Peter Czygan

Determination of bile acids in needle biopsies of human liver.

Abstract A microquantitative procedure is described to determine bile acids in 5–15 mg liver tissue, which can be obtained from the specimen of needle biopsy performed for histological examination. The tissue cylinder is solubilized in KOH and the bile acids are deconjugated by cholylglycine hydrolase, purified by thin-layer chromatography, methylated, and trimethylsililated for gas chromatographic separation and quantitation. Recovery of taurine- and glycine-conjugated bile acid standards is 73–86%; the experimental error of the procedure is less than 15%. The technique permits investigation of hepatic bile acids in liver diseases without requiring surgical exploration.

Mutagenicity of primary and secondary carcinogens altered by normal and induced hepatic microsomes.

To establish the role of the activity of the microsomal biotransformation system in chemical carcinogenesis, the mutagenic effect of primary (ultimate) and secondary (potential) carcinogens after exposure to isolated hepatic microsomes was studied. The principal enzyme system, suggested to be active in the metabolism of carcinogenic compounds, is the nonspecific cytochrome P-450 dependent mixed-function oxidase (1). This microsomal enzyme system is inducible by many substrates, including some environmental contaminants (2). The involvement of the microsomal enzymes in carcinogenesis is suggested by the observed increased carcinogen biotransformation in animals (3) and isolated cells (4) following exposure to inducing substrates. Most carcinogenic compounds are mutagenic for microorganisms (5). The primary are themselves mutagenic, the secondary are inactive until metabolized to a mutagenic form (6). Metabolism of primary carcinogens may reduce mutagenic and carcinogenic properties. Both types of metabolic conversion, activation and deactivation, have been studied by host-mediated assay, which tests the ability of laboratory animals to alter the mutagenicity of carcinogens (6). Similar activity also has been demonstrated in the 30,000g supernatant of mice liver homogenates (7). To demonstrate the precise relationship between the activity of the microsomal enzyme system and the biological activity of carcinogens, an assay is required in which both activities can be measured. For this purpose the ability of isolated normal or induced microsomes to alter the mutagenic activity of two carcinogens, dimethylnitrosamine (DMN) (8) and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) (9) was studied. These compounds were chosen as examples of primary (MNNG) and secondary (DMN) carcinogens. Methods. Hepatic microsomes were isolated from male Swiss-Webster mice untreated or pretreated four days before sacrifice from a single ip injection of 500 mg/kg of the polychlorinated biphenyls (PCB), Arochlor 1254, an inducer of the microsomal enzyme system (10) and a wide-spread polluting agent (1l).

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.

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.