Antigenic determinants in human urinary kallikrein (HUK) and prokallikrein (HUPK) molecules have been investigated. The number of antigenic sites was estimated to be six per mol of HUK from the quantitative precipitin reaction with the anti-HUK antibody and four per mol of HUPK with the anti-HUPK antibody. The values for the association constants were in the order of 10⁷ M^-1. Both antibodies inhibited the enzymatic activity of HUK, and the extents of the inhibition were about 80% and 95% when Pro-Phe-Arg-MCA and kininogen were used as substrate, respectivery.
Among five peptides isolated from CNBr-cleaved S-carboxymethylated (SC) HUK, N-ter-minal peptide, lle (1)-Met (79), and the peptide, Leu (99)-Met (173), were detectable by means of enzyme immunoassay, whereas the other three peptides were undetectable.
Four highly hydrophilic regions as antigenic determinants were identified in HUK molecule by hexapeptide hydropathy analysis of the amino acid sequence of HUK, each region being comprised in the four separate peptides from CNBr-cleaved SC-HUK except C-terminal one. The simulation of three dimensional structure of HUK based on the structure of porcine pancreatic kallikrein revealed that the four regions were located at the surface of HUK molecule.

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We describe a fully automated enzymatic method for the direct measurement of tyrosine and the three branched-chain amino acids (BCAA)-leucine, isoleucine, and valine-in plasma. For the tyrosine assay, we employed tyrosine decarboxylase, tyramine oxidase, and peroxidase; and for the BCAA assay, leucine dehydrogenase. We measured aromatic amino acids and BCAA in plasma from patients with hepatic disorders by this method and by use of an amino acid analyzer (AA). The results obtained by the two procedures correlated well (tyrosine, r=0.957; BCAA, r=0.970). The BCAA/tyrosine ratio by the method was shown to be as good as Fischer's ratio as an indicator for the diagnosis of liver diseases.

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Changes of glycolipids in the brain after formalin fixation were examined. Quantity of lipids in the brain decreased rapidly after formalin fixation. Glycolipids decreased to 50% 24 hours after fixation, and to 10% after 4 months after fixation. Fatty acid composition of glycolipids showed a change characterized by both a diminution of long-chain fatty acids (C: 16-20) and an increase of long-chain fatty acid (C: 23-27) 4months after fixation, and its change was more markedly noted in normal fatty acids than hydroxy fatty acids.

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We evaluated the optimal condition for the assay of volatile hydrocarbons produced by oxidation of red blood cells in vitro by hydrogen peroxide. As a result, we chose the condition of 4.3% packed cell volume (PCV), 37.8mM hydrogen peroxide and for inhibition of catalase activity, 42.8mM sodium azide in final concentration.
Sources of the volatile hydrocarbons were suggested to be red blood cell membranes for n-pentane, but unclear for other volatile hydrocarbons such as n-propane and n-butane.
During aging of red blood cells, production of ethane and n-pentane, which were volatile hydrocarbons specifically produced from red blood cell membranes, did not change signifi-cantly, while that of other uncharacterized hydrocarbons increased. Therefore, it was suggested that there was no change with cell aging in the hydrocarbon production from red blood cell membranes.

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Maillard reaction (glycation) has received attention in relation to diabetic complications. On the other hand, sorbitol which is produced in the polyol pathway has also been pointed out as one of causes of diabetic complications. In this study, we investigated in in-vitro the effect of fructose which is produced from sorbitol, on the Maillard reaction.
Fructose accelerated the fluorescence intensity, amino acid impairment and polymerization of the proteins more and decreased the digestibility of the collagen by collagenase more than glucose. When proteins preincubated with glucose (glucose-protein system) and fructose (fructose-protein system) were incubated continuously for 2 weeks without glucose and fructose under physiological conditions, the fluorescence intensity gradually increased and furosine values conversely decreased in the glucose-protein system. There were no changes in the fructose- protein system. No change of the impairment of amino acid residues was found in either system. Thus, the late-stage of the Maillard reaction in the fructose-protein system is possibly different from that in the glucose-protein system.
These results suggest that fructose more easily produces an increase in the accumulation of the late-stage products of the Maillard reaction than glucose in tissues.
Therefore, it is suggested that fructose in the polyol pathway may accelerate diabetic complications via the Maillard reaction.

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We have developed a reversed phase high performance liquid chromatography for determining serum and urinary creatinine levels using 500mM phosphate buffer (pH 7.0) as a mobile phase and a BSA-ODS column (5μm, TSK: 4.6 I. D.×150mm). Retention times of creatinine (6.3min) in serum and urine samples were well correlated with the pH and concentration of phosphate buffer. Creatinine was well separated from other substances in samples under the conditions of pH 7.0 and 500mM phosphate buffer. This method also enabled the direct injection of serum and urine samples onto column. The minimum detection limit of this method was about 0.10mg/dl of creatinine. Intra-assay coefficients of variation were under 3.0 and 3.1% for serum and urine, respectivery. Recovery rates were about 90-100% for serum and urine, respectivery. Correlation of creatinine values showed r=0.988 for serum samples (n=39) between the proposed HPLC and the creatinine deiminase method, and r=0.997 for urine samples (n=43) between the HPLC and the Jaffé's method, respectivery. This proposed HPLC may be especially recommended for determination of a creatinine clearance rate.

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We have developed an HPLC system for analysis of catecholamines, serotonin, and their related compounds in body fluids. This system consists of an ODS reversed-phase column, a newly developed twin-electrode voltammetric-amperometric detector with two glassy-carbon working electrodes, and an integrator. The lower limits of detection were 8pg for NE, E, DA, DOMA, DOPA, DOPAC, and VMA, 15pg for MHPG, NMN, and 5-HIAA, and 20pg for HVA and 5-HT. Using cerebrospinal fluid (CSF) as sample, the between-day CVs of MHPG, 5-HIAA, and HVA were less than 5%. We have determined MHPG, 5-HIAA, and HVA in CSF from patients with various diseases and characteristic changes in the concentrations of these substances for each disease.

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We investigated the neonatal screening procedure for adenine phosphoribosyltransferase (APRT) deficiency in dried blood, collected on filter paper. We examined APRT activity of bried blood specimen by fluorescent spot test and microfluorometric analysis. APRT spot test was easy to perform and rapid method, and enabled simultaneous measurements of many samples. And, all APRT deficiency samples gave the positive result in this test. On the other hand, the APRT activity measured by the quantitative method using fluorescence microplate reader. The spot test and microfluorometric method should be recommended for the APRT deficiency screening tests in order to pick up lower activity samples.

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We herein describe a procedure for manufacturing the human serum based control specific. f or lipid assay, applying the precipitation method by Burstein et al.
The control serum is prepared by reconstituting a lyophilized LDL and VLDL depleted serum with distilled water and adding a liquefied LDL and VLDL components to it when in use.
The advantageous point this procedure is that the concentration of each lipid component can be changed according to the requirement. The control serum can equally be compared with fresh human serum in its transparency, electrophoretic characteristics and recovery of lipids by ultracentrifugal analysis.
Furthermore the enzymatic analyses of triglycerides, total cholesterol and phospholipids in the control serum the similar reaction kinetics as those of fresh human serum.

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[in Japanese]

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