Sumihiko Okuyama

Determination of serum Δ5-3β-hydroxysteroid sulphates by combined high-performance liquid chromatography and immobilized 3β,17β-hydroxysteroid dehydrogenase in column form

Abstract We studied the use of an immobilized enzyme, covalently bound to aminopropyl-CPG, in the analysis of individual Δ 5 -3β-hydroxysteroid sulphates. A microcolumn with immobilized 3β,17β-hydroxysteroid dehydrogenase was prepared and used together with high-performance liquid chromatogaphy (HPLC). The reduced nicotinamide-adenine dinucleotide produced from Δ 5 -3β-hydroxysteroids by this enzyme was fluorimetrically determined. The immobilized enzyme was sufficiently stable for at least one month or for 180 tests when used repeatedly. A clinical trial demonstrated that this HPLC—immobilized enzyme method is superior to the soluble enzyme method, giving reliable and reproducible results at a low cost.

High performance liquid-chromatographic analysis of individual bile acids: free, glycine- and taurine-conjugated bile acids.

SummaryHigh performance liquid-chromatographic analyses of individual bile acids (cholic acid, chenodeoxycholic acid, deoxycholic acid, and lithocholic acid), free and conjugated with glycine and taurine, are described. The analyses of free and glycine-conjugated bile acids are based on esterification of carboxyl group of bile acids with O-(p-nitrobenzyl)-N, N′-diisopropylisourea (PNBDI). Moreover, ursodeoxycholic acid, hyocholic acid, hyodeoxycholic acid and 3ß-hydroxy-5-cholenoic acid also are able to analyse by this method.These bile acids in biological smaple were extracted by an Amberlite XAD-2 column, and separated by DEAE-Sepharose CL-6B into free, glycine- and taurine-conjugated bile acids. After the separation, free and glycine-conjugated bile acids were esterified with PNBDI directly. Because taurine-conjugated bile acids are unable to be esterified with PNBDI, these bile acids were hydrolyzed by NaOH in order to make free bile acids, and then they were esterified. Because the p-nitrobenzyl ester of bile acids has characteristic ultraviolet absorption, these compounds were separated to individual bile acids by high performance liquidchromatography, and detected by an UV-detector. An analysis of individual bile acids in human bile was demonstrated.

Electron microscopic observation of thr liver in portal hypertension following chronic exposure to vinyl chloride monomer

SummaryTwo patients with portal hypertension following chronic exposure to vinyl chloride monomer were studied histologically and ultrastructurally. Both cases showed non-cirrhotic portal hypertension accompanied by esophageal varices and splenomegaly. They proved hepatic fibrosis with conventional microscope. Ultrastructurally, there were large amounts of small myelin-like lamellar materials with electron density and remarkable proliferation and vesiculation of smooth endoplasmic reticulum in the cytoplasm of hepatocytes. The widened Disse’s spaces, where the sinusoidal lining cells were arranged in multilayers, were plugged by collagenous fiber bundles. The finding may be consistent with the development of portal hypertension.

Simultaneous assay for individual sulphated 3α- and β-hydroxysteroids in serum using high-performance liquid chromatography combined with 3α- and β-hydroxysteroid dehydrogenases immobilized on one column

Abstract A high-performance liquid chromatographic method for the simultaneous determination of individual sulphated 3α- and β-hydroxysteroids in serum using 3α- andβ-hydroxysteroid dehydrogenases (3α-HSD and β-HSD, respectively) immobilized on one column and a fluorimeter to detect the resulting NAD+ to NADH transformation is described. Individual sulphated 3α- and β-hydroxysteroids in serum are extracted with ethanol, solvolysed with sulphuric acid in ethyl acetate and then separated by high-performance liquid chromatography. The hydroxysteroids thus separated are subsequently mixed with NAD+ and then passed through the column in which the following catalytic reaction occurs: 3α- or β-hydroxysteroids + NAD+ 3-oxosteroids + NADH + H+ The detection limits are as low as 0.5-1.0 μg/dl for sulphated 3α- or β-hydroxysteroids in serum. The present assay method is highly specific, reliable and reproducible and is thus applicable to a clinical study on the metabolism of sulphated 3α- and β-hydroxysteroids in patients with adrenal or gonadal diseases.

Clinical significance of mitochondrial glutamic-oxaloacetic transaminase in serum of patients with liver disease

SummarySerum mitochondrial glutamic-oxaloacetic transaminase activity was determined in 83 patients with various liver diseases and 10 healthy adults.1)The average of mitochondrial glutamic-oxaloacetic transaminase value was 1.2 mU in healthy adults, 8.3 mU in patients with acute hepatitis, 13.7 mU in patients with post-transfusion hepatitis, 5.0mU in patients with persistent hepatitis, 4.5mU in patients with chronic inactive hepatitis, 9.6mU in patients with chronic active hepatitis, 5.6 mU in liver cirrhosis, and 295 mU in a patient with fulminant hepatitis.2)While one patient with acute hepatitis showed the highest value in the group of 29 mU, one patient with fulminant hepatitis showed an extremely high value of 295 mU, revealing an obvious difference between them.3)One patient with fresh myocardial infarction also showed an extremely high value of 110 mU.

A case report of benign recurrent intrahepatic cholestasis.

SummaryA case of benign recurrent intrahepatic cholestasis was reported. A man had first experienced jaundice at the age of twenty-five, and suffered three subsequent attacks over the next five years. These attacks were characterized by prodromal severe pruritus.During the icteric phase, serum levels of total bile acids increased, most being conjugates of primary bile acids. Electron microscopy revealed that the bile canaliculi were filled with an increased volume of granular substances. Some bile canaliculi fused together resulting in the formation of abnormally elongated channels which abutted on the Disse space. These observations suggest bile regurgitation through the channel of altered canaliculi. In convalescence, these bile canaliculi disappeared. Every attack was followed by complete recovery of liver structure and function.

Change in serum and biliary esterified bile acids in patients with extrahepatic cholestasis during percutaneous transhepatic cholangiodrainage

SummaryEighteen patients with total extrahepatic cholestasis undergoing PTCD were classified into three groups, depending on the bilirubin decrease rate at two weeks after PTCD. Serum and biliary esterified bile acids in each group were measured before PTCD and at 24 hours, 48 hours, 1 week, and 2 weeks after PTCD. Bile acids were measured by Okuyama’s methods (HPLC), and esterified bile acids were calculated from the difference between samples treated with sulfatase or β-glucuronidase for enzymatic hydrolysis and untreated samples measured at the same time. The following results were obtained.The percentages of biliary esterified bile acids in total bile acids were as follows: before PTCD, in the fair improvement group, sulfate (S) = 6.4 ± 4.6 % (mean ± S.D.), glucuronide (G) = 11.7 ± 9.0 % ; in the poor improvement group, S = 2.8 ± 1.6 %, G = 1.0 ± 0.9 % and at 24 hours after PTCD, in the fair group, S = 9.1 ± 7.5 %, G = 7.5 ± 4.3 % ; in the poor group, S = 2.9 ± 2.4 %, G =1.7 ± 1.1 %. The percentages of esterified bile acids in the fair group were higher than in the poor group, and significant differences were noted in G (p<0.05). Thus PTCD is expected to reduce jaundice in cases with high percentages of biliary esterified bile acids before and shortly after PTCD.

Clinical evaluation of serum 3β-hydroxy-5-cholenoic acid in hepatobiliary diseases

SummarySerum 3β-hydroxy-5-cholenoic acid (3β-OH-Δ5) was analyzed in 100 cases (90 patients with hepatobiliary diseases, 10 normal subjects) and its clinical significance investigated. The measurement of 3β-OH-Δ5 was performed by high performance liquid chromatography (HPLC) with immobilized 3β-hydroxysteroid dehydrogenase (3β-HSD) as the enzyme column. Esterified 3β-OH-Δ5 was measured after enzymatic hydrolysis with sulfatase andβ-glucuronidase. 3β-OH-Δ5 was hardly detected in normal cases. On the other hand, serum 3β-OH-Δ5 levels were remarkably high in cholestatic cases and also high in other cases with high bilirubin levels. The ratio of glycineto taurine-conjugates (G/T ratio) was effective in discriminating cholestasis from hepatocellular damage such as in cases of acute hepatitis or fulminant hepatitis. More than 90% of the 3β-OH-Δ5, which is toxic, was sulfated or glucuronidated, suggesting detoxification by esterified bile acids. Significant increases of taurine-conjugated 3β-OH-Δ5 were observed in cases with pruritus, and a relationship between taurine-conjugates and pruritus was presumed. Therefore, analysis of 3β-OH-Δ5 is considered to be effective in clarifying the pathogenesis of hepatobiliary diseases.

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

Proceedings of the 19th autumn meeting from October 18 to 20, 1977-Nara, Japan

The first step in the formation of bile acids is 7uhydroxylation of cholesterol. Subsequently 7ahydroxycholest-4-en-3-one is formed, which is a common precursor of the two primary bile acids, cholic acid and chenodeoxycholic acid. 5,BCholestane-3~t , 7a -d io l , a p recursor of chenodeoxycholic acid is formed from the A4-3-keto intermediate by a pathway which involves the reduction of the keto group and the saturation of the double bond but not a hydroxylation at C-12, while a pathway involving the 12a-hydroxylation of the A4-3-keto intermediate leads to the formation of 5flcholestane-3~, 7a, 12a-triol, which is a precursor of cholic acid. The C2~-bile alcohol intermediates , 5flcholestane-3a, 7t~-diol and 5,g-cholestane-3tz, 7t~, 1 2 ~ t r i o l , a r e t h e n t r a n s f o r m e d i n t o chenodeoxycholic acid and cholic acid by the degradation of the side chain which entails an moxidation followed by fl-oxidation. Chenodeoxycholic acid is also formed by a number of different pathways differing only with respect to the stage of nuclear transformation at which oxidation of the side chain is initiated. A number of cholestanepolyols such as 5flcholestane-3ct, 70t, 12ct, 25-tetrol are accumulated in the bile and feces of patients with cerebrotendinous xanthomatosis. No 26-hydroxylated bile alcohols are accumulated. The results suggest that there exists an alternative pathway of cholic acid biosynthesis involving the 25-hydroxylated bile alcohol as an intermediate. (2) Analytical methods of bile acids in biological materials