Relatively little is known about the mechanism, regulation, or subcellular localization of the hepatic uptake, storage, conjugation, and excretion of bilirubin. In the work here reported, the ultrastructural localization of the sites of these processes was studied by determining the subcellular distribution and chemical nature of bilirubin after the injection of physiologic amounts of tritiumlabeled unconjugated bilirubin (UCB-3H) and conjugated bilirubin (CB-3H) in normal and glucuronyl transferase-deficient (Gunn) rats (1, 2). Studies were also performed using bilirubin-14C for comparison with the intracellular distribution of tritiated bilirubin. While these studies were in progress, Brown, Grodsky, and Carbone (3) described the subcellular distribution of injected UCB-3H in normal rat liver. MethodsTritium-labeled bilirubin was prepared by the Wilzbach procedure and purified and equilibrated with crystalline bovine albumin to constant specific activity as described by Grodsky, Carbone, Fanska, and Peng (4). The purity of labeled bilirubin was established by diazotization, chro-* Submitted for publication January 28, 1966; accepted April 5, 1966. These studies were supported by research grants from the U. S. Public Health Service (AM 02019 and TI AM 5384) and the New York Heart Association and Heart Fund, Inc.This work was presented in part at the Eastern Sec- Bile obtained from normal rats injected with UCB-'H was stored at -200 C for not more than 48 hours before use and served as a source of CB-8H. Chromatography and subsequent strip scanning of azo pigments obtained from this bile revealed 82 to 96% of the total radioactivity in bile to migrate with the conjugated azo pigment (Azo-B) on paper chromatograms. The mean specific activity of CB-'H was 60% of UCB-'H, and accordingly 85 to 125 ucg containing 1.5 X 107 dpm was used in each experiment.Bilirubin-'4C was prepared according to the technique of Ostrow, Hammaker, and Schmid (6) and was recrystallized to a constant specific activity of 0.91 mc per mmole.At intervals of 1 to 60 minutes after injection of UCB-8H, UCB-14C, or CB-'H, a single lobe of the liver was removed, maintained at 00 C, rinsed in 0.25 M sucrose at pH 7.4, blotted dry, weighed, and homogenized in a Teflon glass homogenizer containing 9.0 vol of the buffered sucrose solution. The order in which lobes were removed was varied in initial experiments, but no significant difference in uptake of radiobilirubin among different lobes was observed.Eight normal rats were injected with UCB-8H, and total intrahepatic radioactivity was estimated in homogenates of liver obtained at 1, 2,4,8,9,15, 30, 45, and 60 minutes after injection. Twenty studies of the subcellular distribution of radiobilirubin were performed using liver obtained from these rats at intervals from 1 to 60 minutes after injection of UCB-3H. Three normal 1194 HEPATIC DISTRIBUTION OF BILIRUBIN TABLE I
The mean activity of glutathione peroxidase (GSH-PX) in erythrocytes of 22 Israeli-Jewish patients with multiple sclerosis (19.3 +_ 4.5 U/gHb) was significantly lower than in a control group of 30 Jewish patients with various neurological disorders (24.3 +_ 5.1 U/gHb). This observation confirms a similar finding of a decreased activity of GSH-Px in erythrocytes of multiple sclerosis patients in Denmark (Shukla et al. 1977). These results are discussed in relation to the possibility of selenium deficiency and to the recently described genetic polymorphism and ethnic variation of GSH-Px activity in human red cells. It is concluded that additional investigations are required in order to elucidate the cause of the decreased activity of this enzyme in red cells of patients with multiple sclerosis.
Red cell glutathione peroxidase (GSH-Px) activity has been measured in samples from seven Jewish population groups in Israel. No significant differences were found between the various communities. The mean GSH-Px activity was similar to that observed by Beutler and Matsumoto in Jewish samples from the USA and Israel. According to a genetic model proposed by the above investigators, the sample was subdivided into three phenotypes with low, intermediate and high activity, suggesting gene frequencies of 0.5072 and 0.4928 for the GSH-PxL and GSH-PxH alleles, respectively.
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