Haruto Takase

Effect of sex hormones on hypoprothrombinemia induced by N-Methyltetrazolethiol in rats

N-Methyltetrazolethiol (NMTT) increased prothrombin time (PT) and decreased plasma factor VII and prothrombin levels only in vitamin K-deficient male rats. In female rats identical treatment with NMTT did not produce hypoprothrombinemia. Conventional and germ-free rats displayed no significant difference in the manifestation of hypoprothrombinemia, but the increase of PT in NMTT-treated vitamin K-deficient rats was greater in the germ-free males. Estradiol administration or castration of male rats retarded manifestation of vitamin K deficient syndromes such as increases of PT and activated partial thromboplastin time (APTT), decreases of plasma factor VII and prothrombin levels, and increases of plasma and liver descarboxyprothrombin (PIVKA) levels, and testosterone injection to the castrated rats restored these changes. In female rats testosterone treatment or castration enhanced the manifestation of hypoprothrombinemia and estradiol treatment to the castrated females retarded it. Gamma-glutamyl-carboxylase activity was increased by vitamin K-deficiency but not inhibited by testosterone or NMTT. These data suggest that estrogen protects the rat against manifestation of hypoprothrombinemia even with NMTT treatment, while androgen enhances vitamin K deficiency, and supplementation of vitamin K prevents its deficiency in NMTT-treated rats.

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.

Transformation of bile acids and sterols by clostridia (fusiform bacteria) in Wistar rats

The effects on bile acid and sterol transformation of clostridia (fusiform bacteria), the dominant intestinal bacteria in rodents (ca. 1010 counts per g wet feces) were examined in Wistar rats. After inoculation of clostridia into germ-free rats and into rats previously inoculated solely with Escherichia coli, most of the endogenous bile acids were deconjugated, and cholic acid and chenodeoxycholic acid were 7α-dehydroxylated to deoxycholic acid and lithocholic acid, respectively. Tauro-β-muricholic acid, another major bile acid in rats, was deconjugated, but only part of it (ca. 30%) was transformed into hyodeoxycholic acid. Cholesterol and sitosterol were also reduced to coprostanol and sitostanol, respectively. Escherichia coli transformed neither bile acids nor sterols. These data suggest that clostridia play an imporant role in the formation of secondary bile acids and coprostanol in rats.

Differential effects of ursodeoxycholic acid and ursocholic acid on the formation of biliary cholesterol crystals in mice

The preventive effect of 3α,7β,12α-trihydroxy-5β-cholanoic acid (ursocholic acid) and ursodeoxycholic acid on the formation of biliary cholesterol crystals was studied in mice. Cholesterol crystals developed with 80% incidence after feeding for five weeks a lithogenic diet containing 0.5% cholesterol and 0.25% sodium cholate. When 0.25% ursocholic acid or ursodeoxycholic acid was added to the lithogenic diet, the incidence as well as the grade (severity) of the gallstones were reduced. Plasma and liver cholesterol levels were decreased by ursodeoxycholic acid but not by ursocholic acid. Gallbladder cholesterol and phospholipid levels were decreased by both bile acids. The biliary bile acid level was decreased by ursocholic acid but not by ursodeoxycholic acid. After feeding ursocholic acid, its level in the bile was about 25% and the levels of cholic acid and β-muricholic acid decreased. Fecal sterol excretion was not changed by ursocholic acid, but was increased by ursodeoxycholic acid. After feeding ursocholic acid, fecal excretion of deoxycholic acid, cholic acid, and ursocholic acid increased. No differences were found between mice, with or without gallstones, in plasma and liver cholesterol levels, biliary phospholipid and bile acid levels, fecal sterol and bile acid levels, and biliary and fecal bile acid composition. The results suggest that the lower incidence of crystal formation after treatment with ursocholic acid is probably by a different mechanism than with ursodeoxycholic acid. In the mouse model, ursodeoxycholic acid exerts its effect at least partially, by decreasing cholesterol absorption. Ursocholic acid is well absorbed and excreted into bile and transformed into deoxycholic acid by the intestinal microflora in mice.

Effects of triton WR 1339 and orotic acid on lipid metabolism in rats

In order to investigate the effect of hepatic cholesterol flux on biliary bile acids, Triton WR 1339 and orotic acid were administered to rats, and the biliary cholesterol, phospholipids and bile acids were analyzed together with serum lipoproteins and hepatic lipids. Triton, which raised serum very low density lipoprotein and lipid levels and decreased serum high density lipoprotein liver lipid levels, increase the biliary cholic acid group/chenodeoxycholic acid group ratio (CA/CDCA) in the bile without affecting the total amount of bile acids and the other biliary lipids. Orotic acid, which decreased serum lipid and lipoprotein concentrations and increased liver lipid levels, increased the biliary excretion of cholesterol and phospholipids, but produced no significant change in the total amount of bile acids and in the CA/CDCA ratio in bile.

Age-related changes in cholesterol and bile-acid metabolism in spontaneously hypertensive rats

Age-related changes in serum and liver cholesterol, phospholipid and triglyceride levels, serum lipoproteins, biliary secretion of cholesterol, phospholipids and bile acids, fecal excretion of sterols and bile acids, and the pool size of bile acids were examined in spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKR). SHR showed distinct age- and sex-related changes when young and marked aged-rat hypercholesterolemia after 1 yr of age. (1) Cholesterol shifted from blood to the liver between 10 and 20 wk only in male SHR and not at all in WKR. (2) Serum lipoprotein percentages changes; alpha-lipoprotein decreased, pre-beta-lipoprotein increased, but beta-lipoprotein did not change. These changes appeared only in male SHR and after 13 to 15 wk of age. (3) Liver enlargement in SHR, although not detected at 5 to 6 wk, progressed more rapidly than in WKR, giving values almost double those in WKR after 13 to 15 wk. Liver enlargement in female SHR was much less than in the male. (4) Bile flow, biliary secretion, and the pool size of bile acids increased. However, when expressed on the basis of liver weight, these values were similar to those in WKR, suggesting that the increases were caused by the hepatomegaly. (5) Differences appeared in the bile acid composition. A large amount of beta-muricholic acid was present in SHR of both sexes and the cholic acid percentage was low in male SHR. (6) Changes were observed in fecal bile acid excretion. Since the daily amounts in male SHR were similar to those in WKR, the hepatic synthesis activity (mg per day per 10 g liver) in male SHR was almost half that in WKR at all ages.

Bile acid metabolism in analbuminemic rats

The bile acid concentrations in the serum, liver, bile, intestines, and feces of 3- and 19-mon-old male and female Nagase analbuminemic (NA) rats were compared with those in Sprague-Dawley (SD) rats. There was no significant difference in the bile acid levels between NA and SD rats. However, increased biosynthesis and pool size of cholic acid (CD) derivatives and decreased levels of chenodeoxycholic acid (CDCA) derivatives (increased CA/CDCA ratio) were detected in male NA rats as compared to SD rats. The CA/CDCA ratio in female NA rats was not different from that in their SD rats in the biliary bile flow, bile acid levels in the small and large intestines, fecal bile acid excretion, bile acid concentration in the portal and systemic circulation, and in the pool size of bile acids. The blood lipid concentrations were significantly higher in the NA rats than in the SD rats. The hepatic levels of lipids were not significantly different between the two rat strains. In conclusion, this study showed that metabolism of bile acids in NA rats is not significantly affected, and that the hypercholesterolemia observed in these strains is not related to abnormalities of bile acid metabolism.

BILE ACID METABOLISM IN YOUNG-OLD PARABIOTIC RATS

Serum cholesterol, triglyceride and phospholipid levels, liver cholesterol concentration, bile flow, biliary cholesterol, phospholipid and bile acid secretion rates, fecal sterol and bile acid levels and their bile acid compositions were examined in young-old parabiotic rats and compared with those in young and old control rats and young-young parabiotic rats. Bile acid composition was expressed in terms of the cholic acid group/chenodeoxycholic acid group (CA/CDCA) ratio. Body weight (BW) gain decreased after parabiosis especially in old rats, but the liver weight (g/100 g BW), diet-intake, feces dry weight, liver cholesterol concentration and fecal sterol level were almost the same in all the groups. The biliary bile acid secretion rate was higher and the fecal bile acid level was lower in old rats than those in young rats but both the levels became comparable with those in young rats after parabiosis of old rats with young rats. Young rats, however, showed no changes in these levels after parabiosis. The serum cholesterol level and the biliary and fecal CA/CDCA ratios in old rats were higher than those in young rats but decreased after parabiosis with young rats, although they were still higher than those in young rats. The serum cholesterol level in young rats increased after parabiosis with old rats, but not after parabiosis with young rats, and the fecal bile acid level and the CA/CDCA ratio were not changed in either case. It is concluded from these findings that the serum cholesterol level and the CA/CDCA ratio increased with age and that these increases were prevented after parabiosis with young rats, while young rats, although their serum cholesterol level was increased, showed no increase in the CA/CDCA ratio after parabiosis with old rats.

Studies on solid phase extraction of bile acids from biological matrix

To quantify bile acids in biological samples, a solid phase extraction method was examined. This method is known as simpler procedures with less contamination compared with solvent extraction methods. Rat bile, feces and urine were used as biological samples. Since Bond Elut C18 and C8 were proved to be more suitable than the other phases (CH and SAX) so far examined using standard bile acids, Bond Elut C18 was used for biological samples. Quantification of biological sample was carried out by gas chromatography after the extracted sample was derivatized to methyl ester by treatment with trimethylsilyldiazomethane then to trifluoroacetyl ester by trifluoroacetic anhydride. On the gas chromatography, two columns (Rtx-50 and Rtx-200) were connected to the injector with Y-tube for elimination of interference. Except for a few bile acids, high recovery with less biological contamination was obtained by this solid phase extraction method.

Trifluoroacetylation of muricholic acids and hyocholic acids

Abstract Trifluoroacetylation of α-muricholic acid and α- and β-hyocholic acids is incomplete under routine conditions (at room temperature with trifluoroacetic anhydride for 30 min), whereas β-muricholic acid reacts completely. Complete trifluoroacetylation of these bile acids was achieved by reaction at room temperature for 16–24 h. Trifluoroacetylation of α-muricholic acid at room temperature for 48 h or at a higher temperature (50°C) for 0.5–1.0 h led to another unknown peak.