Influence of cholesterol feeding on bile acid metabolism in young and aged germ-free rats.

The effects of cholesterol feeding on serum and liver cholesterol levels, fecal and biliary bile acid levels, bile acid pool size and bile acid composition were examined in 2-, 12- and 24-month-old male germ-free rats. The major bile acids in these animals were cholic and beta-muricholic acids. Cholesterol feeding increased synthesis of bile acids by 3- to 4-fold, especially that of chenodeoxycholic acid (mainly beta-muricholic acid in the rat), decreasing the cholic acid/chenodeoxycholic acid (CA/CDCA) ratio in all rats regardless of age, even though the CA/CDCA ratio increased as a linear function of age in both diet groups. Cholesterol feeding increased the serum cholesterol level markedly in aged rats. This hypercholesterolemia may be produced by the increase in CA/CDCA ratio in aged rats.

Purification of a novel serpin-like protein from bovine brain.

We purified a novel serine proteinase inhibitor (serpin)-like protein from the bovine brain and named it B-43 from its molecular mass, 43 kDa. A cleaved peptide from B-43 was copurified with the native B-43. Partial amino acid sequencing of the purified B-43 showed that this protein was homologous to glia-derived nexin/protease nexin-1 (GDN/PN-1), plasminogen activator inhibitor 2, leukocyte elastase inhibitor (LEI) and placental thrombin inhibitor (PTI) among the serpins. Although B-43 had a similar amino acid composition to these serpins, the biochemical features of B-43 were different from them. B-43 did not form sodium dodecyl sulfate (SDS)-resistant serpin-proteinase complexes with thrombin, urokinase, pancreatic elastase and plasmin, suggesting that these proteinases were not the targets of B-43. In contrast to GDN/PN-1, B-43 did not have an affinity for heparin. B-43, having different biochemical properties from GDN/PN-1, appears to be an additional serpin expressed in the brain.

Effect of minute amounts of [D-Ala2,MePhe4,Gly(ol)5]enkephalin injected into the tuberomammillary nucleus of rats on histamine release from the cerebral cortex.

[D-Ala2,MePhe4,Gly(ol)5]enkephalin (DAGO) (10 ng/0.5 microliters saline solution) injected into the tuberomammillary nucleus (TM) of rats in minute amounts decreased the amount of histamine released to approximately 50% of the basal value on measurements taken 20-40 min after administration. This effect of DAGO was inhibited by the simultaneous microinjection of naloxone (320 ng). These results may be explained in two ways. The first is that the stimulation of mu-receptors results in the inhibition of histaminergic cell bodies. The second is that the somatodendritic release of histamine was increased by the stimulation of mu-receptors and as a result of increased histamine concentration in TM, many histaminergic neurons may be inhibited through the stimulation of H3-receptors. Further studies are necessary regarding the influence of mu-agonists on various cellular sites of histaminergic neurons.

Ocular absorption, distribution, and systemic absorption of a novel antiglaucoma medication, prostaglandin derivative, in male white rabbits.

A prostaglandin derivative, (5Z,9 alpha,11 alpha,13E)-9,11-dihydroxyprosta- 5,13-dienoic acid sodium salt (S-1033), that lowers intraocular pressure with little adverse effect, may have clinical value in the treatment of glaucoma. After [14C]S-1033 (0.2% solution) was instilled into the eye of a white rabbit, radioactivity and S-1033 appeared in systemic plasma so rapidly (tmax, 5 min) and S-1033 was eliminated very rapidly with half-lives of 2.8 and 11.0 min at alpha- and beta-phases, respectively. The metabolite, M-1, [1R-[1 alpha,2 beta-(1E),3 alpha,5 alpha]]-3,5-dihydroxy-2-(1- octenyl)-cyclopentanepropanoic acid (tetranor-S-1033), appeared in plasma very rapidly (tmax, 5 min), suggesting that a fast metabolism was a major factor in the rapid elimination of S-1033 from plasma. The values for the ratios of the area under the curve of ocular instillation to intravenous administration for radioactivity and S-1033 were 1.01 and 0.52, respectively, indicating that more than half of the S-1033 instilled was transported into the systemic circulation. To clarify the contributing pathway of the massive and rapid systemic absorption of S-1033 after topical dosing, plasma levels of S-1033 were investigated after instillation to rabbits in which the nasolacrimal ducts were occluded. Plasma concentrations of S-1033 were slightly higher than those in intact rabbits, suggesting that conjunctiva would be as important as nasal mucosae for the systemic absorption under the physiological condition. As for the intraocular distribution, the highest levels of radioactivity were found in the cornea, conjunctiva, and anterior sclera.(ABSTRACT TRUNCATED AT 250 WORDS)

Increase in the extracellular histamine concentration in the rat striatum by mu-opioid receptor activation.

The effects of morphine and selective ligands for mu-, kappa-, and delta-opioid receptors on the extracellular histamine (HA) concentration in the striatum of freely moving rats were examined by in vivo microdialysis. On the day after implantation of the dialysis probe, the HA output per 30-min period was measured using HPLC-fluorometry. Morphine (3.8 mg/kg, s.c.) significantly increased the HA output by approximately 200% 1-3 h after treatment. This effect was completely antagonized by naltrexone (1.6 mg/kg, s.c.). The HA output decreased to a level below 10% of the basal value by 4 h after treatment with (S)-alpha-fluoromethyl-histidine (77 mg/kg, s.c.). In such animals, morphine (3.8 mg/kg, s.c.) had no influence on the HA output. [D-Ala2,MePhe4,Gly(ol)5]Enkephalin (DAGO; 0.2 microgram, i.c.v.), a selective mu-agonist, significantly increased the HA output by approximately 150% 0.5-1.5 h after treatment, and this effect was also completely blocked by naltrexone. A selective kappa-agonist, U-50,488 (3.8 and 7.6 mg/kg, s.c.), and a selective delta-agonist, [D-Pen2,D-Pen5]enkephalin (0.5 and 2 micrograms, i.c.v.), had no effect on the HA output. These findings suggest that the stimulation of mu-opioid receptors by morphine and DAGO increases the extracellular HA concentration by accelerating HA release from nerve endings.

Microdialysis study of the effects of sedative drugs on extracellular histamine in the striatum of freely moving rats.

The effects of some sedative drugs on the extracellular concentration of histamine (HA) in the striatum of conscious freely moving rats were examined by in vivo microdialysis coupled with high-performance liquid chromatography-fluorometry. The HA output did not significantly change until 4 hr after intraperitoneal saline injection. Pentobarbital (46 mg/kg i.p.) significantly decreased the HA output by 76% at 0.5 to 1 hr after treatment. Muscimol (5 mg/kg i.p.) and diazepam (20 mg/kg i.p.) also significantly decreased the HA output at 0.5 to 1.5 hr after treatment. delta 9-Tetrahydrocannabinol (5 mg/kg i.p.) induced biphasic changes in the HA output, i.e., a significant increase and a significant decrease were observed at 0 to 0.5 hr and 1.5 to 2.5 hr after treatment, respectively. Reserpine (5 mg/kg i.p.) significantly increased the HA output by 47% to 58% at 0 to 2 hr after treatment. The HA output decreased to a level below 10% of the basal value by 4 hr after treatment with (S)-alpha-fluoromethylhistidine (77 mg/kg i.p.). Reserpine slightly restored the HA output (to about 25% of the original basal value). These results, taken together with our previous results, suggest the following: 1) pentobarbital, muscimol and diazepam inhibit HA release; 2) tetrahydrocannabinol initially increases HA release for a short period but markedly decreases the release thereafter; and 3) reserpine increases the extracellular HA concentration probably as a result of inhibition of its elimination.

A radioimmunoassay of bile acids.

A radioimmunoassay for beta-muricholic acid was developed using an antiserum which was prepared by injecting beta-muricholic acid conjugated with bovine serum albumin into rabbits. The antiserum reacted with glyco-beta-muricholic, tauro-beta-muricholic and beta-muricholic acids, but not with other bile acids. The radioimmunoassay showed good reproducibility with inter- and intra-assay coefficients of variations of 6% to 15%. When the validity of the method was examined by comparing it with a gas-liquid chromatography method, a linear correlation was obtained.

Age-related changes of bile acid metabolism in rats.

Cholesterol and bile acid leves were examined in young (8 weeks), middle-aged (12 months) and old (24 months) germ-free male rats, and young (8 weeks) and middle-aged (12 months) conventional male rats. The plasma cholesterol levels were higher in the aged rats, being more marked in the conventional rats. The liver cholesterol levels also increased with age and the increases were almost identical for both groups. No age-related changes were found in the biliary bile acid secretion, the pool size and distribution of bile acids in the bile, small intestine and large intestine, nor in the turnover frequency of bile acids, but the pool size in the young and middle-aged germ-free rats was much larger than that in the conventional rats. The turnover frequency was less in the germ-free rats. The bile acid synthesis presumed from the fecal bile acid excretion decreased in the aged germ-free rats but not in the conventional rats. A most remarkable age-related change was found in the bile acid composition; cholic acid increased and beta-muricholic acid derived from chenodeoxycholic acid in the rat decreased by aging, resulting in an increase of the CA/CDCA ratio (bile acids belonging to the cholic acid group/bile acids to the chenodeoxycholic acid group) in the bile, feces and pool. These results suggest that cholic acid synthesis increases while chenodeoxycholic acid synthesis is impaired by aging in rats.

Deconjugation of bile acids by human intestinal bacteria implanted in germ-free rats.

Fecal bile acids in germ-free rats were analyzed after inoculation with Bacteroides vulgatus, Bifidobacterium longum, Escherichia coli or Clostridium ramosum. B. vulgatus preferentially deconjugated tauro-beta-muricholic acid and B. longum taurocholic acid. C. ramosum deconjugated both bile acids, but E. coli deconjugated neither. 7 alpha-Dehydroxylation of bile acids was negligible even after 18 days of inoculation, but a small amount of 7-oxo-bile acids, less than 5%, was formed. Fecal excretion of bile acids increased after inoculation with B. vulgatus, B. longum and C. ramosum, but not with E. coli.

Studies on steroids. CCXXVII. Separation and determination of bile acid 7- and 12-sulphates in urine by high-performance liquid chromatography with fluorescence labelling.

A method for the characterization and determination of bile acid 7- and 12-sulphates in urine without prior deconjugation is described. The sulphate fraction was obtained from an urine specimen by passing it through a Sep-Pak C18 cartridge, followed by group separation by ion-exchange chromatography on a lipophilic gel, piperidinohydroxypropyl Sephadex LH-20. Bile acid 7- and 12-sulphates were derivatized quantitatively into the fluorescent compounds through the hydroxyl group at C-3 by treatment with 1-anthroyl nitrile in the presence of quinuclidine in acetonitrile. Subsequent resolution into individual 7- and 12-sulphates was attained by high-performance liquid chromatography (HPLC) on a Cosmosil 5C18 column using 0.3% potassium phosphate buffer (pH 4.0)-methanol (1:3) as a mobile phase. The 3-(1-anthroyl) derivatives of 7- and 12-sulphates were monitored by fluorescence detection. Taurochenodeoxycholate 7-sulphate in human urine was unequivocally identified on the basis of behaviour in HPLC using mobile phases of different pH values. The present method has proved to be applicable to the characterization and quantification of bile acid 7- and 12-sulphates in human urine.

Studies on steroids. CCXVI. Separation of bile acid 3-glucuronides by high-performance liquid chromatography.

The separation of 3-glucuronides of cholate, chenodeoxycholate, deoxycholate, ursodeoxycholate and lithocholate, and their glyco- and tauro-conjugates, has been carried out by high-performance liquid chromatography on a reversed-phase column. The chromatographic behaviour of bile acid 3-glucuronides was dependent on the type of conjugation. An effect of the pH of the mobile phase on the capacity ratio was observed at higher pH for chenodeoxycholate 3-glucuronide, probably owing to steric interaction of the 7 alpha-hydroxy group with the carboxy group in the glucuronyl moiety. Conversion of the alpha-hydroxy function on the steroid nucleus into an oxo group resulted in a 50% decrease in the capacity ratio. Bile acid 3-glucuronides were efficiently separated on Shodex ODS Pak F-411 using three kinds of ammonium phosphate buffer-acetonitrile systems.

Studies on steroids. CLXXXVII. Determination of serum bile acids by high-performance liquid chromatography with fluorescence labeling.

A method for the simultaneous determination of bile acids in serum by high-performance liquid chromatography (HPLC) with fluorescence labeling is described. The bile acid fraction was obtained from a serum specimen by passing it through a BondElut cartridge. Bile acids were derivatized quantitatively into the fluorescent compounds through the hydroxyl group at C-3 by treatment with 1-anthroyl nitrile in the presence of quinuclidine in acetonitrile. These derivatives were separated into the free, glycine- and taurine-conjugate fractions by ion-exchange chromatography on a lipophilic gel, piperidinohydroxypropyl Sephadex LH-20. Subsequent resolution of each fraction into cholate, ursodeoxycholate, chenodeoxycholate, deoxycholate and lithocholate was attained by HPLC on a Cosmosil 5C18 column using 0.3% potassium phosphate buffer (pH 6.0)--methanol (1:5) and 0.1% potassium phosphate buffer (pH 6.0)--methanol (1:8) as mobile phases. The anthroyl bile acids were monitored by fluorescence detection (excitation wavelength 370 nm; emission wavelength 470 nm), the limit of detection being 20 fmol. The proposed method proved to be applicable to the quantitation of bile acids in serum with satisfactory reliability and sensitivity.