No abstract

The chemical structure of the major conjugate of bilirubin was unequivocally elucidated by structural analysis. The conjugated bilirubins were first separated from the lipid components of human duodenal aspirates or dog gall-bladder bile, and then resolved by t.l.c. into a series of tetrapyrroles. The major tetrapyrrole was then converted into its more stable dipyrrolic azo derivative for further analysis. The conjugated moiety of the azopigment was characterized after methanolysis with sodium methoxide. This reaction yields two types of product, those soluble in water and those soluble in organic solvents. The organic-soluble fraction was shown by t.l.c. and mass spectrometry to contain the methyl esters of the dipyrrolic azo derivatives of bilirubin. The water-soluble materials were analysed by enzymic procedures, t.l.c., n.m.r. spectrometry and combined g.l.c. and mass spectrometry. This analysis showed that the only water-soluble product resulting from the methanolysis was glucuronic acid. The structure was identical with that of pure standards, on both mass spectrometry and n.m.r. spectroscopy. No contaminating moieties were found. Quantitative measurement indicated that the glucuronic acid had been released in a 1:1 molar ratio with the resulting methyl esters of the dipyrrolic azo derivatives of bilirubin. This unequivocally establishes bilirubin diglucuronide as the major pigment present in bile. Past problems with identification of bilirubin diglucuronide were shown to originate from procedures which resulted in incomplete separation and isolation of the azopigments of the conjugated bilirubins, owing to contamination by biliary lipids.

Baboons fed ethanol (50% of total calories) chronically develop ultrastructural alterations of hepatic mitochondria. To determine whether mitochondrial functions are also altered, mitochondria were isolated from nine baboons fed ethanol chronically and their pair-fed controls. At the fatty liver stage, ADP-stimulated respiration was depressed in ethanol-fed baboons by 59.4% with glutamate, 43.2% with acetaldehyde, 45.1% with succinate and 51.1% with ascorbate as substrates. A similar decrease was noted in the ADP/O ratio (14 to 28%) and respiratory control ratio (20 to 44%) with all substrates. Similar alterations of mitochondrial functions were observed in baboons with more advanced stages of liver disease, namely fibrosis. These changes after ethanol treatment were associated with decreases in the enzyme activities of mitochondrial respiratory chain: glutamate, NADH and succinate dehydrogenase (42, 24 and 28%, respectively), glutamate-, NADH- or succinate-cytochrome c reductase (42, 27 and 32%, respectively) and cytochrome oxidase (59.6%). The content of all cytochromes was also decreased in ethanol-fed baboons, especially aa3 (57%). Moreover, [14C]leucine incorporation into mitochondrial membranes was depressed by 21% after ethanol treatment. On the other hand, glutamate dehydrogenase activities of serum and cytosol in ethanol-fed baboons were significantly higher than those in pair-fed controls. Morphologically, mitochondria of ethanol-fed baboons were larger than those of pair-fed controls. However, the mitochondrial protein content per mitochondrial DNA was unchanged. From these results, we conclude that, morphologically and functionally, hepatic mitochondria in baboons are altered by chronic ethanol consumption; it is noteworthy that these changes are fully developed already at the fatty liver stage, and that morphological alteration appears to reflect the damage of mitochondrial membranes rather than an adaptive hypertrophy.

The role of hormones in the regulation of bile secretion is not known; however vasoactive agents, which act via the phosphoinositide signal transduction pathway, may mediate changes in bile flow by altering the hepatic microvasculature. We therefore examined the effects of phorbol esters and diacylglycerol, agonists of the protein kinase C branch of the phosphoinositide cascade, on perfusion pressure and bile flow in a single-pass, hemoglobin-free, isolated perfused rat liver system with constant perfusate flow. The active phorbol ester, 12,13-phorbol dibutyrate, produced a dose-dependent (maximal effect at 10(-6) M), sustained and reversible decrease in bile flow from 1.09 +/- 0.18 to 0.61 +/- 0.09 microliters per min per gm liver (37.2 +/- 5.9%) while simultaneously increasing perfusion pressure from 12.3 +/- 0.7 to 21.5 +/- 2.5 cm H2O (74.0 +/- 4.3%). Both effects were inhibited by the synthetic protein kinase C antagonist H-7. 1,2-Dioctanoyl-sn-glycerol, a diacylglycerol, produced changes in bile flow and perfusion pressure that were similar to, but more marked than, those caused by 12,13-phorbol dibutyrate, whereas the inactive phorbol ester 4 alpha-phorbol didecanoate and the vehicle dimethyl sulfoxide had no effects on either parameter. 12,13-Phorbol dibutyrate infusion resulted in reversible decreases in oxygen consumption (23.3%) and a reversible vascular redistribution of trypan blue dye but did not alter hepatic venous effluent concentrations of K+.(ABSTRACT TRUNCATED AT 250 WORDS)