Steroid metabolism in the rabbit: Biliary and urinary excretion of metabolites of [4-14C]testosterone

1. [4-(14)C]Testosterone was administered to anaesthetized male and female New Zealand White rabbits as a single injection or as a 45-60min. infusion. 2. After a single dose a total of approx. 56-80% of the radioactivity was excreted in bile and urine. After infusion total recovery of radioactivity was approx. 63-75%. 3. The mean ratio of metabolites in urine to those in bile was 0.77+/-0.41 (range 0.3-1.5). 4. Bile and urine samples were hydrolysed successively by beta-glucuronidase, cold acid and hot acid. In both bile and urine neutral metabolites extracted by ethyl acetate-ether were found mainly after beta-glucuronidase hydrolysis, but a considerable proportion of the dose was converted into substances not extractable from alkaline aqueous solution after all forms of hydrolysis used.     

Steroid metabolism in the rabbit. Biliary and urinary excretion of metabolites of [4-14C]progesterone

1. [4-(14)C]Progesterone was administered intravenously to anaesthetized male and female New Zealand White rabbits as a single injection or as a 45-60min. infusion. 2. After a single dose about 60% of the radioactivity was recovered in 6hr., and twice as much radioactivity was present in bile as in urine. After infusion total recovery of radioactivity was only about 40% in 6hr., but the relative proportions of metabolites in bile and urine were about the same as after a single dose. 3. Bile and urine samples were hydrolysed successively by beta-glucuronidase, cold acid and hot acid. 4. In bile the major proportion of metabolites appeared in the glucuronide fraction; in urine beta-glucuronidase hydrolysis yielded the greatest amounts of ether-extractable radioactivity, but the greatest proportion of radioactivity could not be extracted by ether from an alkaline solution of the hydrolysed urine. 5. There was no apparent difference in the quantity or distribution of metabolites excreted by male and female animals.     

Steroid metabolism in the cat. Biliary and urinary excretion of metabolites of [4-14C]cortisone

1. [4-(14)C]Cortisone was administered to anaesthetized male cats as a single injection or as a 45-60min. infusion. 2. After the single dose a total of 69.6-89.6% of the radioactivity was excreted in bile, and 0.5-7.1% in urine. After infusion total recovery in bile was 73.4-92.1%, and 1.2-1.7% in urine. 3. When bile and urine samples were hydrolysed successively by beta-glucuronidase, cold acid and hot acid, most of the radioactivity was converted into substances not extractable from neutral aqueous solution by ethyl acetate-ether. 4. In bile, metabolites hydrolysable by beta-glucuronidase were found in only small proportions (3-4%) of the dose.     

Metabolism of intravenously administered 7 alpha-hydroxycholesterol-3 beta-stearate in the hamster.

In order to investigate the metabolic fate of serum esterified 7 alpha-hydroxycholesterol, [4-14C]7 alpha-hydroxycholesterol-3 beta-stearate was synthesized from labeled cholesterol and administered to bile fistula hamsters intravenously. Bile samples were collected at every 20 min for 7 h. Radioactivity was detected in bile 40 min after the beginning of the infusion of the labeled compound and 56.5 +/- 5.7% (48.7-66.0%) of the administered radioactivity was recovered in bile during 7 h. The liver contained appreciable radioactivity (19.5 +/- 7.6% of the administered dose) at the time of sacrifice. Only a trace amount of radioactivity was detected in urine and blood. Cumulative recovery of the radioactivity was 76.3 +/- 8.6% (63.3-90.4%). Major radioactive metabolites in the bile samples were identified to be taurine- and glycine-conjugated cholic acid and chenodeoxycholic acid by radioactive thin-layer chromatographic analysis of the bile samples before and after enzymatic hydrolysis and 3 alpha-hydroxysteroid dehydrogenase treatment. The conversion was nearly complete and we could not detect neutral metabolites, such as the mother compound, free 7 alpha-hydroxycholesterol and bile alcohols, as well as glucuronidated or sulfated bile acids. It is concluded that serum esterified 7 alpha-hydroxycholesterol could be effectively taken up by the liver, hydrolyzed by cholesterol esterase and metabolized via the normal biosynthetic pathway to taurine- or glycine-conjugated primary bile acids to be excreted into bile.     

Protection of catechol oestrogen from oxidation during enzymic hydrolysis of biliary conjugates

2-Hydroxyethynyloestradiol (2-OHEE2), a major biliary metabolite of 17 alpha-ethynyloestradiol in female rats, is conjugated largely with glucuronic acid. Accurate quantitation of [3H]2-OHEE2 deconjugated by enzymic hydrolysis depends upon co-incubation with ascorbate (5-10 mM). In the absence of ascorbate, the proportion of [3H]2-OHEE2 declines by 30 +/- 7% (x +/- SD, n = 4) during a 3 h incubation of bile with arylsulphohydrolase and beta-glucuronidase. Over 16 h, decomposition of the catechol leads to a decrease in ether-extractable 3H labelled components.     

Metabolism of [6]-gingerol in rats

The metabolic fate of [6]-gingerol, one of the active constituents of Zingiber officinale Roscoe, was investigated using rats. The bile of rats orally administered [6]-gingerol was shown to contain a major metabolite (1) by HPLC analysis. Although the metabolites derived from [6]-gingerol were not detected in the urine, the ethyl acetate extract of the urine after enzymatic hydrolysis was shown to contain six minor metabolites (2-7). Their structures were determined to be (S)-[6]-gingerol-4'-O-beta-glucuronide (1), vanillic acid (2), ferulic acid (3), (S)-(+)-4-hydroxy-6-oxo-8-(4-hydroxy-3-methoxyphenyl) octanoic acid (4), 4-(4-hydroxy-3-methoxyphenyl)butanoic acid (5), 9-hydroxy [6]-gingerol (6) and (S)-(+)-[6]-gingerol (7) based on spectroscopic and chemical data. The total cumulative amount of 1 excreted in the bile and 2-7 in the urine during 60 h after the oral administration of [6]-gingerol were approximately 48% and 16% of the dose, respectively. The excretion of 2-7 in the urine decreased after gut sterilization. On the other hand, the incubations of [6]-gingerol with rat liver showed the presence of 9-hydroxy [6]-gingerol, gingerdiol (8), and (S)-[6]-gingerol-4'-O-beta-glucuronide (1). These findings suggest that the gut flora and enzymes in the liver play an important part in the metabolism of [6]-gingerol.     

Bile acids. XXXV. Metabolism of 5 -cholestan-3 -ol in the Mongolian gerbil

The principal bile acid of Mongolian gerbil bile is cholic acid, although small amounts of chenodeoxycholic and lesser amounts of deoxycholic acids are identified. Muricholic acids were not found in gerbil bile. The ratio of trihydroxy to dihydroxy bile acids in gerbil bile is approximately 11:1. After administration of [4-(14)C]5alpha-cholestan-3beta-ol to gerbils with bile fistulas, 4-7% of the administered (14)C was recovered in bile and 16% in urine on the first 6 days. Alkaline hydrolysis of the bile afforded the biliary acids which were separated by partition chromatography. The (14)C ratio of trihydroxy to dihydroxy bile acids was 11:1. Allocholic acid was identified as the major acidic biliary metabolite. From analysis of (14)C retained in selected tissues, the adrenal gland appears to be an important site for retention of cholestanol or its metabolites.     

Steroid metabolism in the cat. Biliary and urinary excretion of metabolites of [4-14C]testosterone

1. [4-(14)C]Testosterone was administered intravenously to anaesthetized male cats as a single injection or as a 45-60min. infusion. 2. Most of the administered radioactivity was excreted in the bile (70-80%); only 2.9-5.5% of the dose was excreted in the urine. 3. Bile and urine samples were hydrolysed successively to yield glucuronide, ;cold-acid-hydrolysed' and ;hot-acid-hydrolysed' fractions. 4. The proportion of glucuronides in bile decreased in successive samples, but cold-and hot-acid-hydrolysed metabolites showed no consistent change. 5. After hydrolysis most of the radioactivity in both bile and urine could not be extracted by ether from neutral aqueous solution.     

Excretion of cholate glucuronide

[3-3H]Cholic acid glucuronide [7 alpha,12 alpha-dihydroxy-3 alpha-O-(beta-D-glucopyranosyluronate)-5 beta- cholan-24-oate] was synthesized and administered to rats prepared with either an external biliary fistula or a ligated bile duct. When bile fistula animals were given either microgram or milligram amounts of the glucuronide, biliary secretion of label was rapid and efficient: greater than 90% of the administered label was secreted within 60 min and total recovery of label in bile was 98.6 +/- 1.2%. Studies in which [14C]taurocholate was included in the dose indicated that this bile acid was secreted into bile significantly more rapidly than was the glucuronide. In animals with ligated bile ducts, urinary excretion was the major route of elimination: after 20 hr, 83.4 +/- 9.3% of the administered dose had been excreted in urine. Urinary excretion of cholate glucuronide was significantly more rapid than that of taurocholate. Gas-liquid chromatographic analysis of the methyl ester acetate derivatives of labeled compounds isolated from bile and urine by chromatography established that the bulk (greater than 70%) of the administered material was secreted in bile or excreted in urine as the intact cholate glucuronide. From these results, we conclude that the glucuronidation of cholic acid produces a derivative which is rapidly and effectively cleared from the circulation and excreted.     

Conjugated forms of [3H] alpha, 25-dihydroxyvitamin D3 in rat bile

When small doses of [3H]D3, [3H]25-OHD3 and [3H]alpha, 25-diOHD3 were administered intravenously to rats 6.3 +/- 1.1% (means +/- SEM, n = 4), 9.7 +/- 0.9% (n = 6) and 12.8 +/- 2.6% (n = 8), respectively, of the administered radioactivity was excreted in bile. The radioactive biliary conjugated metabolites were analysed by ion exchange chromatography: in the case of all 3 substrates about 30% of metabolites were found to be cationic on the basis of their being retained on sulphopropyl-Sephadex G-25 (H+-form) when applied in 70% methanol. The balance of the metabolites were neutral and anionic and were analysed on TEAP-Lipidex: in the case of 1 alpha, 25-diOHD3 the following metabolite classes were detected (on the basis of co-elution with authentic standards) (in order of quantitative importance): taurine conjugates, neutral metabolites, monosulphates, glucuronides, carboxylic acids, glycine conjugates and disulphates. Alkaline hydrolysis of the taurine and glycine conjugates yielded products 60% of which now chromatographed as carboxylic acids. Hydrolysis of the glucuronide and monosulphate fractions indicated significant levels of mixed conjugation yielding some products which now chromatographed as glycine and taurine conjugates, respectively. The nature of the cationic conjugates was not elucidated but they had the following properties: they could be hydrolysed by alkali to yield non-cationic radioactive metabolites (these released metabolites were heterogeneous as judged by TEAP-lipidex chromatography); they were partially hydrolysed to non-cationic forms by beta-glucuronidase; and on reverse-phase HPLC they had an elution profile that was significantly different to that of histidyl-, ornithyl- or lysyl-calcitroic acid.     

Bile acids of patients with renal failure receiving chronic hemodialysis

Bile acids in serum, urine and dialysate of 8 patients with renal failure in chronic hemodialysis were analyzed by gas chromatography and gas chromatography-mass spectrometry. The following results were obtained: 1. Lithocholic acid, 3 beta-hydroxy-5-cholen-24-oic acid, deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, and cholic acid were identified in hemodialysate as well as in serum and urine. 2. The serum bile acid concentration of the patients was 2.78 +/- 0.57 micrograms/mL before hemodialysis and 1.34 +/- 0.48 micrograms/mL after a 5-h period hemodialysis with cuprophane membrane. The proportions of secondary bile acids in predialysis and postdialysis serum of patients were significantly higher than those of healthy subjects. 3. Two out of 8 patients excreted urine. But the amounts of bile acids in urine of the patients were very small compared to those of healthy subjects. 4. The amount of bile acids removed from blood by hemodialysis was 0.70 +/- 0.25 mg. In dialysate, cholic acid constituted a larger proportion of the total bile acids, and lithocholic acid a smaller proportion, when compared to those in urine of patients and healthy subjects.     

Metabolism of cysteinyl leukotrienes in monkey and man

The proinflammatory cysteinyl leukotrienes are inactivated in primates by (a) intravascular degradation, (b) hepatic and renal uptake from the blood circulation, (c) intracellular metabolism of leukotriene E4 (LTE4), and (d) biliary and renal excretion of LTC4 degradation products. We have analyzed cysteinyl leukotriene metabolites excreted into bile and urine of the monkey Macaca fascicularis and of man. In both species, hepatobiliary leukotriene elimination predominated over renal excretion. In a representative healthy human subject at least 25% of the administered radioactivity were recovered from bile and 20% from urine within 24 h. In monkey and man intravenous administration of 14,15-3H2-labeled LTC4 resulted in the biliary and urinary excretion of labeled LTE4, omega-hydroxy-LTE4, omega-carboxy-LTE4, omega-carboxy-dinor-LTE4, and omega-carboxy-tetranor-dihydro-LTE4. Small amounts of N-acetyl-LTE4 were detected in human urine only. Oxidative metabolism of LTE4 proceeded more rapidly in the monkey resulting in the formation of higher relative amounts of omega-oxidized leukotrienes in this species as compared to man. [3H]H2O amounted to less than 2% of the administered dose in monkey and human bile and urine samples. Incubation of isolated human hepatocytes with [3H2]LTC4, [3H2]LTD4, and [3H2]LTE4 showed that only [3H2]LTE4 underwent intracellular oxidative metabolism resulting in the formation of omega- and beta-oxidation products. N-Acetylated LTE4 derivatives were not detected as products formed by human hepatocytes. By a combination of reversed-phase high-performance liquid chromatography and radioimmunoassay, endogenous LTE4 and N-acetyl-LTE4 were detected in human urine in concentrations of 220 +/- 40 and 24 +/- 3 pM, corresponding to 12 +/- 1 and 1.5 +/- 0.2 nmol/mol creatinine, respectively (mean +/- SEM; n = 10). Endogenous LTD4 and LTE4 were detected in human bile (n = 3) in concentrations between 0.2-0.9 nM. Our results demonstrate that LTD4 and LTE4 are major LTC4 metabolites in human bile and/or urine and may serve as index metabolites for the measurement of endogenously generated cysteinyl leukotrienes. Moreover, omega-oxidation and subsequent beta-oxidation from the omega-end contribute to the metabolic degradation of LTE4 not only in monkey but also in man.     

Steroid metabolism in the cat. Biliary and urinary excretion of metabolites of [4-14C]oestradiol

1. [4-¹⁴C]Oestradiol was administered to seven male, seven female and two castrated male cats as a single intravenous injection. Bile and urine were collected for 6h. 2. The radioactivity was excreted mainly in the bile of all animals (53–60%); only approx. 1% of the dose appeared in the urine. 3. Bile and urine samples were hydrolysed successively by β-glucuronidase, cold acid and hot acid. There were significant differences (P<0.005) between the percentage of the dose present in the bile fractions hydrolysed by β-glucuronidase (male, 9.0±1.7%; female, 18.6±1.45%) and by cold acid (male, 18.9±1.44%; female 12.1±1.02%). The excretion of radioactivity in these fractions by the castrated male cats was closer to that of female cats. 4. Approx. 20–27% of the dose could not be extracted from aqueous solution (pH10.5) by ethyl acetate–ether after hydrolysis.     

Behaviour of the NO-β-d-glucosiduronic acid of N-acetyl-N-phenylhydroxylamine as a substrate for β-glucuronidase

1. Biosynthetic sodium (N-acetyl-N-phenylhydroxylamine NO-beta-d-glucosid)-uronate is hydrolysed completely by purified mouse urinary beta-glucuronidase into the products N-acetyl-N-phenylhydroxylamine and glucuronic acid. The hydrolysis is inhibited by saccharo-(1-->4)-lactone. These results not only confirm the identity and purity of the substrate but also establish it as a substrate for beta-glucuronidase. 2. Mammalian and bacterial beta-glucuronidase preparations hydrolysed the substrate at a rate one-fifth of that for (phenolphthalein beta-d-glucosid)uronic acid under the optimum conditions of hydrolysis for each source. 3. The pH optimum is 4.1 and the Michaelis constant, K(m), is 3.3x10(-4)m with purified mouse urinary beta-glucuronidase as the enzyme source acting on the NO-beta-d-glucosiduronic acid. The aglycone after extraction into chloroform was quantitatively determined spectrophotometrically at its absorption maximum (256mmu). 4. The hydrolysis was studied as a function of time and temperature. 5. From a consideration of the chemical and enzymic properties of this NO-beta-d-glucosiduronic acid it is possible to suggest its catabolism in vivo.     

Studies on flavonoid metabolism. Biliary and urinary excretion of metabolites of (+)-[U-14C]catechin

1. The biliary and urinary excretion of (+)-[U-(14)C]catechin was studied in normal male rats after a single injection of the flavonoid. 2. In rats large amounts of radioactivity (33.6-44.3% of the dose in 24h) were excreted in the bile as two glucuronide conjugates [one of which was a (+)-catechin conjugate] and three other unconjugated metabolites. 3. Excretion of radioactivity in the urine when the bile duct was not cannulated amounted to 44.5% of the dose. 4. In both the urine and bile the new metabolites showed maximum excretion in the (1/2)-1(1/2)h after intravenous injection of [(14)C]catechin. 5. The metabolites m-hydroxyphenylpropionic acid, p-hydroxyphenylpropionic acid, delta-(3-hydroxyphenyl)-gamma-valerolactone and delta-(3,4-dihydroxyphenyl)-gamma-valerolactione originate from the action of the intestinal micro-organisms on the biliary-excreted metabolites of (+)-catechin. These phenolic acid and lactone metabolites are then reabsorped and excreted in the urine. 6. It is proposed that, depending on the route of administration of (+)-catechin, there exists an alternative pathway, involving biliary excretion, for the metabolism of (+)-catechin.     

Tissue distribution, elimination, and metabolism of [3H]leukotriene C4 by the conscious marine toad, Bufo marinus.

Tissue distribution, elimination, and metabolism of 3H-labelled leukotriene (LT) C4 were studied in ureter-catheterized conscious marine toads, Bufo marinus. Six and 24 h after injection, organs containing the highest percent of injected radioactivity were small intestine, liver, and kidney. Radioactivity declined in these organs at 24 h by approximately threefold. Peak elimination time for radioactivity in the urine was between 2 and 4 h after the injection. During the 24-h collection period, 55.2 +/- 0.2% of the injected radioactivity was eliminated in the urine. Polar metabolites represented 40.3 +/- 1.1, 57.3 +/- 5.6, and 62.8 +/- 1.6% of the radioactivity at 2, 4, and 6 h, respectively. The primary urinary polar metabolite was 20-carboxy-LTE4, with 18-carboxydinor-LTE4 and 20-hydroxy-LTE4 also present. [3H]LTE4 decreased from 37.2 +/- 1.8% at 2 h to 15.8 +/- 3.3 and 15.0 +/- 2.1% of the radioactivity at 4 and 6 h, respectively. Bile radioactivity was low. N-Acetyl-LTE4 was not detected in urine or bile samples. Radioactivity in the pan water was 14.3 +/- 2.4 and 15.8 +/- 2.5% of the injected radioactivity, at 6 and 24 h, respectively, suggesting that the skin was a route for excretion of leukotrienes. The marine toad is an interesting model demonstrating both similarities and differences from mammals in distribution, elimination, and metabolism of peptide leukotrienes.     

Optimized glucuronide hydrolysis for the detection of psilocin in human urine samples

In order to develop a sensitive and reliable analytical method for psilocin (PC) in urine samples, the hydrolysis conditions including the acid, alkaline and enzymatic hydrolyses have been investigated by monitoring not only PC but also psilocin glucuronide (PCG) by liquid chromatography tandem mass spectrometry (LC-MS-MS); PCG was initially identified in a "magic mushroom (MM)" user's urine by liquid chromatography mass spectrometry (LC-MS) and LC-MS-MS. The proposed conditions optimized for the hydrolysis are as follows: hydrolysis, enzymatic hydrolysis; enzyme, Escherichia coli beta-glucuronidase (5000 units/ml urine); incubation, pH 6 at 37 degrees C for 2h. The complete hydrolysis of PCG in urine was obtained under these conditions, while the enzymatic hydrolyses with three types of beta-glucuronidases originated from bovine liver (Type B-1), Helix pomatia (Type H-1) and Ampullaria provided uncompleted hydrolysis of PCG. Also, neither the acid nor alkaline hydrolysis was found to be applicable. According to the present method, 3.55 microg/ml of psilocin was detected in the "magic mushroom" user's urine after the enzymatic hydrolysis, though psilocin was not detected without hydrolysis.     

Human beta-glucuronidase. Studies on the effects of pH and bile acids in regard to its role in the pathogenesis of cholelithiasis

Human bile contains a considerable amount of endogenous beta-glucuronidase. The effects of pH and bile acids on its activity have been studied in regard to its role in the pathogenesis of cholelithiasis. beta-Glucuronidase, purified from human liver to homogeneity, was structurally stable between pH 4 and 10, but was active only over a much narrower range of pH, with a pH optimum of 5.2. The inactivation below pH 4 was due to its irreversible denaturation, whereas the inactivation at higher pH was due to a true reversible pH effect on the enzyme velocity. Kinetic studies revealed that hydrogen ion acted as a substrate-directed activator of the free enzyme, but not the enzyme-substrate complex, with a molecular dissociation constant of 4 X 10(-6). The enzyme activity was not affected by unconjugated bile acids, primarily due to their extremely low water solubility. Conjugated bile acids, on the other hand, exerted heterogeneous and pH-dependent effects on the enzyme. At pH 5.2, taurocholic acid and glycocholic acid were substrate-directed activators of the enzyme; taurochenodeoxycholic acid and taurodeoxycholic acid, competitive inhibitors; and glycochenodeoxycholic acid and glycodeoxycholic acid, mixed inhibitors. At pH 7.0 all taurine and glycine conjugates behaved as substrate-directed activators. Though beta-glucuronidase activity at pH 7 was only 23% of its maximal activity at pH 5.2, conjugated bile acids tended to restore its activity to a certain extent at pH 7. Thus, endogenous beta-glucuronidase could play a significant role in pigment cholelithiasis.     

Metabolism of lithocholic acid in the rat: formation of lithocholic acid 3-O-glucuronide in vivo

Milligram amounts of [3 beta-3H]lithocholic (3 alpha-hydroxy-5 beta-cholanoic) acid were administered by intravenous infusion to rats prepared with a biliary fistula. Analysis of sequential bile samples by thin-layer chromatography (TLC) demonstrated that lithocholic acid glucuronide was present in bile throughout the course of the experiments and that its secretion rate paralleled that of total isotope secretion. Initial confirmation of the identity of this metabolite was obtained by the recovery of labeled lithocholic acid after beta-glucuronidase hydrolysis of bile samples. For detailed analysis of biliary metabolites of [3H]lithocholic acid, pooled bile samples from infused rats were subjected to reversed-phase chromatography and four major labeled peaks were isolated. After complete deconjugation, the two major compounds in the combined first two peaks were identified as murideoxycholic (3 alpha, 6 beta-dihydroxy-5 beta-cholanoic) and beta-muricholic (3 alpha, 6 beta, 7 beta-trihydroxy-5 beta-cholanoic) acids and the third peak was identified as taurolithocholic acid. The major component of the fourth peak, after isolation, derivatization (to the methyl ester acetate), and purification by high pressure liquid chromatography (HPLC), was positively identified by proton nuclear magnetic resonance as lithocholic acid 3 alpha-O-(beta-D-glucuronide). These studies have shown, for the first time, that lithocholic acid glucuronide is a product of in vivo hepatic metabolism of lithocholic acid in the rat.     

Orally administered rosmarinic acid is present as the conjugated and/or methylated forms in plasma, and is degraded and metabolized to conjugated forms of caffeic acid, ferulic acid and m-coumaric acid

Rosmarinic acid (RA) is contained in various Lamiaceae herbs used commonly as culinary herbs. Although RA has various potent physiological actions, little is known on its bioavailability. We therefore investigated the absorption and metabolism of orally administered RA in rats. After being deprived of food for 12 h, RA (50 mg/kg body weight) or deionized water was administered orally to rats. Blood samples were collected from a cannula inserted in the femoral artery before and at designated time intervals after administration of RA. Urine excreted within 0 to 8 h and 8 to 18 h post-administration was also collected. RA and its related metabolites in plasma and urine were measured by LC-MS after treatment with sulfatase and/or beta-glucuronidase. RA, mono-methylated RA (methyl-RA) and m-coumaric acid (COA) were detected in plasma, with peak concentrations being reached at 0.5, 1 and 8 h after RA administration, respectively. RA, methyl-RA, caffeic acid (CAA), ferulic acid (FA) and COA were detected in urine after RA administration. These components in plasma and urine were present predominantly as conjugated forms such as glucuronide or sulfate. The percentage of the original oral dose of RA excreted in the urine within 18 h of administration as free and conjugated forms was 0.44 +/- 0.21% for RA, 1.60 +/- 0.74% for methyl-RA, 1.06 +/- 0.35% for CAA, 1.70 +/- 0.45% for FA and 0.67 +/- 0.29% for COA. Approximately 83% of the total amount of these metabolites was excreted in the period 8 to 18 h after RA administration. These results suggest that RA was absorbed and metabolized as conjugated and/or methylated forms, and that the majority of RA absorbed was degraded into conjugated and/or methylated forms of CAA, FA and COA before being excreted gradually in the urine.