Anti-bovine serum albumin (BSA) and anti-ovalbumin (OA) antibody concentrations in human sera were determined by the end point method using double antibody solid phase enzyme immunoassay (SP-EIA). The concentration of standard IgG at the end point was measured with a sandwich SP-EIA, and used as the standard for determining antibody concentrations in human sera. Error extents of the sensitivity of double antibody SP-EIA as well as solid phase radioimmunoassay (SP-RIA) showed within a 10-fold range, and its minimum detection levels gave values from 0.10 to 0.92ng/ml using 1% BSA-phosphate buffered saline (PBS) as the blocking reagent, from 1.02 to 9.37ng/ml using 0.8% gelatin-PBS as the blocking reagent. When SP-EIA was applied to determine the human sera antibody, 7 of the 24 samples were judged to be anti-BSA antibody positive samples, and 11 of the 24 samples were judged to be anti-OA antibody positive samples. The positive distribution ranges revealed the values from 41.1 to 370.2ng/ml of anti-BSA antibody concentrations and from 41.1 to 1110.5ng/ml of anti-OA antibody concentrations. However, the titers of anti-BSA and anti-OA antibodies had no significant correlation.

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We discovered and reported that the intravenous infusion of maltose interfered with the Lana AG^® kit used for the determination of 1, 5-anhydroglucitol in plasma or serum. The principle of the kit consists of eliminating the interfering substances with a mini-column, an oxidation of the recovered 1, 5-anhydroglucitol by pyranose oxidase and a detection of the hydrogen peroxide with a color development reaction. In normal samples, glucose is a major interfering sugar in the detection system, but it is completely trapped in the mini-column. While a slight quantity of the infused maltose leaked out of the mini-column, the leaked maltose was hydrolyzed into glucose with a slight amount of maltase being present in pyranose oxidase and the formed glucose was oxidised with pyranose oxidase and changed color. To activate the affinity of the mini-column for maltose, we tried some conditioning methods for the mini-column. Washing the mini-column with a 10% ethylene glycol solution was the most effective method for the activation. The infused maltose was complete ly trapped in the conditioned mini-column and did not interfere with the determination of 1, 5-anhydroglucitol.

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An amidolytic assay using a new substrate, H-D-Pro-Phe-Arg-ρ-acetylanilide (KL-12), was designed to measure prekallikrein in plasma, Prekallikrein was converted to kallikrein with Pseudomonas aeruginosa elastase, and the amidolytic response of kallikrein to KL-12 was subsequently measured.
The kinetic parameters of plasma kallikrein as a function of KL-12 or of the ordinary substrate, H-D-Pro-Phe-Arg-ρ-nitroanilide (S-2302), were Km=2.8×10^-4M and k_cat=208s^-1, or Km=2.5×10^-4M and k_cat=176s^-1, respectively, A comparative study of the automated methods employing plasma samples showed good correlation between the assays using KL-12 and S-2302 (r=0.997, n=30, y=0.95x+4.5). The coefficients of variation for multiple measurements with pseudomonal elastase and KL-12 of a plasma sample with low level prekallikrein (106nM, 30% of the normal pooled plasma) within a run (n=10) and between runs (n=10) were CV=2.51% and CV=4.24%, respectively. Prekallikrein concentrations in plasma samples from 80 healthy individuals (M=40, F=40) were measured with the automated method and the normal range obtained by the parametric method was 270-426nM (77-121% of the normal pooled plasma). We have reported that the determination of plasma prekallikrein can be easily automated by the activation of prekallikrein with Pseudomonas aeruginosa elastase and by using a new chromogenic substrate of kallikrein.

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A simplified fluorometric method used for the determination of selenium in serum was described. The serum (0.1ml) was digested with 0.3ml of concentrated nitric acid and 0.5ml of 0.75mol/l perchloric acid (at 160°C, for 60-90min), and reduced with 0.5ml of 6 mol/l hydrochloric acid (at 160°C, for 15-30min). The fluorescence reaction was induced by the addition of 2, 3-diaminonaphthalene reagent to the digested sample, and heating at 50°C for 15min. The produced piazselenol was extracted as cyclohexane and its fluorescence was measured (excitation at 377nm, emission at 520nm). All procedure were performed in a single-test-tube and about 50 samples could be assayed within three hours.
With this method, the detection limit of selenium was 7μg/l (0.7ng/tube), within-series accuracies for a 500μg/l standard solution and serum (133.7μg/l selenium) were 1.79% and 2.11%. and that between-series for serum was 5.94%.
Analytical recoveries of 100ng selenium added to 0.1ml of serum were 90.0-112.0% (n=10). Normal levels of selenium in serum obtained from 42 normal adults (21-60yr, male=8, female=34) were 156.7±42.6μg/l (mean±2SD).

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We described the new enzymatic assay for serum sialic acid, using N-acetyl-D-mannosamine dehydrogenase.
The following results were obtained.
1. Sialic acid could be quantified accurately and precisely.
2. This assay was well suited for screening programs when evaluating significant risk factors for the development of acute myocardial infarction and also when measuring sialic acid in serum used in clinical laboratories.
We recommended that this assay be calculated based on the increase in absorbance at 340 nm without any standard calibration.

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Transfers of cholesteryl esters from high density lipoproteins (HDL) to very low density lipoproteins (VLDL) were examined using VLDL and d>1.063 fractions glycated in vitro. From these results, it was found that the transfer of HDL cholesteryl ester to VLDL was accelerated by glycation. The accelerated transfer of HDL cholesteryl ester was believed to be caused by glycation of the higher density fraction of serum proteins with a density of more than d>1.063, but not by glycation of VLDL.
When sera were incubated with 200mmol/l glucose for 70hr, every lipoprotein fraction was glycated while the HDL fraction was reduced significantly. Therefore. it is suggested that the increased transfer of HDL cholesteryl ester might be related to the reduction in serum HDL levels.
Since HDL₂ concentrations decreased and HDL₃ concentrations did not change after incubation with glucose, the ratios of HDL₂/HDL₃ decreased.

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When sera were incubated at 37°C for 70 hr, cholesterol and phospholipids in high density lipoproteins (HDL) were reduced compared with control sera kept at 4°C, but triglycerides increased significantly. Cholesterol and phospholipids in HDL further decreased concomitantly with HDL levels after incubation with glucose, though HDL triglyceride did not change.
Concentrations of HDL₂ were not affected by incubation and there were no significant effect on other constituents in HDL₂ excluding increased triglyceride. Triglycerides in HDL₂ further increased by the addition of glucose but other HDL₂ constituents and HDL₂ concentrations decreased reversely.
HDL₃ concentrations decreased in both incubated sera with or without glucose because of the decreases of their constituents except increased triglyceride contents. The compositions of HDL₃ were almost similar in both incubated sera.
Cholesterol in d>1.21 fractions decreased and triglycerides as well as phospholipids increased after the incubation with or without glucose. But apoproteins in these fractions were not changed after the incubation, except increased apo C-III protein.
It is concluded that the most remarkable change in HDL by the addtion of glucose to the incubated sera is the decrease of HDL₂ fraction, which might be derived from increased transfer of cholesteryl esters to VLDL. Reduction of serum HDL levels is considered to be not caused from fragility of HDL particles, because lipids and apoproteins in d>1.21 fractions are not influenced by the addition of glucose.

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Measurements of the biochemicals in the serum of patients with liver diseases using the liver function tests involving guanase, multivariate analysis was performed in order to compare such measurements. By principal component analysis, these tests could be divided into the characteristic groups. The first principal component was considered to be relate to the severity of hepatocellular damage which belongs to guanase, aspartate aminotransferase, alanine aminotransferase, mitochondrial aspartate aminotransferase and lactate dehydrogenase. Multiple regression analysis revealed also that aspartate aminotransferase (p<0.001), alanine aminotransferase (p<0.01) and mitochondrial aspartate aminotransferase (p<0.05) were hepatic factors related to guanase. The equation of the multiple regression line was as follows: y_(Gua)=2.43+8.68×10^-3x_(AST)+3.39×10^-3x_(ALT)+6.92×10^-3x_(m-AST).

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Serum bilirubin concentration in a patient with type I Crigler-Najjar syndrome (CNJ) was determined by means of a Kodak Ektachem 700N analyzer, and a questionable presence of delta bilirubin (Bd) was observed. HPLC was used to ascertain whether or not the derived Bd concentration was artifactual. Total bilirubin concentration in the patient was correctly evaluated by the Ektachem method, whereas the measured unconjugated bilirubin was underestimated as compared with that obtained by the HPLC method. Therefore, a questionable Bd value, derived from these results, was observed.

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A simple method for the measurement of the catalase activity, which uses an ordinary platinum electrode covered with a dialysis membrane is proposed. The anodic current measured at+0.6 V vs. saturated-calomel electrode (SCE) is proportional to the concentration of hydrogen peroxide in a solution and is decreased by the addition of catalase to the solution. The initial slope of the time-dependent current decrease after the addition of human blood cells to a test solution is directly proportional to the catalase activity. The catalase activity of normal human blood cells was measured as 4.53 mol/hr·ml^-1 by this method. This method has an advantage over the previously proposed methods using an oxygen electrode, whereby no deaeration of the test solution is required with the present method.

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