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Institute for Biological Chemistry and Nutrition, University of Hohenheim, D-70593 Stuttgart, Germany
1To whom correspondence should be addressed. E-mail: >andlauer@uni-hohenheim.de" locator-type="email">locator-type="email">andlauer@uni-hohenheim.de locator="" locator-type="email">
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSSampling and sample preparationAnalytical proceduresTofuChemicals and solventsRESULTSDISCUSSIONREFERENCES |
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45 d old). A synthetic
emulsion free from blood components was used as vascular medium, with a
perfluorocarbon as oxygen carrier. Luminal media consisted of tofu,
predigested with pepsin and pancreatin and emulsified with bile acids,
containing 39.5 µmol/L genistein compounds and 19.1
µmol/L daidzein compounds. Viability of the organ
preparation was maintained during the entire perfusion, confirmed by
lack of significant differences between tofu and control perfusion
experiments for arterial pressure, glucose consumption, oxygen uptake,
lactate-pyruvate ratio and acid-base homeostasis. Daidzein
(8.9%) and genistein (8.0%) compounds from tofu exhibited almost the
same (P > 0.05) absorption rate during small
intestinal passage. The majority of the absorbed genistin appeared
vascularly as genistein (4.4%), in addition to minor amounts of
unchanged genistin (2.1%) and genistein glucuronide (1.5%). In the
luminal effluent, a considerable increase of genistein (338%) as well
as daidzein (190%) as cleavage products of the glucosides and
malonyl-glucosides was observed. The distribution of daidzein
compounds in the small intestine was not different from that of
genistein compounds (P > 0.05), except for the
blood vessels, which had extremely low total amounts. Sulfate
derivatives of genistein and daidzein compounds were not detectable. An
effect of tofu ingredients was observed on absorption rate of genistin,
on glucuronidation and on distribution of genistein glucuronide in the
intestine.
KEY WORDS: • intestinal absorption • intestinal metabolism • tofu • isoflavone • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSSampling and sample preparationAnalytical proceduresTofuChemicals and solventsRESULTSDISCUSSIONREFERENCES |
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, Wang and Murphy 1994
) have
recently attracted great attention because of their proposed
health-related and clinical benefits such as estrogen receptor
binding (Adlercreutz 1990
, Barnes et al. 1994b
), radical scavenging (Vedavanam et al. 1999
), and antiproliferative and growth inhibiting effects on
cancer cells (Booth et al. 1999
). There have been
numerous investigations into the antioxidative and anticancer
activities of soy and its bioactive isoflavones (Coward et al. 1993
, Herman et al. 1995
, Record et al. 1995
, Wei et al. 1995
). Intestinal absorption is
a prerequisite for a possible causal relationship between isoflavone
intake and its proposed chemopreventive action. To assess intestinal handling of isoflavones from tofu, we employed an ex vivo isolated vascularly and luminally perfused rat small intestine. The isolated organ preparation facilitates direct investigation of luminal disappearance and venous appearance of administered compounds, thereby allowing the assessment of intestinal absorption under strictly controlled conditions. Chemically defined perfusion media facilitate a constant supply of substrates and phytochemicals to the intestine. The linear oxygen dissociation and chemical inertness render the perfluorotributylamine emulsion an ideal oxygen carrier for the intestinal mucosa with high oxygen turnover.
In preceding studies, we investigated absorption and intestinal
metabolism of genistin and its aglycone genistein from buffered saline
using the isolated rat small intestine (Andlauer et al. 2000b and 2000d
). However, food structure and food components that
are able to bind isoflavones such as dietary fiber or proteins might
influence the extent of absorption and metabolism. To assess the
influence of the food matrix, in the present study, we investigated the
intestinal handling of predigested tofu. Soybeans and therefore
soy-based foods such as tofu have an extremely variable isoflavone
concentration [ranging from 200 to >3500 µg/g
(Tsukamoto et al. 1995
)], depending on variety and
environmental conditions (Tsukamoto et al. 1995
,
Wang and Murphy 1994
). Therefore, isoflavone
concentration of the digested tofu was verified before perfusion.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSSampling and sample preparationAnalytical proceduresTofuChemicals and solventsRESULTSDISCUSSIONREFERENCES |
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Male Sprague-Dawley rats (CD-rats), 30 d old and weighing
130 g, were obtained from Charles River (Sulzfeld, Germany). Rats
were fed a cornstarch-based isoflavone-free synthetic diet
(Altromin
C-1000,2
Altromin International GmbH, Lage, Germany) for 14 d to allow
elimination of isoflavones. Rats were provided with free access to tap
water and food.
Vascularly and luminally perfused rat small intestine.
The small intestine (duodenum, jejunum, ileum) was prepared in rats as
described elsewhere (Hartmann et al. 1984
, Plauth et al. 1991
). Briefly, small intestine was prepared in seven
rats for perfusion with digested tofu (n = 3; 222.0
± 14.7 g) and for control perfusions with basic luminal
media (n = 4; 226.0 ± 11.1 g) in
narketan (Chassot AG, Bern, Switzerland)-xylazin (Vetimex, Bladel, The
Netherlands) narcosis after overnight food deprivation. After
cannulation of the superior mesenteric artery and the portal vein, the
small intestine was vascularly perfused with an artificial oxygen
carrier (see below). Subsequently, the intestine was excised, and the
intestinal lumen was cannulated and rinsed free with warm saline (155
mmol/L NaCl). The isolated small intestine was transferred to a 37°C
tissue bath and allowed to equilibrate for 30 min. The experiment was
started after filling the intestinal lumen with a 7-mL bolus of luminal
media [1.0 g digested tofu (see below) or buffered saline containing
135 mmol/L NaCl and 20 mmol/L NaHCO3, pH 7.2, in the case
of controls, respectively], with sampling over 60 min. Perfusion was
carried out according to a single-pass technique. In this mode, the
flow rates were 5 mL/min vascularly (venous) and 0.5 mL/min luminally.
The vascular perfusion medium consisted of a perfluorotributylamine (ABCR, Karlsruhe, Germany) emulsion in Krebs-buffer containing 10 mmol/L glucose and an additional 0.6 mmol/L glutamine, gassed with 5% carbon dioxide in oxygen (pH 7.4). The perfluorotributylamine (200 g/L) was emulsified with polyoxyproylene-polyoxyethylene copolymer (25.6 g/L Pluronic, F-68, BASF, Ludwigshafen, Germany) in sterile, pyrogene-free water, using a high pressure homogenizer (Mouton-Gaulin LAB 60/60–10TBS, APV Gaulin GmbH, Lübeck, Germany) to an average diameter of 0.2 µm.
The viability of the model was carefully controlled by repeatedly measuring oxygen uptake and acid-base homeostasis using a Clark pO2-electrode and a pH-electrode integrated with an ABL 30 Acid-Base Analyzer (Radiometer, Copenhagen, Denmark). Glucose, lactate and pyruvate were determined photometrically using enzymatic test kits (Monotest; Boehringer Mannheim, Mannheim, Germany). The following kits were used: for glucose, the MPR3 Glucose/GOD-Perid test kit (glucose oxidase, peroxidase; ABTS; Boehringer Mannheim); for lactate, the MPR3 lactate test kit (lactate dehydrogenase; NAD+); and for pyruvate, the MPR1 pyruvate test kit (lactate dehydrogenase; NADH). The study was approved by the Regierungspräsidium Stuttgart, Germany
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Sampling and sample preparation |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSSampling and sample preparationAnalytical proceduresTofuChemicals and solventsRESULTSDISCUSSIONREFERENCES |
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Vascular samples.
Of each vascular (venous) sample, 2 mL was spiked with the internal standard 4-nitrophenol (50 µL of a 170 µmol/L solution) and centrifuged at 2800 x g for 40 min. The supernatant was separated and the pellet was extracted with 0.4 mL ethanol by sonication for 20 min and centrifuged at 2800 x g for 20 min (Hermle ZK 364; Kontron, Zürich, Switzerland). The combined supernatants were analyzed by HPLC.
For assessment of recovery, isoflavone-spiked perfluorocarbon emulsions (2 mL) were prepared using the same procedure. Genistin (0.2, 0.3, 0.6 nmol spiked), genistein (0.4, 0.4, 0.8 nmol spiked), daidzin (three times 3.0 nmol spiked) and daidzein (three times 4.4 nmol spiked) exhibited a recovery of 100.1 ± 3.9, 97.2 ± 3.0, 102.5 ± 5.0 and 98.0 ± 3.2%, respectively (means ± SD, n = 3).
Luminal samples.
4-Nitrophenol (50 µL of a 14.7 mmol/L solution) as an internal standard was added to the luminal effluent of a 10-min period and centrifuged at 2800 x g for 20 min. The supernatant was separated and the pellet extracted with ethanol by sonication for 20 min and centrifuged again at 2800 x g for 20 min. The combined supernatants were analyzed by HPLC.
Genistin (three times 7.1 nmol spiked), genistein (16.7, 16.7, 17.0 nmol spiked), daidzin (three times 3.0 nmol spiked) and daidzein (three times 4.4 nmol spiked) recovery from the spiked luminal media (4 mL) was 100.3 ± 1.8, 99.9 ± 1.2, 98.1 ± 3.0 and 101.7 ± 1.4%, respectively (means ± SD, n = 3).
Small intestinal tissue.
Dry weight of the small intestines was determined after lyophilization (tofu perfusions: 0.68 ± 0.26 g, n = 3; control perfusions: 0.69 ± 0.06 g, n = 4). Then the tissue was powdered using a mortar pestle and defatted by extraction with 10 mL hexane twice. As an internal standard, 4-nitrophenol (25 µL of a 14.7 mmol/L solution) was added. The pellet was extracted three times with 3 mL methanol/acetic acid (3%) (1:1) and centrifuged at 2800 x g for 20 min. The extracts were pooled and adjusted to 10 mL. Genistin (0.6, 2.6, 2.8 nmol spiked), genistein (three times 14.8 nmol spiked), daidzin (three times 3.0 nmol spiked) and daidzein (three times 4.4 nmol spiked) recovery from a spiked entire small intestine was 100.4 ± 3.9%, 100.0 ± 4.8%, 99.0 ± 7.0% and 99.9 ± 5.8%, respectively (means ± SD, n = 3).
Blood vessels.
Blood vessels were lyophilized and defatted as described for the small intestinal tissue. As an internal standard, 4-nitrophenol (5 µL of a 14.7 mmol/L solution) was added. Genistein and genistin were extracted three times according to the intestinal tissue, with 1 mL of methanol/acetic acid (3%) (1:1). Genistin (three times 2.0 nmol spiked), genistein (three times 4.4 nmol spiked), daidzin (three times 3.0 nmol spiked) and daidzein (three times 4.4 nmol spiked) recovery from spiked blood vessels was 99.4 ± 4.4, 96.4 ± 9.9, 101.2 ± 4.7 and 102.6 ± 5.1%, respectively (means ± SD, n = 3).
Clean up for liquid chromatography (LC)-MS identification of isoflavones and metabolites.
The vascular perfusate (12 mL) was acidified (hydrochloric acid) to pH 6.5 and centrifuged at 11,600 x g for 30 min. The clear supernatant was evaporated to 3 mL at 22°C. After addition of 60 µL phosphoric acid (16 mol/L), the concentrate was drawn through a nonconditioned Nexus column (Varian GmbH, Darmstadt, Germany) under low vacuum. After column rinsing with 1 mL water, the conjugates were eluted with 2 mL methanol. The eluate was diluted with 0.25 mL water, then concentrated under a gentle flow of nitrogen to 0.5 mL. This concentrate was used for LC-MS analyses.
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Analytical procedures |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSSampling and sample preparationAnalytical proceduresTofuChemicals and solventsRESULTSDISCUSSIONREFERENCES |
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The HPLC-system (Sykam, Gilching, Germany) consisted of a microsolvent delivery system S 1100, a low pressure gradient mixer S 8110, equipped with an autosampler (Spark Triathlon, Emmen, The Netherlands; 50 µL filling loop). Gradient control, continuous on-line monitoring and data quantitation were performed with Pyramid-Software (Axxiom Chromatography, Moorpark, CA). UV-absorbance was monitored with a UVIS 200 (Linear, Freemont, CA) at 262 nm with a flow cell of 10 µL.
A 125 mm long, 2.0 mm i.d. Grom-Sil ODS-3 (particle size 3
µm) column was used (Grom, Herrenberg, Germany). The
column was at 40°C (column oven S 4110; Sykam), with a flow rate of
0.3 mL/min. The eluents were composed of 0.2% acetic acid in
H2O (A) and 0.2% acetic acid in acetonitrile (B). The
elution conditions were as follows: 0–2 min, 5% B; 2–9 min, 5–15%
B; 9–22 min, 15–52% B; 22–25 min, 52–5% B. An injection volume of
25 µL resulted in detection limits of 11 nmol/L
genistin, 12 nmol/L daidzin, 9 nmol/L daidzein and 5 nmol/L genistein,
and quantitation limits of 20 nmol/L genistin, 23 nmol/L daidzin, 17
nmol/L daidzein and 9 nmol/L genistein. These isoflavones were analyzed
directly, without preceding cleavage. Because Kudou et al. (1991)
showed that the molar extinction coefficients of the
daidzein and genistein malonyl-glucosides approximated those of
daidzin and genistin, respectively, the concentrations of the
malonylated conjugates were calculated as the corresponding glucoside.
Gradient HPLC-system with MS-detection (LC-MS).
For the identification of isoflavones and metabolites we used a gradient HP HPLC system series 1100 (Hewlett-Packard, Böblingen, Germany) combined with an autosampler ALS G1313A, a quat pump G1311A, a degasser G1322A and a column oven ColComp G1316A at 40°C. The mass spectrometric detector was a Micro Mass Platform II (Mass Lynx 4.0, Manchester, UK) equipped with a cross-flow interface. The tuning parameters for negative ion spray (ES-) were 3.0 kV for capillary and 45 V for cone at a source temperature of 120°C. Negative ion characterization was performed in the m/e range of 100–800 at a scan rate of 0.5 scans/s and a multiplier voltage of 650 V.
Separation was carried out with a Inertsil ODS-2 column (250 mm x 4.6 mm i.d., 5 µm, VDS-Optilab, Berlin, Germany) at 40°C, with a flow rate of 0.9 mL/min using ammonium formiate buffer (5 mmol/L, solvent A) and acetonitrile/100 mmol/L ammonium formiate (95:5, solvent B). The elution conditions were as follows: 0–2 min, 5% B; 2–9 min, 5–25% B; 9–15 min, 25–52% B; 15–28 min, 52–70% B; 28–30 min, 70–5% B. Injection volume was 50 µL.
Cleavage of isoflavone glucuronide and sulfate conjugates.
Genistein and daidzein glucuronide and sulfate conjugates were analyzed
as aglycones after enzymatic cleavage according to (Sfakianos et al. 1997
), with modifications as described in the following.
Potassium phosphate buffer (0.25 mL; 0.2 mol/L, pH 6.8 for
glucuronidase and pH 7.1 for sulfatase) and 0.1 mL glucuronidase
solution (220 Fishman units) or 0.02 mL arylsulfatase solution (0.3
U), respectively, were added to 0.5 mL of sample solution.
The applicability of the enzymatic cleavage in cleaned-up fluorocarbon emulsion was confirmed by the conversion of 4-nitrophenol glucuronide and 4-nitrophenol sulfate with ß-glucuronidase and arylsulfatase, respectively. The cleavage of 4-nitrophenol glucuronide resulted in 4-nitrophenol recovery of 100.1%; the recovery after cleavage of 4-nitrophenol sulfate was 99.2%.
Glucuronides in the vascular effluent and in the tissues were calculated from genistein (daidzein, respectively) after enzymatic hydrolysis minus genistein and genistin (daidzein and daidzin, respectively) from direct HPLC analysis before enzymatic hydrolysis. It should be noted that glucuronidase possesses glucosidase activity and therefore cleaves the isoflavone glucosides as well as glucuronides.
Additionally, the prepared luminal effluent (see above) was hydrolyzed with acid to hydrolyze all of the isoflavones (including the glucosides). A mixture of 0.2 mL of the luminal effluent, 0.2 mL methanol and 0.4 mL HCl (9.5 mol/L) was heated to 90°C for 60 min. Before HPLC analysis of aglycones, the reaction mixture was diluted with water (1 + 4).
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Tofu |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSSampling and sample preparationAnalytical proceduresTofuChemicals and solventsRESULTSDISCUSSIONREFERENCES |
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Tofu digestion.
Chewing was simulated by pressing the tofu through a sieve (0.7 mm).
Tofu (8.1 g) was suspended with 170 mL of 0.1 mol/L HCl in a
flat-bottomed glass flask and stirred magnetically for 5 min in a
37°C water bath. The pH was adjusted to 1.9, and 25 mL pepsin
solution (7 g/L pepsin in 0.1 mol/L HCl) was added. After 1 h, the
digestion was stopped by increasing the pH to 7.4 with 1 mol/L NaOH. To
the reaction mixture, 10 mL NaHCO3 (3.4 g/L) solution and
25 mL pancreatin solution (7 g/L in buffered saline containing 135
mmol/L NaCl, 20 mmol/L NaHCO3 at pH 7.4) were added.
Pancreatin from porcine pancreas is a mixture of many enzymes,
including amylase, trypsin, chymotrypsin, lipase, ribonuclease and
carboxypeptidase. The pancreatin digestion was carried out for 1 h
at 37°C. After digestion, bile salts (to a concentration of 7.4 g/L;
cholic acid/deoxycholic acid, 50:50) were added. This enzymatic
hydrolysate containing 1.0 g tofu in 30 mL was used for luminal
perfusion. During the digestion procedure, protein digestion was
controlled and confirmed by amino acid HPLC-analysis employing
precolumn derivatization with o-phthaldialdehyde (LKB, Bromma, Sweden)
(Graser et al. 1985
) and analysis of soluble nitrogen by
elemental analyzer (Elemental Analyzer Antek 7000 V; Antek Instruments,
Houston, TX) (Grimble et al. 1988
). The stability of
isoflavones was confirmed during the whole digestion.
Isoflavone analysis from digested tofu.
Digested tofu (2 mL) was prepared in the same way as the luminal samples and analyzed by HPLC.
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Chemicals and solvents |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSSampling and sample preparationAnalytical proceduresTofuChemicals and solventsRESULTSDISCUSSIONREFERENCES |
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Calculations.
Fluxes [nmol/(min · g dry intestine), means ± SD)], were calculated from arteriovenous and proximodistal
concentration differences (
C), respectively, the corresponding flow
rates and the dry weight (DW) of the entire small intestine used in the
experiment according to the following equation:
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Differences between fluxes were determined using ANOVA and subsequent Tukey’s range test for paired observations at a procedure-wise error rate of 5%. Viability parameters, isoflavone (genistein, daidzein) distribution in the small intestine and percentage values for glucuronides and total absorption of genistin and tofu were compared using ANOVA and subsequent Student’s t test of the unpaired observations. P-values < 0.05 were considered to indicate significant differences.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSSampling and sample preparationAnalytical proceduresTofuChemicals and solventsRESULTSDISCUSSIONREFERENCES |
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) increased significantly, indicating an increasing extent of protein
digestion. After addition of the exopeptidase pancreatin, both
nitrogen-containing soluble compounds and free amino acids
increased significantly and reached a plateau after 2 h. During the
enzymic incubation, the stability of isoflavones was confirmed by HPLC.
Neither the acidic nor the enzymatic conditions of the simulated
predigestion hydrolyzed the glucosides and malonyl-glucosides.
After addition of bile acids, the isoflavone content of digested tofu
(n = 3) was analyzed. The isoflavone content of
1.0 g digested tofu was 760.7 nmol genistin, 84.8 nmol genistein,
339.1 nmol malonyl-genistin, 366.7 nmol daidzin, 47.5 nmol daidzein
and 158.6 nmol malonyl-daidzin (a total of 1184.6 nmol genistein
compounds and 572.8 nmol daidzein compounds). For the assessment of
intestinal absorption and metabolism of isoflavones, this predigested
tofu was perfused through the isolated rat organ preparation.
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As in earlier studies (Andlauer et al. 2000c
,
Hummel 1998
), viability and functional integrity of the
organ preparation were monitored continuously in terms of maintenance
of regular perfusion pressure, stable lactate-pyruvate-ratio, regular
oxygen uptake, glucose consumption and acid-base homeostasis. No
significant differences in viability data were observed between
perfusions with digested tofu and control perfusion
experiments.4After luminal perfusion of predigested tofu, 91.2% of genistein
compounds and 92.0% of daidzein compounds were eliminated via luminal
efflux (Table 1
). Of administered malonyl-genistin and malonyl-daidzin, 65.0
and 68.3%, respectively, were found unchanged in the luminal effluent.
Genistein (337.7%) and daidzein (189.7%) content of the luminal
effluent strongly increased as a result of cleavage of the
corresponding glucosides and malonyl-glucosides, which concurrently
decreased. In the luminal effluent, 12.2% of total genistein and
11.6% of total daidzein compounds were conjugated with glucuronic
acid, calculated from genistein (daidzein, respectively) after acid
hydrolysis minus genistein, genistin and malonyl-genistin
(daidzein, daidzin and malonyl-daidzin, respectively) from direct
HPLC analysis before hydrolysis. For cleavage of conjugates in the
luminal effluent, we used acid hydrolysis because enzymatic cleavage
was incomplete.
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18% of the absorbed isoflavones appeared vascularly as
glucuronide conjugates. Only small amounts of genistein (1.4%) and
daidzein (1.6%) compounds were located in the small intestinal tissue.
Total recoveries of genistein and daidzein compounds were 100.6% and
102.6%, respectively. In the luminal and vascular perfusates as well as gut tissue extracts, no mixed glucurono-sulfo-conjugates, no sulfate conjugates of isoflavones, no glucuronide or sulfates conjugates of the glycosides, no malonylated isoflavones, equol, o-desmethylangolensin or p-ethylphenol were detectable by LC-MS.
Considering the variations of the small intestinal weights, the actual fluxes (transport rates) were calculated on the basis of the small intestinal dry weight. Luminal disappearance rates of genistin [7.95 nmol/(min · g)] and malonyl-genistin [3.38 nmol/(min · g)] were constant over the whole perfusion time. The total vascular appearance rate of genistein compounds was 2.48 nmol/(min · g), composed of genistein [0.78 nmol/(min · g)], genistin [1.25 nmol/(min · g)] and genistein glucuronide [0.45 nmol/(min · g)]. The secretion of genistein glucuronide [3.15 nmol/(min · g)] to the luminal side was constant during perfusion, whereas genistein secretion [mean flux: 4.95 nmol/(min · g)] showed a significant increase until the 20- to 30-min time point, when an apparent steady state was established.
In the luminal effluent, daidzin [2.34 nmol/(min · g)] and malonyl-daidzin [1.40 nmol/(min · g)] disappeared, whereas daidzein [0.89 nmol/(min · g)] and daidzein glucuronide [1.49 nmol/(min · g)] appeared. The vascular appearance of daidzein [0.67/(min · g)] exceeded the appearance of daidzin [0.46 nmol/(min · g)] and daidzein glucuronide [0.22 nmol/(min · g)]. No significant differences over the perfusion time were observed in any fluxes of daidzein compounds.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSSampling and sample preparationAnalytical proceduresTofuChemicals and solventsRESULTSDISCUSSIONREFERENCES |
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). The functional integrity and
viability of the intestinal preparations were monitored carefully and
could be fully confirmed considering data from earlier studies using
the same model (Andlauer et al. 2000b, 2000c and 2000d
,
Hummel et al. 1997
, Hummel 1998
). Oxygen
uptake of the preparations [3.8 and 5.9 µmol/(min · g)]
conformed well with earlier published data [3.7–8.6
µmol/(min · g) (Hummel 1998
, Kavin et al. 1967
, Stevenson and Weiss 1988
,
Windmüller et al. 1970
)]. Oxygen consumption of
the small intestine in vivo was apparently higher, corresponding to 10
µmol/(min · g) (Anzeto et al. 1984
). Glucose
consumption [6.7 µmol/(min · g) (Hanson and Parsons 1976
)] agreed well with that in the present study [5.9 and
6.9 µmol/(min · g)]. Arterial pressure in the gut of the
narcotized rat varied from 80 to 100 mm Hg (Anzeto et al. 1984
), slightly higher than in the present perfused intestine
(70.8 and 79.5 mm Hg) and highly dependent on the viscosity of the
vascular medium. From studies with isolated small intestinal
preparations, 58–90 mm Hg were reported (Hartmann et al. 1984
, Hummel 1998
, Plauth et al. 1991
). Control perfusions with basic media without isoflavones confirmed that the small intestine of the experimental rats did not contain any isoflavones sequestered from food. Importantly, a nearly complete recovery of isoflavones from luminal and vascular perfusion media as well from the intestinal tissue was obtained. Mean recoveries from three experiments for daidzein and for genistein compounds were somewhat over 100%, which might be explained by small amounts of acetyl-glucosides of genistein and daidzein, observable with LC-MS, but not quantifiable with HPLC-UV.
The measured absorption rate of genistein compounds derived from tofu
in the present study (8.0%) is in fair agreement with earlier
observations gained from human feeding experiments with soy milk,
corresponding to 14.6% (Lu et al. 1995
), 9% (Xu et al. 1994
), 10% (Xu et al. 1995
) and 16%
with tofu, calculated from the urinary recovery (Xu et al. 2000
). Absorption rate of pure genistin investigated at the
same concentration and with the same intestinal model in a previous
study was 17.2% (Andlauer et al. 2000b
). Thus it
appears that the tofu matrix decreases the genistin absorption rate. On
the other hand, the relative absorption rate of genistin is difficult
to calculate because malonyl-genistin is partly cleaved to yield
both genistin and genistein. In previous studies, daidzein revealed a
better bioavailability than genistein in human studies [16%
(Xu et al. 1995
), 21% (Xu et al. 1994
),
51% (Xu et al. 2000
)]. In this study, however,
daidzein showed about the same absorption rate (P > 0.05) as genistein compounds (8.9 and 8.0%, respectively); the
absolute absorption of daidzein compounds was much lower because of the
lower content of daidzein compounds in the tofu. The similar absorption
rates of genistein and daidzein in the small intestinal preparation
might be explained by the greater gut microbial degradation of
genistein compared with daidzein in in vivo models (Xu et al. 1995
). Food-derived differences in isoflavone absorption
rates might be explained by the varying conjugation patterns of
isoflavones (not conjugated, glucosylated, malonyl-glucosylated) in
different soy products (Coward et al. 1993
, Wang and Murphy 1994
). In this respect, it is notable that flavonoid
and isoflavonoid glycosides are poorly absorbed in the small intestine
compared with their aglycones, due to higher hydrophilicity and greater
molecular weight (Hutchins et al. 1995
, Xu et al. 1995
). Results from previous studies support this notion by
showing a considerably higher vascular uptake of genistein (41%;
Andlauer et al. 2000d
) compared with genistin (17.2%;
Andlauer et al. 2000b
) (Table 2
).
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and Figure 3
, respectively. It is obvious that genistin, the main genistein
compound, is either absorbed from the lumen partly unhydrolyzed and
transported directly (by an unknown transporter or diffusion), or
partly hydrolyzed in the intestinal tissue and subsequently transported
to the vascular side as genistein (Fig. 2)
. Genistein is conjugated
with glucuronic acid and the glucuronide secreted chiefly by an active
transport process. It seems unlikely that charged and therefore highly
polar glucuronides (pKa = 2–3;
Dutton 1980
) are released from the metabolizing
compartment by passive diffusion. The possible underlying absorption
and secretion mechanism of glucuronides might be explained by specific
transport carriers for phenol glucuronides in the brush border and the
basolateral membrane as assumed by some investigators (De Vries et al. 1989
, Koster and Noordhoek 1983
).
Actually, for glucuronides of the flavonoid chrysin, an active
transport via the multidrug resistance protein MRP2 pump could be
described in the human intestinal cell line, Caco-2 (Walle et al. 1999b
). Malonyl-genistin, as well as part of
tofu-derived genistin, is presumably hydrolyzed in the lumen by
enzymes bound to the brush border membrane, e.g., lactase phloridzin
hydrolase (Day et al. 2000
). Genistein secretion from
intestinal tissue can be excluded because of the high luminal
concentration of genistein provided, assuming that no active transport
exists (Walle et al. 1999a
). Daidzin is partly
absorbed from the intestinal tissue by an unknown transport process
(Fig. 3)
. Furthermore, daidzin and daidzein, both cleavage products of
malonyl-daidzin, are partly absorbed during small intestinal
passage. In the small intestine, daidzin might be partly cleaved to
daidzein, which is readily conjugated with glucuronic acid. Daidzin,
daidzein and daidzein glucuronide are transported to the vascular side,
the glucuronide certainly by an active transporter (for active
transport of glucuronides, see genistein discussion above). Most of the
daidzein glucuronide is secreted and found in the luminal effluent. The
predominant compound in the luminal effluent is daidzin coming from
digested tofu and conceivably from malonyl-daidzin, which might be
converted during intestinal passage (also to daidzein). A secretion of
daidzein into the higher concentrated luminal perfusate is not likely,
considering that there is no active transport.
|
|
, Manach et al. 1996
, Setchell et al. 1998
, Yasuda et al. 1996
). In support of this,
phloretin and quercetin have recently been shown to occur as glycosides
in human plasma (Paganga and Rice-Evans 1997
). Yet
there are no reports of isoflavone glucosides in human plasma, despite
several studies in which such isoflavones could have been detected.
Genistin was hydrolyzed to a greater extent than daidzin,
resulting in a considerable increase in genistein (338%) in the
luminal perfusate. Consistent with the fact that genistin is stable in
the luminal effluent and thus any microbial degradation can be
excluded, we propose therefore a glycosidic cleavage as repeatedly
reported (Booth et al. 1957
, Day et al. 1998
, Griffiths 1982
, Ioku et al. 1998
). ß-Glucoside–cleaving enzymes include the
intracellularly located ß-glucuronidase (Andlauer et al. 2000a
) and a broad-specificity cytosolic ß-glucosidase
(Day et al. 1998
, McMahon et al. 1997
) as
well as the lactase phloridzin hydrolase, which is present on the
luminal side of the brush border membrane (Day et al. 2000
). From the low aglycone concentrations on the blood side
and the high luminal aglycone concentrations, we exclude a back
transport of genistein and daidzein from the intestinal tissue to the
luminal side. Therefore, the high luminal genistein and daidzein
concentrations more likely result from the luminal cleavage of
glucosides by the lactase phloridzin hydrolase than from secreted
aglycones coming from cytosolic cleavage.
Isoflavones are extensively transformed by phase II enzymes,
especially by UDP glucuronosyltransferase (EC 2.4.1.17) (Lundh 1990
). From earlier studies, the glucuronidation of isoflavones
was thought to be liver specific as is the case with most steroidal
estrogens (Axelson et al. 1984
). However, several
investigators have shown recently that this phase II biotransformation
also occurs in the gut tissue (Chowdhury et al. 1985
,
Koster and Noordhoek 1983
, Mizuma and Awazu 1998
). Our results, as well as previous studies with genistin
and genistein in the same model (Andlauer et al. 2000b and 2000d
) and with everted intestinal sac preparations
(Sfakianos et al. 1997
), provide evidence that
isoflavone aglycones are glucuronidated in the small intestinal tissue.
No other conjugates or conjugates of the 7-glucosides were found in any
perfusion experiment, in contrast to results obtained from a study
after oral administration of daidzin to rats, reporting sulfate,
disulfate and sulfoglucuronide conjugates of daidzein (Yasuda et al. 1994
). The distribution of genistein and daidzein
glucuronide did not differ in the compartments of the small
intestine with the exception of the blood vessels, which contained only
small amounts of both isoflavones. Glucuronide conjugates were
preferentially secreted into the luminal perfusate, whereas only
12% of total glucuronides was transported to the vascular side
(Fig. 2)
. In previous experiments with genistin, about a third
of the genistein glucuronide was transported to the vascular side
(Table 2)
. We assume that tofu compounds influence glucuronide
transport and the extent of glucuronidation via the UDP
glucuronosyltransferase or as a result of a limited glucuronidation
capacity of the small intestinal tissue, as calculated from perfusion
experiments with genistin (Andlauer et al. 2000b
),
genistein (Andlauer et al. 2000d
) and tofu isoflavones.
Indeed, the amounts of glucuronide substances were practically
identical (303–329 nmol/g dry intestine) despite the considerably
higher isoflavone content of tofu.
Intestinal handling of phytochemicals derived from tofu was
investigated for the first time in this study. The results were
compared with data acquired from studies of absorption and metabolism
of phytochemicals from buffered saline (Table 2)
. Tofu ingredients
influenced genistin uptake, the extent of glucuronic conjugation and
distribution of glucuronides in the intestine. Indeed, this study
suggests that simulated digestion and the use of the isolated perfused
rat small intestine are suitable tools with which to investigate and
evaluate the influence of the food matrix on intestinal handling of
phytochemicals.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
3 Abbreviations used: C, concentration
differences; DW, dry weight; LC-MS, liquid chromatography-mass
spectrometry; RP-HPLC, reversed-phase HPLC.
4 Viability parameters (means ± SD) for control perfusion (n = 4) and
tofu (n = 3) experiments: oxygen consumption: 3.8
± 0.3 (5.9 ± 2.4) µmol/(min · g);
lactate-pyruvate ratio: 27.4 ± 5.3 (32.9 ± 17.1);
glucose consumption: 6.9 ± 1.5 (5.9 ± 1.4)
µmol/(min · g); arterial pressure 79.5 ± 21.0 (70.8 ± 10.9) mm Hg; arterial pH: 7.5 ± 0.1 (7.5
± 0.0); venous pH: 7.3 ± 0.0 (7.4 ± 0.1).
Manuscript received June 26, 2000. Initial review completed August 3, 2000. Revision accepted September 4, 2000.
|
REFERENCES |
|---|
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