitative data, an LC-MRM-MS procedure was developed for genistein, genistin, and genistein 7ß-O-glucuronide. The method was found to be linear over the range 0.1–50 µmol/L. The correlation coefficient of each standard curve was >0.999. This allowed selective and specific quantitative data to be obtained for each genistin metabolite (Fig. 2A–C). Using this method, it was shown that both genistin and genistein 7ß-O-glucuronide were excreted into bile (Fig. 3A). The areas under the concentration-time curves of each compound were approximately equal in both the femoral and portal infusion experiments (data not shown). Integration of the total biliary output combined with small amounts of genistin and genistein 7ß-O-glucuronide in urine samples recovered at the end of the experiment revealed that all the infused genistin could be accounted for (infused amount = 30.3 ng; recovered amount = 37.8 ± 9.5 ng). This suggests that any other metabolites that were not detected by this procedure are minor.


Figure 2
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  		Figure 2  LC-MS-MRM analysis of genistin (431/269), genistein 7ß-O-glucuronide (GenGlcA 445/269), and genistein (269/133) in bile (A) before and (C) after infusion of genistin for 60 min into the femoral vein in female rats. The 3 parent ion-product ion pairs were added together to produce a single ion chromatogram. The chromatogram obtained with a standard mixture of genistin, genistein 7ß-O-glucuronide, and genistein is shown in B.

 

Figure 3
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  		Figure 3  Time-dependent mean biliary excretion of genistin, genistein, and genistein 7ß-O-glucuronide following infusion of genistin into the femoral and portal veins (A) and intestine (B) in female rats. Values are presented as means ± SD, n = 6. Rates of excretion of genistin, genistein, and genistein 7ß-O-glucuronide were considerably different from each other in the 2-h period after the start of genistin infusion into the femoral or portal veins. In the case of intraduodenal infusion of genistin, only genistein 7ß-O-glucuronide was significantly different after the start of infusion.

 
The identification of biliary genistin as a marker of genistin that enters the blood stream enabled examination of the question of whether genistin can be absorbed intact from the intestinal compartment. However, when genistin was infused into the upper small intestine, no genistin was detected by LC-MS-MRM in biliary or urine samples. The major biliary metabolite was genistein 7ß-O-glucuronide, with very small amounts of genistein (Fig. 3B). Because bile was quantitatively collected, this implies that conversion of genistin to genistein 7ß-O-glucuronide occurs in the intestinal compartment, probably in the intestinal wall. Genistein 7ß-O-glucuronide was the only observed metabolite in the blood serum. We also analyzed urine samples and only genistin metabolites, genistein, and genistein 7ß-O-glucuronide were detected (data not shown).


   Discussion
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
LITERATURE CITED
 
The data from this study demonstrate that the ß-glucoside genistin can be transported by the liver into bile if directly introduced into the bloodstream. It is unlikely that genistin is first converted to genistein and then glucosylated in the liver, because we have previously shown that when genistein is infused intravenously in rats, only the 7ß-O-glucuronide is excreted into bile (12). However, the presence of large amounts of biliary genistein 7ß-O-glucuronide suggests that hydrolysis of genistin in this model occurs before it is excreted into bile. The liver contains ß-glucosidase activity toward flavonoid glycosides (22). An alternative, but unlikely, explanation is that the glucose moiety is directly oxidized to the glucuronic acid in the liver.

Although genistin is rapidly absorbed from the intestinal compartment when introduced into the upper small intestine in the intact rat, nonetheless, no genistin appeared in bile. Instead, the biliary metabolite was genistein 7ß-O-glucuronide. We interpret this result to mean that genistin did not undergo significant intestinal absorption prior to hydrolysis in the intact rat. This is in contrast to the data showing that the glucosides of the flavonoid quercetin not only have higher bioavailability than the aglycone (23–27) but are also detected in the blood and urines of treated subjects. The expectation that genistin might exhibit carrier-mediated transport in the small intestine (and hence direct uptake) came from its structural similarity to phloridzin, an inhibitor of intestinal Na+-dependent glucose transport, which is the ß-glucoside of the flavonoid phlorizin. In addition, a phytochemical extract of soy protein rich in isoflavone glucoside conjugates has been shown to inhibit glucose transport in rat small intestinal brush border membrane vesicles (28). However, genistein is an inhibitor of the GLUT 1 transporter (29). Thus, once hydrolysis of genistin occurs in the lumen of the small intestine by bacteria or the intestinal wall by lactose-phlorizin hydrolase (30), the newly formed genistein may interfere with the transport of genistin. However, the model used in this study cannot distinguish luminal hydrolysis of genistin and uptake of genistein from uptake of unchanged genistin into the intestinal cell followed by further metabolism of genistin to genistein. What the study shows is that following intraduodenal infusion, unchanged genistin does not enter the mesenteric venous drainage and is taken up by the liver and excreted in bile.


   ACKNOWLEDGMENTS
 
A genistin concentrate, used in the isolation and purification of genistin and genistein, was kindly donated by Protein Technologies International (now the Solae Company).


   FOOTNOTES
 
1 Supported by grants from the National Cancer Institute (5R01 CA-61668 and U54 CA-100949), the National Center for Complementary and Alternative Medicine (5P50 AT-00477 to the Purdue University-UAB Botanicals Center for Age-Related Disease, Connie Weaver, PI), and the United Soybean Board (7312). The mass spectrometer was purchased by funds from a NIH Instrumentation Grant (S10RR06487) and from this institution. Operation of the UAB Comprehensive Cancer Center Mass Spectrometry Shared Facility has been supported in part by a NCI Core Research Support Grant to the UAB Comprehensive Cancer (P30 CA13148). Back

6 Present address: Department of Cell Biology, Yale University, New Haven, CT 06520. Back

Manuscript received 31 July 2006. Initial review completed 25 August 2006. Revision accepted 18 September 2006.


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 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
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3. Hedlund TE, Maroni PD, Ferucci PG, Dayton R, Barnes S, Jones K, Moore R, Ogden LG, Wahala K, et al. Long-term dietary habits affect soy isoflavone metabolism and accumulation in prostatic fluid in caucasian men. J Nutr. 2005;135:1400–6.[Abstract/Free Full Text]

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