Unconjugated Fraction Research Articles

Metabolism of [ 3H] 17β-dihydroequilin and [ 3H] 17β-dihydroequilin sulfate in normal postmenopausal women

The metabolism of 17β-dihydroequilin and 17β-dihydroequilin sulfate was investigated after intravenous administration of [ 3H] 17β-dihydroequilin and [ 3H] 17β-dihydroequilin sulfate to postmenopausal women Urine was collected for 3 days and 46.2±10.5% and 54.5±8.7% of the injected dose of [ 3H] 17β-dihydroequilin and [17β- 3H]dihydroequilin sulfate was excreted in the urine respectively. The estrogens present in urine were extracted and fractionated into unconjugated, sulfate, and glucuronide conjugated forms. With both precursors, the major amount (63–64%) of metabolites were excreted in the urine conjugated with glucuronic acid. From the unconjugated, sulfate, and glucuronide fraction, 17β-dihydroequilenin, 17β-dihydroequilin, equilenin, and equilin were isolated. The conversions with both precursors were similar and 17β-dihydroequilenin was the major metabolite isolated from all three fractions; however, the highest levels of all four metabolites were present in the glucuronide fraction. Along with these identifiable metabolites, a large amount (51–81%) of radioactivity was present in the form of metabolites which are more polar than any of the known ring-B unsaturated estrogens. These appear to by polyhydroxy 17β-reduced ring-B unsaturated estrogens which remain to be identified. The in-vivo formation of equilenin and 17β-dihydroequilenin indicates the presence of the enzyme 6,8(9) steroid dehydrogenase in humans. (Steroids 59:389–394, 1994)

Absorption, Distribution, Metabolism, Excretion and Effects on the Hepatic Drug Metabolizing Enzymes of 1-(Bis(4-fluorophenyl)methyl)-4-(2,3,4-trimethoxybenzyl)piperazine dihydrochloride(KB-2796) after Multiple Oral Administration in Rats.

The absorption, distribution, metabolism, excretion and the effect on the drug-metabolizing enzyme activities of 1-[bis (4-fluorophenyl) methyl]-4 (2, 3, 4-trimethoxybenzyl) piperazine dihydrochloride (KB-2796) were studied in male rats during and after multiple oral administration at a dose of 2 mg/kg/day. 1. The blood radioactivities at 24 hr after daily administration increased progressively with an increase in the number of dosing, but remained practically constant after the 15th administration. 2. The blood radioactivity reached Cmax of 188.8 ng/ml at 8 hr after the 21st administration, and decreased gradually with the half-life of 67.3 hr. The Cmax and AUC after the 21st administration were 4.4 and 5.7 times greater than those after the 1st dosing, respectively. 3. The extents of cumulative excretion in the urine and feces were almost constant after the 15th administration, and those until 120 hr after the 21st administration were about 9 and 86%, respectively, which were similar with those after the 1st administration. 4. The radioactivity in tissues at 24 hr after daily administration increased with an increase in the number of dosing. After the final administration, a high level of radioactivity was observed in the fat, lung, liver, skin, adrenal and kidney. The elimination of radioactivity from tissues was slow, especially from the aorta. 5. The radioactivity in the brain, liver, lung, spleen, thymus and aorta were present predominantly in the unconjugated fraction. The main metabolites in the tissues were bis (4-fluorophenyl) methylpiperazine (M6) and 4, 4'-difluorobenzophenone (M10), and the unchanged drug was confirmed to be present in all their tissues. 6. The drug-metabolizing enzyme activities were increased after 7 day consecutive oral administration of KB-2796.

Pharmacokinetics of 17 beta-dihydroequilin sulfate and 17 beta-dihydroequilin in normal postmenopausal women.

The MCRs of 17 beta-dihydroequilin sulfate and 17 beta-dihydroequilin were determined in normal postmenopausal women by single iv injection of either 17 beta-[3H]dihydroequilin sulfate ([3H]17 beta-EqS) or 17 beta-[3H]dihydroequilin ([3H]17 beta-Eq). After the administration of [3H]17 beta-EqS, blood was drawn at various time intervals, and the plasma obtained was fractionated into the unconjugated, sulfate, and glucuronide fractions. The bulk of radioactivity was present in the sulfate fraction, and from this [3H]17 beta-EqS, [3H]equilin sulfate, [3H]equilenin sulfate, and 17 beta-[3H]dihydroequilenin sulfate were isolated and purified, and their concentrations were measured. The disappearance of [3H]17 beta-EqS from plasma can be described as a function of two exponentials. The half-life of the initial fast component was 5 +/- 0.2 min; this component represents the distribution and transfer from a space, with a mean volume (V1) of 6 +/- 0.5 L. The value for the rate constant (k) of total removal from this space was 300 +/- 20 U/day, of which 35 +/- 2% was irreversible. The mean half-life of the slower component of 17 beta-EqS was 147 +/- 15 min, and the mean MCR was 376 +/- 93 L/day.m2. Similarly, after the administration of [3H]17 beta-Eq, the disappearance of radioactivity as 17 beta-Eq from plasma also had two components. The half-lives of the fast and slow component were 5.5 +/- 0.8 and 45 +/- 2.0 min, respectively. The MCR of 17 beta-Eq was 1252 +/- 103 L/day.m2. From both series of experiments, unconjugated and sulfate-conjugated equilin, equilenin, and 17 beta-dihydroequilenin were isolated and purified, and their concentrations were measured. No 17 alpha-reduced metabolites were detected. These results indicate that 17 beta-EqS is cleared twice as fast as equilin sulfate (MCR, 176 L/day.m2), whereas the more potent estrogen 17 beta-Eq is cleared 2 times slower than equilin. The slower elimination and greater estrogenic activity of 17 beta-Eq support the hypothesis that the major in vivo activity of equilin sulfate present in conjugated equine estrogen preparations is expressed via its metabolites 17 beta-EqS and 17 beta-Eq.

Separation and Characterization of Ursodeoxycholate 7-N-Acetylglu-Cosaminides in Human Urine by High-Performance Liquid Chromatography with Fluorescence Detection

Abstract The separation and characterization of unconjugated, glycine- and taurine-conjugated ursodeoxycholate 7-N-acetyl-glucosaminides in human urine without prior deconjugation have been carried out by high-performance liquid chromatography (HPLC) on a reversed phase column. The bile acid fraction was obtained from a urine specimen by the combined use of a Sep-Pak C18 cartridge for solid phase extraction and lipophilic gel, piperidinohydroxypropyl Sephadex LH-20 (PHP-LH-20), for ion-exchange chromatography. Bile acid N-acetyl-glucosaminides were derivatized quantitatively into the corresponding fluorescent esters through the inherent primary hydroxyl group by treatment with 9-anthroyl cyanide. The derivatives were separated into unconjugated, glycine- and taurine-conjugated fractions on PHP-LH-20. Subsequent resolution into individual N-acetylglucosaminides was attained by HPLC with fluorescence detection on a Cosmosil 5C18 column using 0.3% potassium phosphate buffer-methanol as a mobile phase. Unconjugated, glycine- and taurine-conjugated urso-deoxycholate 7-N-acetylglucosaminides in human urine from a patient with primary biliary cirrhosis were unambiguously identified on the basis of their chromatographic behavior using mobile phases of different pHs and organic modifiers. It has proved that ursodeoxycholate 7-N-acetylglucosaminides in urine were dominantly conjugated with glycine, while any 3-N-acetylglucosaminides of ursodeoxycholates as well as other common bile acids were not present.

In vitro synthesis of 20α-reduced and of 11- and 21-oxygenated steroids and their sulfates by testes of the goldfish (Carassius auratus): Testicular synthesis of corticosteroids

Testes from spermiating goldfish were incubated with [(3)H]17-hydroxyprogesterone. The major products in the unconjugated fraction were identified as androstenedione, androstenetrione, 11-β-hydroxyandrostenedione, 11-ketotestosterone, 17,20α-dihydroxy-4-pregnen-3-one (17,20αP) and 11-deoxycortisol. Testosterone was present predominantly in the glucuronide fraction, but yields were low (1-3%). The major components of the sulfate fraction were 17,20αP and 11-deoxycortisol. The identification of cortisone in low yield (< 2.5010) in both the free and sulfate fractions is the first report of corticosteroid biosynthesis by a teleost testis. The high yields of 17,20αP and 11-deoxycortisol and their sulphates suggests that their possible role in spermiation of the goldfish should be further investigated.

Secretion of 19-hydroxyandrostenedione and 19-hydroxytestosterone by porcine Leydig cells in vitro and in vivo

19-Hydroxytestosterone and 19-hydroxyandrostenedione have been identified as secretory products of the testes in the mature male domestic pig. Their isolation and identification were made by reverse-phase high-performance liquid chromatography and capillary gas chromatography-mass spectrometry (CGC-MS) of extracts from testicular vein blood and media of incubations with Leydig cells. Blood was collected from veins on the surface of the testes of anaesthetized boars. Collagenase-dispersed Percoll-purified cells (> 90% pure) were incubated (20 x 10(6) cells/flask) with androstenedione (8.75 mumol/l) or [3H]androstenedione (5 x 10(6) c.p.m.) for < 60 min. Steroids were recovered from plasma or media by solid-phase extraction and the unconjugated fractions chromatographed isocratically in two solvent systems (acetonitrile:water, 37:63 (v/v) and methanol:water, 70:30 (v/v)) before CGC-MS analysis. 19-Hydroxytestosterone was present in greater quantities than 19-hydroxyandrostenedione in testicular vein blood; it was also seen as a quantitatively significant metabolite of unlabelled and radioactive androstenedione in the incubation studies. The demonstration of the secretion of 19-hydroxyandrogens from porcine testes thus raises questions concerning the physiological significance of a testicular, rather than an adrenal, secretion of these compounds.

Liquid chromatographic separation and gas chromatographic—mass spectrometric determination of 17α-methyltestosterone residues extracted from rainbow trout tissues

The residues of [ 3 H]-17-methyltestosterone (17MT) in rainbow trout after a single intragastric dose (200 μg, 0.37 MBq) were investigated. The metabolites were extracted from liver and carcass with chloroform—methanol (2 + 1, v/v). After enzymatic hydrolysis of the glucuronides, unconjugated steroids and aglycones were chromatographed by reversed-phase liquid chromatography. Each labelled fraction was collected and analysed by gas chromatography—mass spectrometry. After 24 h, the liver contained 4% of the administered dose. In the hepatic unconjugated and aglycone fractions the major metabolite was an isomer of 17α-methylandrostane-3ξ,16ξ,17β-triol. 7α-Hydroxy-17α-methylandrostane-3α,17β-diol was also observed in the unconjugated fraction. In addition to these metabolites, unchanged 17MT, 17α-methyl-5β-androstane-3α,17β-diol and 17β-hydroxyandrosta4,6-dien-3-one were identified in the unconjugated fraction extracted from carcass samples. After 72 h, 7α-hydroxy-17α-methylandrostane-3α,17β-diol persisted to a significant extent in carcass samples whereas no trace of 17MT was detected. This metabolite could therefore be used as an indicator of the administration of 17MT in fish.

High-Performance Liquid Chromatography of Clomipramine and Metabolites in Human Plasma and Urine

An isocratic, high-performance liquid chromatography method has been developed for simultaneous determination of the tricyclic antidepressant clomipramine and six metabolites: desmethylclomipramine, 2- and 8-hydroxy-clomipramine, 2- and 8-hydroxydesmethylclomipramine and didesmethyl-clomipramine in plasma and urine. In addition, clomipramine N-oxide was determined in the unconjugated fraction of urine. The method is based on a three-step liquid-liquid extraction, the chromatographic eluent was 30% acetonitrile and 70% aqueous sodium perchlorate solution, pH 2.5, and the UV detection was performed at 220 nm. The method was applied to the analysis of samples followed by oral doses of clomipramine to patients and healthy volunteers.

In vitro biosynthesis of steroids, including 11-deoxycortisol and 5α-pregnane-3β,7α,17,20β-tetrol, by ovaries of the goldfish Carassius auratus during the stage of oocyte final maturation

To examine the production of steroids with potential oocyte maturation-inducing or pheromonal activity in the goldfish ( Carassius auratus) we have incubated mature ovaries of this species with 17-[ 3H]hydroxyprogesterone. The metabolites in the unconjugated, glucuronide, and sulfate fractions were identified by chromatography, microchemical reaction, and, in most cases, crystallization to constant specific activity. A major metabolite, present in all three fractions, was tentatively identified as 5α-pregnane-3β,7α,17,20β-tetrol. Although 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) was found in only low yield (as a sulfate), the presence of the tetrol indicates that it is synthesized in high yield but very rapidly metabolized. The relative proportions of 17,20α-dihydroxy-4-pregnen-3-one (17,20α-P), 11-deoxycortisol (17,21-dihydroxy-4-pregnen-3,20-dione) and 17,20β,21-trihydroxy-4-pregnen-3-one (17,20β,21-P) varied significantly between incubations and may be affected by the maturational state of the ovary or the method used to stimulate oocyte maturation. Testosterone was present predominantly as its glucuronide. Significant production of glucuronides and sulfates was observed in all incubations. Twenty-five to 30% of the radioactivity remained associated with the tissue, but the distribution of activity between the metabolites did not differ greatly from that found in the medium. These results indicate that 11-deoxycorisol and its 20β-reduced derivative (17,20β,21-P) may be significant in spawning female goldfish.

Changes in bile acid composition in patients with primary biliary cirrhosis induced by ursodeoxycholic acid administration

We describe a detailed study of the effects of ursodeoxycholic acid administration on bile acid composition of the serum and bile of patients with primary biliary cirrhosis. Gas chromatography-mass spectrometry was used to analyze bile acids from 10 patients with primary biliary cirrhosis before and during ursodeoxycholic acid administration (500 mg/day, corresponding to approximately 8 mg/kg body wt), after group separation of the unconjugated and conjugated fractions by lipophilic anion exchange chromatography. These studies were directed at assessing whether the beneficial role of ursodeoxycholic acid in primary biliary cirrhosis was the consequence of a shift in the hydrophobic/hydrophilic balance of the bile acid pool and whether the hypercholeresis might result from the cholehepatic circulation of unconjugated ursodeoxycholic acid in bile. In basal conditions, the unconjugated bile acids accounted for only 5.5% and 2.5%, respectively, of the total bile acids of serum and bile; cholic acid was the major component of the conjugated fraction of serum and bile (56.0% ± 4.0%, mean ± S.E.M.), and ursodeoxycholic acid was present in only trace amounts. The conjugated fraction contained many unusual bile acids (representing 16.5% ± 1.3% of total) including C 25 bile acids, iso-chenodeoxycholic acid and several oxo-bile acids. After ursodeoxycholic acid administration biochemical indices of liver function all improved, but the proportions of the unconjugated bile acids in serum and bile did not significantly change. Ursodeoxycholic acid became the predominant biliary bile acid of conjugated bile acid fraction (33.4% ± 1.4%) and significant decreases occurred (p < 0.05) in biliary levels of cholic acid (28.3% ± 2.3%) and the unusual bile acids (9.2% ± 0.8%), but no changes occurred in the proportions of chenodeoxycholic acid (from 19.4% ± 2.7% to 19.4% ± 1.3%) and deoxycholic acid (from 5.4% ± 1.4% to 6.8% ± 1.3%). The proportions of biliary lithocholic acid increased significantly after ursodeoxycholic acid administration (from 0.7% ± 0.1% to 2.9% ± 0.9%; p < 0.05). In serum, ursodeoxycholic acid concentrations attained levels of 64 μmol/L. Serum lithocholic acid concentrations increased, whereas cholic and chenodeoxycholic acids decreased. Relative percentages of individual biliary bile acids in the unconjugated fraction were similar to the conjugated fraction before and after ursodeoxycholic acid treatment. The proportions of biliary unconjugated ursodeoxycholic acid did not change significantly after its administration (from 0.6% to 0.9% of the total). These data provide little support for a significant shift in the hydrophilicity of the circulating bile acid pool after ursodeoxycholic acid administration. However, because the detergency and hepatotoxicity of a bile salt appears to correlate well with the retention index determined by reverse-phase liquid chromatography, the qualitative changes observed in the biliary pool (i.e., a displacement of cholic acid by ursodeoxycholic acid), suggests that a relative lowering of the hepatotoxicity index of the bile acid pool occurs after ursodeoxycholic acid therapy. Furthermore, our findings indirectly suggest that a hypercholeresis related to the biliary secretion of unconjugated ursodeoxycholic acid does not play a role in the beneficial effects of ursodeoxycholic acid therapy in primary biliary cirrhosis.

Microanalysis of bile acid composition in intrahepatic calculi and its etiological significance

Brown pigment stones in the intrahepatic bile ducts were compared with those found in the extrahepatic bile ducts with special reference to the bile acids modified by bacterial intervention, that is, unconjugated, glucuronidated, secondary, and ketonic bile acid fractions. The former showed significantly lower amounts of total bile acids (P < 0.01) and lower proportions of unconjugated bile acid fraction (P < 0.01), secondary bile acid fraction (P < 0.05), and ketonic bile acid fraction (P < 0.05) to total bile acids than the latter. The discriminant analysis using these bile acid parameters led to complete separation between intrahepatic and extrahepatic stones in the case of brown pigment stones. In contrast, cholesterol stones in the intrahepatic bile ducts showed the bile acid composition close to those found in the extrahepatic ducts and gallbladder. The above data show that the bacterial infection plays a less important role in the formation and ensuing growth of most intrahepatic brown pigment stones than in extrahepatic stones, and that factors other than or in addition to bacterial infection are involved.

Studies on anabolic steroids—6. Identification of urinary metabolites of stenbolone acetate (17β-acetoxy-2-methyl-5α-androst-1-en-3-one) in human by gas chromatography/mass spectrometry

The metabolism of stenbolone acetate (17β-acetoxy-2-methyl-5α-androst-1-en-3-one), a synthetic anabolic steroid, has been investigated in man. Nine metabolites were detected in urine either as glucuronic or sulfuric acid aglycones after oral administration of a single 50 mg dose to a male volunteer. Stenbolone, the parent compound, was detected for more than 120h after administration and its cumulative excretion accounted for 6.6% of the ingested dose. Most of the stenbolone acetate metabolites were isolated from the glucuronic acid fraction, namely: stenbolone,3α-hydroxy-2-methy-5α-androst-1-en-17-one, 3α-hydroxy-2ξ-methyl-5α-androstan-17-one; 3 isomers of 3ξ,16ξ-dihydroxy-2-methyl-5α-androst-1-en-17-one; 16α and 16β-hydroxy-2-methyl-5α-androst-1-ene-3,17-dione; and 16ξ,17β-dihydroxy-2-methyl-5α-androst-1-en-3-one. Only isomeric metabolites bearing a 16α or a 16β-hydroxyl group were detected in the sulfate fraction. Interestingly, no metabolite was detected in the unconjugated steroid fraction. The steroids identities were assigned on the basis of their TMS ether, TMS enol-TMS ether, MO-TMS and d 9-TMS ether derivatives and by comparison with reference and structurally related steroids. Data indicated that stenbolone acetate was metabolized into several compounds resulting from oxidation of the 17β-hydroxyl group and/or reduction of A-ring δ-1 and/or 3-keto functions with or without hydroxylation at the C 16 position. Finally, comparison of stenbolone acetate urinary metabolites with that of methenolone acetate shows similar biotransformation pathways for both δ-1-3-keto anabolic steroids. This indicates that the position of the methyl group at the C 1 or C 2 position in these steroids has little effect on their major biotransformation routes in human, to the exception that stenbolone cannot give rise to metabolites bearing a 2-methylene group since its 2-methyl group cannot isomerize into a 2-methylene function through enolization of the 3-keto group as previously observed for methenolone.

Metabolism of metandienone in man: Identification and synthesis of conjugated excreted urinary metabolites, determination of excretion rates and gas chromatographic-mass spectrometric identification of bis-hydroxylated metabolites

After oral administration of metandienone (17α-methyl-androsta-1,4-dien-17β-ol-3-one) to male volunteers conjugated metabolites are isolated from urine via XAD-2-adsorption, enzymatic hydrolysis and preparative high-performance liquid chromatography (HPLC). Four conjugated metabolites are identified by gas chromatography-mass spectrometry (GC/MS) with electron impact (EI)-ionization after derivatization with N-methyl- N-trimethyl- silyl-trifluoroacetamide/trimethylsilyl-imidazole (MSTFA/TMS-Imi) and comparison with synthesized reference compounds: 17α-methyl-5β-androst-1-en-17β-ol-3-one (II), 17α-methyl-5β-androst-1-ene-3α,17β-diol (III), 17β-methyl-5β-androst-1-ene-3α,17α-diol (IV) and 17α-methyl-5β-androstane-3α,17β-diol (V). After administration of 40 mg of metandienone four bis-hydroxy-metabolites—6β,12-dihydroxy-metandienone (IX), 6β,16β-dihydroxy-metandienone (X), 6β,16α-dihydroxy-metandienone (XI) and 6β,16β-dihydroxy-17-epimetandienone (XII)—were detected in the unconjugated fraction. The metabolites III, IV and V are excreted in a comparable amount to the unconjugated excreted metabolites 17-epimetandienone (VI), 6β-hydroxy-metandienone (VII and 6β- hydroxy-17-epimetandienone (VIII). Whereas the unconjugated excreted metabolites show maximum excretion rates between 4 and 12 h after administration the conjugated metabolites III, IV and V are excreted with maximum rates between 12 and 34 h.

Disposition of 17β-trenbolone in humans

The urinary excretion and metabolic pattern of 17β-trenbolone, a synthetic anabolic steroid hormone used as a growth promotor for beef cattle in several countries, has been studied in a human subject. For the separation of the metabolites of 17β-trenbolone, a reversed-phase high-performance liquid chromatographic method was established. The method was tested with metabolites obtained from incubation of 17β-trenbolone with rat liver microsomes. Fifteen metabolites could be well separated in one run by using a concave acetonitrile—water—methanol gradient. After ingestion of the tracer-labelled hormone at a dose of 0.04 mg/kg body weight 54% of the administered radioactivity was found in the urine after 26 h and 63% after 72 h. Of the urinary material 54% was present as glucuronides, which contained mostly 17α-trenbolone, 17β-trenbolone and trendione. At least five other polar metabolites, presumably hydroxylated products, were found in smaller amounts, mostly in the unconjugated and sulphated fractions. Thus, the disposition of 17β-trenbolone in humans differs significantly from that in rats, which may have a bearing on the toxicological evaluation of the hormone.

Bile Acids and Biliary Carcinoma in Pancreaticobiliary Maljunction.

Pancreaticobiliary maljunction is frequently associated with biliary carcinoma, whether or not there is choledochal dilatation. In this anomalous condition, pancreatic juice regurgitates and the mixture of bile and pancreatic juice stagnates in the biliary tree. In cystic choledochal dilatation, cancers arise, mainly in the dilated bile ducts, while in patients not having cystic dilatation, tumors arise in the gallbladder. Gallbladder bile and/or bile duct bile from fifteen cases of pancreaticobiliary maljunction, including five cancer patients, was analysed biochemically and compared with control bile from 6 patients with a normal pancreaticobiliary junction. Bile levels of pancreatic enzymes were extremely high in the anomalous junction group. In the bile duct bile from patients with cystic choledochal dilatation with pancreaticobiliary maljunction, the concentrations of deoxycholic acid (DCA), lithocholic acid (LCA) and unconjugated bile acid fractions were increased regardless of the presence of cancer. Increases of these bile acid fractions, which are known to have a cancer-promoting effect, were also seen in gallbladder bile from the cancer patients without cystic dilatation.

Metabolism of stanozolol: Identification and synthesis of urinary metabolites

Urinary metabolites of stanozolol (17α-methyl- 17β -hydroxy-5α-androst-2-eno(3,2-c)-pyrazole) following oral administration were isolated by chromatography on XAD-2 and by preparative high-performance liquid chromatography (HPLC) and identified by gas chromatography-mass spectrometry (GC/MS) with electron impact (EI)-ionisation. Stanozolol is excreted as a conjugate but is metabolized to a large extent. All identified metabolites are hydroxylated, namely at C-3' of the pyrazole ring and at C-4β, C-16α and C-16β of the steroid. Less than 5% of the metabolites are found in the unconjugated urine fraction: 3'-hydroxystanozolol ( II) and 3'-hydroxy-17-epistanozolol ( III). Conjugated excreted metabolites are 3'-hydroxystanozolol ( II), stanozolol ( I), 4β-hydroxystanozolol ( IV), 16β-hydroxystanozolol ( V), 16α-hydroxystanozolol ( VI), two isomers of 3',16-dihydroxystanozolol ( VII, VIII), two isomers of4β,16-dihydroxystanozolol ( IX, X) and a 3',?-dihydroxystanozolol ( XI). 3'-Hydroxystanozolol, 4α-hydroxystanozolol, 4β-hydroxystanozolol, 16α-hydroxy-, 16α-hydroxy-17-epi- and 16β-hydroxystanozolol were synthesised to confirm the structural assignment of the main metabolites.

Identification of unconjugated bile acids in human bile.

Unconjugated bile acids in the bile of healthy and diseased humans were determined qualitatively and quantitatively by means of gas-liquid chromatography and gas-liquid chromatography-mass spectrometry, after their isolation by ion-exchange chromatography. In a healthy person and three patients with cholelithiasis, unconjugated bile acids comprised 0.1-0.4% of total biliary bile acids. The bile acid composition of the unconjugated fraction was quite different from that of the glycine- or taurine-conjugate fraction, in that it contained a relatively large proportion of unusual bile acids including C23 and C27 bile acids. In two patients with cerebrotendinous xanthomatosis, C22 and C23 bile acids were the major constituents of the biliary unconjugated bile acids, and comprised about 0.8% of total bile acids; no detectable amounts of C27 bile acids were found in their bile. The analysis of biliary unconjugated bile acids may be useful for the diagnosis of metabolic diseases concerning bile acids, particularly the accumulation or disappearance of unusual bile acids.

７種硬骨魚におけるオキソリン酸の胆汁中代謝物の比較

Biliary metabolites of a synthetic antibacterial agent, oxolinic acid were compared in seven species of teleosts after its oral administration. The teleosts tested were rainbow trout Salmo garidneri, Japanese eel Anguilla japonica, tilapia Tilapia mossambica, red sea bream Pagrus major, Japanese mackerel Trachurus japonicus, yellowtail Seriola quinqueradiata, and Japanese flounder Paralichthys olivaceus. Biliary metabolites were separated into unconjugated (FRACTION A) and conjugated fractions (FRACTION B) by extracting the biles with chloroform, the metabolites of each fraction were identified by thin-layer chromatography with the authentic samples. In the unconjugated fractions, only intact oxolinic acid was detected in all the species. However, in the conjugated fractions, there were some differences between the results for the freshwater fish (rainbow trout, Japanese eel, tilapia) and these for the seawater fish (red sea bream, Japanese mackerel, yellowtail, Japanese flounder). Three glucuronides of oxolinic acid, 7-hydroxy-6-methoxy derivative, and 6-hydroxy-7-methoxy derivative were detected in the bile of the former and only two glucuronides of 7-hydroxy-6-methoxy derivative and 6-hydroxy-7-methoxy derivative were detected in that of the latter.

Bartter's Syndrome Associated with Indirect Hyperbilirubinemia: A Possible Clinical Variety.

This report deals with three cases of Bartter's syndrome whose symptomatology was associated with indirect hyperbilirubinemia. The bilirubin disorder was suggestive of Gilbert's syndrome, with no pathological findings being detected as far as the liver function was concerned. Furthermore, the unconjugated fraction of bilirubin increased after fasting. The therapy with indomethacin exerted beneficial effects on both electrolytes and bilirubin disorders, and the patients recovered a good healthy state. These findings suggest the possibility that Bartter's syndrome may coexist in a variety associated with indirect hyperbilirubinemia.

Studies on Steroids CCXXXXIV. Separation and Characterization of C-25 Epimers of Unconjugated and Conjugated Trihydroxycholestanoic Acids in Urine from a Patient with Zell Weger Syndrome by High-Performance Liquid Chromatography

Abstract The separation and characterization of C-25 epimers of unconjugated and glycine- and taurine-conjugated 3α, 7α, 12α - trihydroxy-5β-cholestanoic acid (THCA) in biological fluids by high-performance liquid chromatography (HPLC) are described. The 5β-cholestanoic acid fraction was obtained from a urine specimen from a patient with Zellweger syndrome by passing it through a Sep-pak C18 cartridge. Bile acids were derivatized quantitatively into the fluorescent compounds through the hydroxyl group at C-3 by treatment with 1-anthroyl nitrile. The derivatives were separated into the unconjugated, glycine- and taurine-conjugated fractions by ion-exchange chromatography on alipophilicgel, piperidinohydroxypropyl Sephadex LH-20. Sub-sequent resolution of each fractionin to (25s)- and (25R)-THCA was attained by HPLC on a Cosmosil 5C column. The C-25 epimers of unconjugated and conjugated Tk%A were unequivocally identified on the b asis of the irbehaviors in HPLC using mobile phases of different pHs. The ratios of the unconjugated, glycine- and taurine-conjugated (25RbTHCA to the corresponding (25S)-epimers were 16:1, 5:4 and 3:2, respectively .