Optimal reaction conditions for assaying human serum lactate dehydrogenase (LDH EC 1.1.1.27)(lactate-to-pyruvate) were established for purified isoenzymes 1, 3, 5 and whole sera at 30°C in diethanolamine (DEA) buffer.(1). The optimal reaction conditions with equal efficiency for each isoenzyme were pH 8.8, 60 mmol/L lactate, 6mmol/L NAD⁺, and 300 mmol/L DEA at 30°C.(2). The pH optima were within the range of 8.8-9.4 for LDH 3 and 5, >9.0 for LDH 1.(3). Optimal concentration of DEA buffer ranged from 200 to 400 mmol/L for all isoenzymes.(4). Optimal concentration of L-lactate were 20 mmol/L for LDH 1, 60 mmol/L for LDH 3, and>100 mmol/L for LDH 5, respectively.(5). Optimal activity was obtained at a NAD⁺ concentration of 6 mmol/L for all isoenzymes.(6). Initial velocities were most accurately determined within 75 sec after a lag time of 25 sec.(7). Higher LDH activity was obtained.when the reaction was initiated with the enzyme.(8). The standard curve for this assay is linear to 350 U/L of serum.(9). Coefficients of variation within a run and between days were 1.6% (379.6 U/L) and 2.1% (367.4 U/L), respectively.(10), Normal range of LDH activity in fresh serum was 60-120 U/L.(11). The order of storage stability of serum LDH was-60°C>-20°C>25°C>4°C.

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A rapid method for measuring serum cortisol has been developed by reversed phase high performance liquid chromatography (HPLC) with a column switching technique. The precolumn and analytical column used were BSA-ODS columns (20μm, TSK; 4.6 I.D. ×10mm) and (5μm, TSK;4.6 I.D.×150mm), respectively.Both columns were connectd to a high pressure switching valve (7000, Rheodyne). A serum sample (200μl) was directly injected onto the precolumn, from which serum cortisol was eluted. The eluate was transferred to the analytical column and separated, monitoring the absorbance at 245 nm. A peak corresponding to cortisol was observed at the retention time of 28.2min on the chromatogram and well separated from other glucocorticoids except for prednisolone. In the precision tests, the coefficients of variation (C.V.) for the intra-run and inter-run assays were found to be 3.48% and 2.84%, respectively. The recovery rate of cortisol was almost quantitative. The detection limit of cortisol in serum was 1μg/dl. The standard curve showed linearity in the range of 20 to 200μg/dl.
The proposed method for measuring serum cortisol requires only a small volume of the sample and no internal standard.

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In order to analyze immunoreactive tissue kallikrein (TK) in human plasma, three kinds of enzyme-linked immunosorbent assay (ELISA) systems for human urinary kallikrein (HUK) were developed. All three types of TK in the plasma, i.e., active kallikrein, proka-Ilikrein and kallikrein-aiantitrypsin (α₁AT) complex could be measured by competitive ELISA (C-ELISA). However, sandwich ELISA (S-ELISA) showed more strict specificity. Namely, both active and prokallikreins were measured by S-ELISA, while kallikrein-aiAT complex was hardly measurable. In this ELISA system, kallikrein-α₁AT complex bound to anti-HUK antibody fixed on the solid phase. Horseradish peroxidase (HRP)-labeled anti-HUK Fab'(second antibody), however, hardly reacted with this antigen-antibody complex on the solid phase probably due to some steric hindrance. Measurement of this kallikrein-aiAT complex was made possible by using HRP-labeled anti-α₁AT IgG instead of HRP-labeled anti-HUK Fab' as the second antibody (HS-ELISA).
Using these ELISAs we analyzed TK in the plasma and the following observations were made. First, the greater part of TK in the plasma was found in a form of a complex with α₁AT.Second, the amount of free type TK was very small and the most part of this type TK in normal plasma was prokallikrein.

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We evaluated an automated clinical analyzer, the Du Pont aca® SX with two commercial control sera (Du Pont Liquid Multi-analyte Calibrator and Ortho Tri-level TDM Control). Analytical recovery was 80-113% throughout the assays of 16 drug items [Du Pont Liquid Multi-analyte Calibrator (6 items), 80-113%; Ortho Tri-level TDM Control (16 items), 88-110%]. Within-run reproducibilities for the assays of 14 items in both commercial control sera were less than 10% of the coefficient of variation (CV), whereas the CV's for amikacin and gentamicin were 14.8% and 25%, respectively. Between-day reproducibilities for the assays of 14 items were small, and were not significant in comparison with those of the within-run assays. The concentrations of none of the items in either control serum changed during storage at 4 or -220°C for 30 days.
We concluded that the automated clinical analyzer Du Pont aca® SX is useful for routine assay of serum drugconcentrations based on the results obtained with the Du Pont Liquid Multi-analyte Calibrator and Ortho Tri-level TDM Control. Moreover, the Du Pont aca® SX was found to be a useful instrument for emergency assays of serum drug concentrations because is based on a rapid, simple, and accurate method.

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We prepared two control sera for lipoprotein fractionation testing. The first was frozen in liquid nitrogen and the other was lyophilized with high concentrations of sucrose (wieland's method). This paper presents the electrophoretic and ultracentrifugal characteristics of these two sera.
1) The freezing temperature was critical in the liquid nitrogen method. In ser sera frozen at temperatures above-80°C, partial degradation of the lipoproteins was observed accompanied by a decrease in the pre-beta lipoprotein fraction and an increase in the beta lipoprotein fraction. Furthermore, parts of both fractions remained at the electrophoretic origin as an extra band.
No changes in electrophoretic patterns were observed, even in sera with abnormal lipoproteins, except for lipoprotein X. Liquid nitrogen freezing kept serum lipoproteins stable for 12 months.
2) Wieland's method required over 400mmol/l of sucrose to preserve the electrophoretic patterns of serum lipoproteins. Large quantities of lipoprotein X degraded and moved to the beta-lipoprotein fraction. Anyone who uses this must correct the concentrations of determinants by flame photometry.
Moreover, sera prepared by this method were unsuitable for HDL-cholesterol measurement by the heparin-manganese method. Lypohilized sera were stable for 10 months at -4°C.

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Ascorbic acid, a strong reductant in serum or urine, is known to interfere with the determination of hydrogen peroxide (H₂O₂).
This report describes the redox reaction between H₂O₂ and ascorbic acid in various buffer solutions, and the chemical approaches to protect the H₂O₂ from the above reaction.
It was found that H₂O₂ was reduced by ascorbic acid and ascorbic acid was oxidized by H₂O₂ in phosphate (pH 6.0-8.0) or tris-HCl (pH 7.5-8.5) buffer solution, while the redox reaction did not occur in citrate (pH 5.5-6.5) buffer solution. The redox reaction in the phosphate or tris-HCl buffer was shown to proceed more rapidly at an acidic pH than at an alkaline pH.
The degradation of H₂O₂ and ascorbic acid in these buffer solutions was terminated by the addition of chelating agents such as citrate, oxalate or EDTA, or by passing the buffer solutions through a metal chelating adsorbent.
The reaction was found to be accelerated by trace amounts of metals such as Cu²⁺, Fe³⁺or Fe²⁺, contaminants in conventional buffer solutions.

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A method for assaying diamine oxidase (DAO) by high performance liquid chromatography (HPLC) is described. The method is based on a pre-column reaction: DAO oxidizes putrescine or cadaverine of peroxidase, hydrogen peroxide oxidizes serotonin (5-hydroxytryptamine; 5-HT) to an oxidized compound. 5-HT is analyzed by HPLC, and DAO activity is measured from decreased 5-HT values. The assay is quantitative and reproducible with a detectable range up to 70 mU/ml. The intra-assay coefficient of variation (C.V.) was 5.5% with a recovery of 97.0% at the DAO activities of 11.4, 28.9, and 45.6 mU/ml. A close correlation was found between the results of the present method and the 2, 2'-azino-di (3-ethylbenzthiazoline-6-sulfonic acid) method.

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