A specific method for determination of cytosol leucine aminopeptidase (CLAP) was devised, in which the ammonia libelated from L-leucine amide (LA) added as the substrate was measured with glutamate dehydrogenase added as the aid of the coupling enzyme at pH 7.6 in the presence of 1 mM o-phenanthroline.
The principle of the method is due to the present finding that o-phenanthroline inhibited arylamidase (AA) and cystyl aminopeptidase (CAP) completely at 0.6mM or higher, but not CLAP even at 1 mM.
The present method showed good correlations to the method using LA and that using Lleucyl-p-nitroanilide (L-PNA) as the substrate, except that there were remarkable discrepancies to the former method in the cases of hepatobiliary disorder and pregnancy, and those to the latter method in the cases of hepatitis, malignant lymphoma and leukemia in addition to hepatobiliary disorder and pregnancy.
This suggests that the present method was specific for CLAP, useful for clinical diagnosis.

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We purified human apolipoprotein (apo) A-IV from pooled serum of healthy subjects. The amino acid analysis and the pl of this apo A-IV agreed closely with other published data. In addition, we prepared specific antisera to apo A-IV and used them for immunophoretic quantitation of apo A-IV levels in serum. Serum samples were diluted two times with saline containing 1% Triton X-100. Within-and between-assay CVs, studied with five serum samples, were both not greater than 6%. There was no influence of freezing and thawing of serum on the measured values for apo A-IV. The mean value of serum apo A-IV in fasting healthy subjects was 10.9±2.5 mg/dl. In the study of apo A-IV distribution in serum by this study, apo A-IV was found only in the ultracentrifugal bottom fraction of serum from normolipidemic individuals, whereas a small amount of apo A-IV was also detected in the chylomicron fraction from hyperlipidemic ones. The apo A-IV concentrations could not be detectable in the other lipoprotein fractions by this assay technique.

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Method of determination of neutral glycosylceramides in human plasma and erythrocytes by high performance liquid chromatography was studied.
Neutral glycosylceramides extracted by chloroform; methanol (2;1) were separated from lipids such as glycerides and phospholipids by 50% methanol inactivated Unisil column and derivatized by 4-nitrobenzoyl chloride in pyridine at 37°C overnight. After excess reagents were removed by solvent extraction, pernitrobenzoylated neutral glycosylceramides were injected onto TSK Silica 60 column (4×250mm) and eluted with linear gradient of n-hexane; methylene chloride; acetonitrile=10; 4; 0.5 to 10; 4; 4 for 20min at a flow rate of 2.0 ml/min at room temperature. Eluent was monitored at 254 nm.
N-Palmitoyl-D, L-dihydro-glucosyl- and lactosyl-cerebroside were obtained as single peaks of their pernitrobenzoylated derivatives. A good calibration curve was obtained by injecting 0.1 to 2.0 μg of the samples. Recoveries of Glc- and Lac-cermides from plasma were 89% and 78 % and those from erythrocytes were 88% and 66%, respectively.
Every glycosyl ceramide in plasma and erythrocytes was separated as multiple peaks. In the family of Fabry's disease, ceramide trihexoside concentrations in plasma and erythrocytes of hemizygote were 3 to 5 times higher than those of normal and heterozygotes diagnosed by α-D-galactosidase activities in leukocytes.
This method will be useful for the biochemical diagnosis of other neutral glycosylceramide and ganglioside storage diseases.

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p-Amidinophenylester derivatives were prepared and their inhibitory effects on chymotrypsin, trypsin, thrombin, plasmin, tissue plasminogen activator (TPA), urokinase, factor Xa, tissue kallikrein, plasma kallikrein, and elastase were examined. Among the various synthetic inhibitors tested, aromatic carboxylates of p-amidinophenol were the most effective inhibitors of chymotrypsin, trypsin, thrombin, plasmin, TPA, urokinase, factor Xa, tissue kallikrein, and plasma kallikrein.

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Six species of ‘middle molecules (MMs) ’ were separated from crude fractions of both hydrophobic and hydrophilic MMs obtained from the hemofiltrate of a previously reported dialysis patient. With phosphate buffer and Tris buffer as the incubation media, their action on the level of both oxygen consumption and oxidative phosphorylation in rat liver mitochondria was investigated polarographically with a Clark oxygen electrode. hydrophobic MMs species and some of acidic and neutral hydrophilic MMs species were found to have no effect on mitochondrial respiration. On the other hand, a weakly acidic species, B-MMs, stimulated the respiration of state 4 mitochondria in phosphate medium, but not in Tris medium. They also caused an approximately 3 to 4-fold release of the state 3 respiration in oligomycin inhibited mitochondria. These facts indicate that the B-MMs play a role of phosphate dependent uncoupler of mitochondrial function. Based on the effect of a phosphate-transport inhibitor, N-ethylmaleimide, we confirmed the uncoupling action of B-MMs is associated with the phosphate-transport process via a phosphate carrier. This action suggests a close relationship between MMs and the hyperphosphatemia seen during the uremic state of the patient.

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Cholinesterase activities (ChE) with various substrates and in the presence and absence of inhibitors were measured in sera from human and animals, and the following results were obtained.
1. True ChE, which is inhibited by caffeine but not ethopropazine with acetylthiocholine as substrate, was present to a greater extent in animals than in human. Differences of the inhibition by caffeine and ethopropazine suggest that proportions of true ChE to total ChE were about 60% in rabbits, 40% in male rats, 16% in dogs, 10% in monkeys and 7% in human.
2. In rabbits, some (type I) hydrolysed butyrylthiocholine to a significantly higher extent than others (type II). Since this hydrolysis was mostly inhibited by DFP but hardly inhibited by EDTA or eserine, it was probably due to carboxyesterase.
3. In female rats, ChE with acetylthiocholine as substrate showed age-dependent differences in activity, Km value, and inhibtions by caffeine and ethopropazine. True ChE was constant regardless of age, indicating that the differences were due to the change of pseudo-ChE.

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To study the occupational exposure to hepatitis virus in health care personnel, the occurrence of hepatitis B surface antigen and antibody was examined in 1020 hospital employees.
The personnel were divided into two groups comprising doctors, nurses, and technicians who were exposed to hepatitis virus (exposed group), and office workers and cooking and laundry workers who were not exposed to hepatitis virus (non-exposed group).
Activites of guanine deaminase, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) in hepatitis B antigen-positive employees of the exposed group were compared with those in nonexposed employees who showed negative results for hepatitis B antigen and hepatitis B antibody.
ln the exposed group, the Odds ratio and attributable risk percent (ARP) were the highest among technicians.
The guanine deaminase and ALT activities were significantly higher in doctors and technicians, whereas AST activity was significantly higher only in technicians. The results suggest that guanine deaminase should be included among the enzymes tested in screening health care personnel for prevention of infection from hepatitis and for determination of the onset of hepatitis B.

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Ascorbic acid is well known to give false negative results in the clinical determination of urinary glucose or occult blood (blood/hemoglobin) using commercially available reagent test strips. We describe herein a procedure to remove the ascorbic acid interference with use of a newly deviced enzyme reactor, where ascorbate oxidase is immobilized on the inner surface of polystyrene test tubes treated with a silane coupling reagent. It was clearly shown that even the highest possible concentration of ascorbic acid in urine can be completely oxidized within a few minutes at room temperature by the enzyme reactor, and therefore urinary glucose or occult blood can be determined without any false negative results.
This type of enzyme reactor may be applicable for use in many clinical fields and in many testing situations in clinical laboratories to improve the accuracy and reliability of clinical tests.

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