Ferenc Hutterer

Mechanism of Cholestasis: 4. Structural and biochemical changes in the liver and serum in rats after bile duct ligation

To determine whether changes in the function of the hepatocellular microsomal biotransformation system play a role in the genesis of cholestasis or result from it, the biochemical alterations in the biotransformation system and the appearance of the endoplasmic reticulum were compared in rats during the 1st 4 days after bile duct ligation. The time course of the changes in the structure of the hepatocytes was also compared with the results of commonly used hepatic tests. Cholestasis is characterized by decreased metabolism of substrates bound to the lipoprotein of cytochrome P-450 despite an increase in the amount of smooth endoplasmic reticulum. This increase occurs after cholestasis is apparent and, therefore, results from it. The changes in the endoplasmic reticulum with hypertrophy of the smooth form may be adaptive in nature. The high but transient elevation of transaminase activity soon after bile duct ligation was not associated with morphological signs of injury. The later injurious effects of cholestasis are thought to be caused, at least in part, by the accumulation of bile salts, which by their detergent action decrease the function of the hypertrophic smooth endoplasmic reticulum.

Determination of α-keto acids as silylated oximes in urine and serum by combined gas chromatography-mass spectrometry☆

Abstract Nine α-keto acids in serum and urine were analyzed by gas chromatography as their oxime-trimethylsilylated derivatives, following oxime formation with hydroxylamine in pyridine and silylation with BSA containing 10% TMCS. The oxime-TMS derivatives were identified by both low- and high-resolution mass spectrometry. Recoveries from urine were greater than 90% and from serum greater than 80%. Loads of valine, leucine and isoleucine are shown to result in increased serum concentrations of corresponding α-keto acids in healthy adults. In a child with maple-syrup-urine disease under treatment by dietary restriction of the branchedchain amino acids, administration of leucine results in a large excretion of α-keto isocaproic acid and a less striking excretion of α-keto-β-methylvaleric and α-keto isovaleric acid. The excretion of the branched-chain α-keto acids is reported in two patients with maple-syrup-urine disease before and after institution of dietary treatment. Nine α-keto acids and at least 15 phenolic acids and related compounds can be determined in a single analysis using the method described. The ‘metabolic profile’ thus obtained may be used in screening for abnormalities in the metabolism of amino acids. The gas Chromatographic analysis alone is adequate for screening; when an abnormal quantity of a constituent is found, mass spectrometry is used for confirmation of identity.

Alteration of Microsomal Biotransformation in the Liver in Cholestasis

Summary In cholestasis, 4 days after bile duct ligation, the activity of aminopyrine demethylase was greatly decreased, while the content of cytochrome P-450 and the activities of aniline hydroxylase, NADPH-cytochrome c reductase, and cytochrome P-450 reductase were only slightly decreased in the microsomes of the rat livers. Binding of type II substrate to cytochrome P-450 was unimpaired and its modifier effect on P-450 reductase was intact. The binding of type I substrate was greatly decreased, and its stimulating effect on P-450 reductase was abolished, suggesting that the effect of cholestasis on the hepatocellular smooth endoplasmic reticulum is to alter the type I binding sites, which is responsible for the hypoactivity of the biotransformation system.

HEPATIC FIBROGENESIS AND DISTURBANCE OF HEPATIC CIRCULATION

A vicious circle appears to connect hepatic fibrosis and disturbances of hepatic circulation in chronic and acute liver diseases. Alterations in the intrahepatic microcirculation favor development of fibrosis, whereas the main consequences of hepatic fibrosis are the disturbance of nutrition of the hepatocytes by pericellular fibrosis, reduction of the parenchymal blood flow, and portal hypertension. Understanding the processes involved in hepatic fibrogenesis may thus contribute to the appreciation of the disturbed hepatic function in chronic liver disease and may eventually provide therapeutic approaches by influencing specific processes in fibrogenesis. Fibrogenesis is of general importance in aging, arteriosclerosis, and wound healing. In the following, the specific processes in hepatic fibrogenesis will be briefly reviewed, particularly as they apply to disturbances of hepatic circulation.

Hydroxylation of taurolithocholate by isolated human liver microsomes. I. Identification of metabolic product.

Abstract Human liver microsomes isolated from surgical biopsy specimen hydroxylate taurolithocholic acid in the 6α position. The reaction requires NADPH. The identification of the reaction product as hyodeoxycholic acid is based on data obtained from gas chromatographic, thin-layer chromatographic, and mass spectrometric analyses.

Mechanism of cholestasis. 2. Effect of bile acids on the microsomal electron transfer system in vitro

Abstract 1. 1. Dihydroxy bile acids, which give a Type I binding spectrum with cytochrome P-450, increase the initial rate of P-450 reductase in concentration below 1.0 mM. 2. 2. At 1.0 mM they dissociate the flavoprotein P-450 reductase from P-450, resulting in a concentration-dependent decrease of the initial rate of P-450 reductase and of the amount of enzymatically reducible P-450. 3. 3. Degradation of P-450 by dihydroxy bile acids is concentration and time-dependent. 4. 4. Trihydroxy bile acids do not produce these changes.

Turnover of hepatic cytochrome P-450 in experimental cholestasis

Abstract In mechanical experimental chllestasis, hypertrophy of smooth microsomal membranes was observed. In contrast to typical induction, the membranes were deficient in cytochrome P-450. The total cytochrome P-450 content of the liver, however, as determined in the liver homogenate remained unchanged. To clarify the mechanism of the development of cytochrome P-450 deficient membranes in cholestasis, the half life of the heme portion of cytochrome P-450, and the initial rate of synthesis of cytochrome P-450 and b 5 hemes were compared in bile duct ligated rats and in control animals after labeling the heme by injection of the precursor δ-[4- 14 C]aminolevulinic acid. The half lives were not significantly different, which eliminates the possibility that selective destruction of cytochrome P-450 has occurred. Depression of cytochromal heme synthesis was not observed. During mechanical cholestasis, the relative cytochrome P-450 deficiency is probably caused by proliferation of components of the endoplasmic reticulum other than the hemoprotein.

Effect of Experimental Portacaval Shunt on Hepatic Drug Metabolizing Enzymes

Summary Diversion of the portal flow from the liver in rats results in a reduction of the activities of hepatic drug metabolizing enzymes. Such animals still respond to phenobarbital with an increase in hepatic enzyme levels and hypertrophy of smooth endoplasmic reticulum in hepatocytes. It is suggested that a defect in the detoxifying ability of the liver itself may play an additional role in portasystemic encephalopathy in man.

Effect of Cyclic Amp on the Phenobarbital Induced Increase in Cytochrome P-450 and Hypertrophy of the Endoplasmic Reticulum of the Rat Liver

It has been known for almost a decade that catecholamines depress hepatic drug metabolism (Kato and Gillette, 1965). The action of these hormones is mediated by cyclic adenosine -3’, 5’- monophosphate (cAMP) and the cAMP level in the liver becomes elevated after hormone administration (Exton et al., 1971). However, there is relatively little information available concerning the direct involvement of cAMP in hepatic drug metabolism. Weiner et al., (1972a) have recently shown that cAMP, or rather its dibutyryl derivative, N6, O2’-dibutyryl cAMP (DBcAMP), increases sleeping time after hexobarbital administration. Subsequently it was demonstrated that DBcAMP treatment decreases the activities of hepatic aniline hydroxylase and aminopyrine demethylase and the concentration of cytochrome P-450 (Ross et al., 1973) and partially inhibits the induction of cytochrome P-450 stimulated by phenobarbital (Dressler et al., 1973).

Hydroxylation of taurolithocholate by isolated human liver microsomes II. Cytochrome P-450 dependency

Abstract The conversion of taurolithocholate to taurohyodeoxycholate by 6α-hydroxylation by isolated human liver microsomes and NADPH is inhibited by CO and the inhibition is maximally reversedby monochromatic light at 450 nm indicating that the reaction is cytochrome P-450 dependent.