Biochemical medicine最新文献

Serum concentrations of nonglucuronidated-nonsulfated, glucuronidated, and sulfated bile acids in 9 control children and 16 children with cholestasis were quantitated by mass fragmentography. Total bile acid levels in control children were 19.55 ± 2.78 μmole/liter (mean ± SEM), and glucuronidated and sulfated bile acids comprised 2.6 ± 0.5 and 17 ± 3.1%, respectively. In 9 patients with congenital biliary atrasia, total bile acid levels were 167.34 ± 11.18 μmole/liter of which 2.1 ± 0.3% were glucuronidated and 15 ± 1.4% were sulfated. Lithocholic and 3β-hydroxy-5-cholenoic acids, which have hepatotoxic effects, were presented in only small amounts in cholestatic children, and they were almost all glucuronidated or sulfated. The percentages of glucuronidated bile acids in control and cholestatic children were lower than in healthy and cholestatic adults, which may be explained by the lower activity of UDP-glucuronyltransferase in neonatal liver.

A spectrophotometric method for the determination of arginase (EC 3.5.3.1) is presented. Arginase is coupled to urease and glutamate dehydrogenase and the decrease in absorbance at 340 nm due to the oxidation of NADPH is followed. The method is rapid, is sensitive, is economical and permits continuous monitoring. The initial velocities were directly proportional to the enzyme concentrations between 0.06 and 0.30 units per 0.5 ml. The Lineweaver-Burk plot yielded positive allosteric behavior for the tetrameric enzyme (16). The K′ and the Hill coefficient, n, calculated from Hill plot were found to be 4.7 mm and 1.26 (r = 1.00), respectively. These values are in good agreement with the literature (12,16,17).

A simple modification of the immunological sandwich method of Muilerman et al. (4) for the identification of denatured enzyme proteins in sodium dodecyl sulfate-polyacrylamide gels is described, enabling the method to be used in principle for any enzyme whose activity is not inhibited by binding to antibodies. An immunological sandwich consisting of denatured enzyme, antibodies, and native enzyme is formed on a nitrocellulose filter blot of the gel, the filter is divided into strips, and each strip is tested for enzyme activity. The presence of enzyme activity serves to identify the region in the gel containing denatured enzyme protein. Experiments with human lysosomal α-glucosidase as a model system are described. The method was applied to identify a protein of M_r 125,000 as the main component with UDPgalactose pyrophosphatase activity in a partially purified preparation of the enzyme from rat liver.

The effect of a 0.25% clofibrate diet for 2 weeks on peroxisomal and mitochondrial β-oxidation in chicken liver was studied. The activities of antimycin A-insensitive palmitoyl-CoA oxidation (peroxisomal β-oxidation) and carnitine acetyltransferase increased about two-fold. The activities of palmitoyl-CoA-dependent O₂ consumption (mitochondrial β-oxidation) and carnitine palmitoyltransferase were also slightly activated by the administration of clofibrate, but not significant. Thus, clofibrate may be a typical drug which activates the peroxisomal β-oxidation more than the mitochondrial one in various species. The effect of clofibrate on peroxisomal carnitine acetyltransferase was the same as that on the mitochondrial one in chicken liver. Serum lipids were not lowered, but hepatomegaly was observed in the present experiment with chicken.

3β-Hydroxy-5-cholenoic acid in the serum of control subjects and 62 patients with various hepatobiliary diseases was quantitated by mass fragmentography after separation into nonglucuronidated-nonsulfated, glucuronidated, and sulfated fractions. Deuterium-labeled deoxycholic acid and its glucuronide and sulfate were used as internal standards. Mean concentrations of total 3β-hydroxy-5-cholenoic acid in serum (μmole/liter) were as follows: Control subjects (14), 0.184; obstructive jaundice (15), 6.783; liver cirrhosis, compensated (12), 0.433, and decompensated (12), 1.636; chronic hepatitis (12), 0.241; and acute hepatitis (11), 2.364. Most of the 3β-hydroxy-5-cholenoic acid was glucuronidated or sulfated. Only in patients with obstructive jaundice did glucuronidation (60 ± 14%) exceed sulfation (31 ± 14%), sulfation exceeding glucuronidation in the others. The UDP-glucuronyltransferase might have different substrate specificities for 3β-hydroxy-5-cholenoic acid and other common bile acids, especially in the cholestatic state.

Previous work from our laboratory indicated that pancreatic islets contain myosin light chain kinase, a calcium- and calmodulin-activated enzyme. This enzyme catalyzes phosphorylation of myosin which, in tissues containing smooth muscle, is believed to permit the ATPase of myosin to be activated by actin. The current report shows that incubating islet cytosol with ATP under conditions that should permit phosphorylation of myosin markedly enhances islet myosin ATPase activity in the presence of actin. It has been suggested that contractile proteins power insulin granule movements in the β cell. Phosphorylation of myosin may be one of the means of coupling stimuli to insulin secretion.

Our studies have revealed that the primary lesion of GSD type Ib exists in the G6P transport system in the microsomal membrane. Distinct evidence for the existence of a specific G6P transport system in microsomal membrane was obtained through these studies. This is the first example of a genetic disorder involving the transport system of an intracellular membrane. HHH syndrome (hyperornithinemia, hyperammonemia, and homocitrullinuria), in which the transport of ornithine to the mitochondria is presumed to be defective, may be another example belonging to this category of genetic disorders (18–20). A possibility exists that there are many other disorders due to defects in the membrane transport of intracellular organelles.

The future role of pharmacologists in the evaluation of drugs will increase as scientific knowledge and our understanding of drugs and disease processes increase. In addition, political issues and public fears will place further demands on the scientific community to try to influence the drug regulatory process. The FDA continually issues directives which guide all phases of drug development, from the identification of a chemical as being of potential interest on therapy, through all animal tests and clinical phases (4).

A method for determination of inorganic sulfate is described which represents the terminal step of an automated assay of organic sulfur compounds in biological fluids. Nanomole quantities of inorganic sulfate were applied to a barium chloranilate column. Corresponding amounts of chloranilate ion, released as a result of an exchange reaction, were then measured by uv absorption at 313 nm. The reproducibility and sensitivity of the method and a calibration curve are reported.