Jiro Yanagisawa

Microanalysis of bile acid in human liver tissue by selected ion monitoring

A method of microquantitative determination of bile acid in 5–30 mg of human liver tissue was developed. Bile acids were converted to their ethyl ester dimethylethylsilyl ether derivatives and analyzed by capillary gas chromatography-selected ion monitoring, using [2H4]-lithocholic (LCA), [2H5]deoxycholic (DCA), [2H4]chenodeoxycholic (CDCA), [2H4]-ursodeoxycholic (UDCA), and [2H3]cholic (CA) acids as internal standards. Precision and reproducibility of the present method were tested using surgically obtained liver specimens. The results were statistically analyzed according to one-way layout and the orthogonal polynomial equation. Bile acids except LCA were determined with 2.3 to 11.4% of the coefficient of variation. Recoveries of conjugated bile acids ranged from 72.2 to 96.0% with a mean of 84.3%. The amount of bile acids present in histologically normal liver specimens (n = 10) was found to be 29.56 ± 8.62 μg/g liver. The relative compositions (%) of CDCA and CA were 38.8 ± 8.9 and 41.1 ± 11.0, respectively.

Bile acid metabolism in cirrhotic liver tissue ― altered synthesis and impaired hepatic secretion

Bile acid analysis of mild and severe cirrhotic liver showed that with the advancement of cirrhosis the concentration of chenodeoxycholic acid in liver tissue becomes higher, resulting in the lower ratio of cholic to chenodeoxycholic acid probably due to the progressive alteration of cholic and chenodeoxycholic acid synthesis with the advancement of liver cirrhosis. Bile acid analysis of paired liver and bile of severe cirrhosis showed that the ratio of cholic to chenodeoxycholic acid in liver was lower than that in bile or even with that in bile. This can be explained by postulating the impaired hepatic secretion of bile acids, especially chenodeoxycholic acid. The impaired secretion together with the relatively well preserved chenodeoxycholic acid synthesis results in the accumulation of chenodeoxycholic acid in liver tissue with cirrhosis.

Composition of intrahepatic calculi

Gallstones in intrahepatic (N=42 and extrahepatic (N=22) bile ducts and gallbladder (N=23were subjected to chemical analysis modified to suit the analysis of brown pigment stones with the aim of determining if stone location at surgery influenced stone composition. Dimethylsulfoxide—acetone—1 N HCl (90:9:1, v/v/v) was used to dissolve gallstone specimens. Intrahepatic calculi were divided into two groups, ie, nine cholesterol stones and 33 brown pigment stones. Cholesterol stones in the intrahepatic bile ducts had a similar composition to those in the gallbladder and extrahepatic bile ducts, suggesting a similar pathogenesis wherever formed throughout the biliary tract. Intrahepatic brown pigment stones contained significantly less bilirubin (P <0.001) and more cholesterol (P <0.05) by chisquare analysis than brown pigment stones found in the extrahepatic bile ducts, suggesting that the site of formation affects stone composition and modifies stone pathogenesis.

Absorption of Bile Acids in Dog as Determined by Portal Blood Sampling: Evidence for Colonic Absorption of Bile Acid Conjugates

The intestinal phase of enterohepatic circulation, such as site and state of bile acid absorption, along the length of the intestinal tract has been speculated but not directly quantitated. In order to gain insight into the actual state of intestinal absorption of bile acid, the bile acid composition of portal blood from various segments of the intestinal tract was studied in dogs after loading endogenous bile acid by injection of caerulein. Total and unconjugated bile acids were determined with and without enzymatic hydrolysis, respectively. The amount of conjugated bile acids was calculated by subtracting unconjugated from total bile acids. Quantitation of cholic, chenodeoxycholic, deoxycholic and lithocholic acids and their conjugates was carried out by gas chromatography/mass spectrometry/selected ion monitoring with deuterated bile acids as internal standards. The major site of absorption of taurine-conjugated bile acid, a major conjugate form in the dog, was the distal small intestine. In addition, a considerable amount of cholic acid was found to be absorbed from the distal large intestine, the majority of which was still in the conjugated form. The pronounced absorption of the unconjugated secondary bile acid from the large intestine suggests the very active formation of the secondary bile acid in situ.

Effects of bile duct obstruction and decompression on hepatic microsomal mixed function oxidase system in rats

The effects of biliary obstruction and drainage on the hepatic microsomal mixed function oxidase system were studied in rats. Bile duct obstruction produced a significant reduction in the hepatic cytochrome P-450 dependent mixed function oxidase system. After release of the bile duct obstruction, the reduction in microsomal enzymes was practically reversible; however, the process of recovery was slow and differed with the microsomal enzymes in question. Increases in cytochrome b5 content and NADH-cytochrome b5 reductase activity were slower than increases in cytochrome P-450 content and NADPH-cytochrome c reductase activity. Aniline hydroxylase activity increased more rapidly and corresponded to cytochrome P-450 contents more so than did the aminopyrine demethylase activity. After the release of bile duct obstruction, however, the bile acids which had accumulated in the liver during cholestasis were reduced rapidly, to a normal range. These results suggests that there is a discrepancy between reductions in hepatic bile acids and those in the hepatic microsomal mixed function oxidase system after biliary decompression.

Biliary bile acids in the gall-bladder and the common bile duct of patients with anomalous pancreaticobiliary ductal junction

The high incidence of biliary tract carcinoma in patients with anomalous pancreaticobiliary ductal junction (APBDJ) with or without choledochal cyst (CC) has been well documented. Twenty‐two patients with APBDJ were divided into three groups: Group A, four patients not associated with CC and biliary tract carcinoma; Group B, 13 patients with CC but without biliary tract carcinoma; and Group C, five patients with biliary tract carcinoma (four with and one without CC). Profiles of bile acids in the gall‐bladder and/or common bile duct were analysed in these patients and compared with those in the control patients with cholecystlithiasis to examine the hypothesis that the levels of deoxycholic acid (DCA) and lithocholic acid (LCA) are elevated in patients with APBDJ because these secondary bile acids are mutagenic. Bile acids were quantified by gas—liquid chromatography. Total bile acid concentration in the gall‐bladder bile was significantly lower in any group with APBDJ than that of controls. In the gall‐bladder, increased proportion of chenodeoxycholic acid (CDCA) in Groups A and B, decreased proportion of DCA in Group B and increased proportion of cholic acid (CA) in Group C were found in bile. In the bile duct, total bile acid concentration and proportion of DCA were significantly low in bile from Group C and decreased proportion of DCA and increased proportion of CDCA were found in bile from Group B. In both the gall‐bladder and hepatic bile, proportion of LCA was not significantly different between any intergroups. Thus no increase of DCA and LCA was found in either the bile from the gall‐bladder and the bile duct of APBDJ patients. It is concluded that bile acid plays little role, if any, in the pathogenesis of biliary tract carcinoma in patients with APBDJ.

Bile acid composition in primary hepatocellular carcinoma

Bile acid composition in human primary hepatocellular carcinoma (HCC) tissues from eight non‐hyperbilirubinaemic patients was compared with that in the neighbouring liver tissues. Quantitation of bile acids was carried out using selected ion monitoring. Significant amounts (>14 pmol/mg liver) of bile acids were found to be present in HCC tissues of all patients. In four patients, the concentration of chenodeoxycholic acid was higher in HCC than the corresponding neighbouring liver tissues, whereas those of other bile acids were less. The ratio of cholic to chenodeoxycholic acid was lower in HCC than in adjacent liver tissues in seven out of eight patients. This may indicate predominant synthesis of chenodeoxycholic acid in HCC tissues. The large inter‐individual variation in bile acid concentration and composition of HCC tissues may result from the grade of anaplasia of HCC cells. 3β‐Hydroxy‐5‐cholenoic acid, thought to be excreted in increased amounts in the urine of HCC patients, was shown to be a minor constituent of HCC tissues and of the neighbouring liver tissues in most patients, indicating that the C26‐hydroxylation pathway plays a minor role. Unusual bile acids, such as 23‐nor‐3α,7α,12α‐trihydroxy‐5β‐cholanoic, 3β,7β‐dihydroxy‐5β‐cholanoic, 7‐oxo‐3α,12α‐dihydroxy‐5β‐cholanoic and 12‐oxo‐3α,7α‐dihydroxy‐5β‐cholanoic acids, were found in trace amounts in HCC tissues but their significance is not clear at present.

Lithocholate in liver tissue with obstructive jaundice

SummaryThe existence of tissue-bound lithocholate in liver tissue was first suggested by Nair et al. (Lipids 1988;12:922–929). Since lithocholate is well known to be cholestatic, we investigated whether tissue-bound lithocholate, if present, can be correlated to liver dysfunction in obstructive jaundice. Tissue-bound lithocholate in liver tissues of 7 patients with obstructive jaundice was analyzed using gas chromatography-mass spectrometry-selected ion monitoring. We found no accumulation of lithocholate, including tissue-bound form, indicating that the role of lithocholate in inducing hapatic dysfunction in obstructive jaundice is minimal, if any.

Proceedings of the 23rd Autumn Meeting from October 14–16, 1981-Yonago City, Tottori, Japan

The methodology currently used in the field of physiology of intestinal absorption was reviewed and important progresses in our knowledge of mechanisms of intestinal absorption brought about by introduction of new methods were also summarized. The physiological methods currently employed can cover a broad range of investigations from those at an organ level, e.g. perfusion of intestinal segments, to those at a molecular level, e.g. transport studies in reconstituted systems with purified membrane proteins. By these methods, Na +-dependent mechanism of uphill uptake of various organic solutes and electrolytes across the brush border membrane have been largely clarified and active transport of various solutes is now explained on the basis of the concept of the secondary active transport. The mechanism of exit of solutes from the enterocytes have also been investigated in isolated cell suspensions and purified basolateral membrane vesicles, and some carriers responsible for the exit have been characterized. The charge transfer associated with organic solute transport has been studied by electrophysiological techniques. These studies indicate that organic solutes induce a Na + pathway and resultant Na + flow across the membrane causes a coupled flow of the cosubstrate. A relatively new problem is the transport of small peptides in intact form. Its physiological significance, comparative and developmental aspects are now under investigation in several laboratories. Vira l hepatitismRecent advances of its fundamental research