Horseradish peroxidase (HRP)-labeled porcine insulins (GlyA1-HRP-insulin, Lys^B29-HRP-insulin and Gly^A1, Lys^B29-diHRP-insulin) have been evaluated for their efficiency in solidphase antibody competitive enzyme-immunoassay (EIA) of human insulin using a mouse anti-human insulin monoclonal antibody that recognizes the A8-A10 loop determinant of insulin. The amount of Lys^B29-HRP-insulin bound to the antibody coated on a polystyrene ball is greater than that of GlyA1-HRP-insulin and much greater than that of Gly^A1, Lys^B29-diHRP-insulin. Two EIA methods, by simultaneous additiion and delayed addition of HRPlabeled insulin, are described for the assay of insulin. Lys^B29-HRP-insulin and Gly^A1-HRPinsulin afforded higher sensitivity in detecting insulin than the diHRP insulin. Using Lys^B29-HRP-insulin, which provides the highest sensitivity, the detection limits for insulin are 1.0 and 0.1 μU, respectively, in the simultaneous and delayed addition method. HRP activity is measured fluorimetrically with 3-(p-hydroxyphenyl) propionic acid and hydrogen peroxide.

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A simple and highly sensitive high-performance liquid chromatographic method for the determination of free cholestanol and cholesterol in human serum is described. After extraction of the steroids from serum sample (5μl), spiked with 1-eicosanol (internal standard) with n-hexane, these steroids and the internal standard are converted into the corresponding fluorescent derivatives by reaction with 3, 4-dihydro-6, 7-dimethoxy-4-methyl-3-oxo-quinoxaline-2-carbonyl azide. The derivatives are separated on a reversed-phase column (YMC Pack C₈) with isocratic elution using acetonitrile-methanol-water (81: 9: 10, v/v/v). The detection limits for; cholestanol and cholesterol were 162 fmol and 144 fmol/5μl in serum, respectively, at a signal-to-noise ratio of 3. The concentrations of free cholestanol and cholesterol in normal sera and a cerebrotendious xanthomatosis serum were compared with those of total cholestanol and cholesterol.

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A novel enzymatic simultaneous assay of phenylalanine and tyrosine using phenylalanine-and tyrosine-decarboxylases was developed. The two enzymes were absorbed in a small piece of filter paper, dried and placed in a test tube, followed by the addition of sample solution. After the whole quantity of the sample was absorbed into the filter paper, the tube was incubated, then t-butylmethylether containing diethylamine was added to stop the reaction and to extract the reaction products, β-phenylethylamine and tyramine. Fluorescence intensities of β-pheylethylamine and tyramine in the organic phase were then measured at 278 nm and 305 nm of emission with excitation at 255 nm, and 275 nm, respectively.

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We have developed a highly sensitive system of high-performance liquid chromatography (HPLC) consisting of an electrochemical detector and an automatic pretreatment device.
Using this system, basic studies on the mesurement of plasma free catecholamines (CA). i.e., noradrenaline (NA), adrenalin (A), and dopamine (DA) were carried out, their normal values were determined, and their clinical significance in various diseases such as hypertension was evaluated. The coefficient of variation (CV) for the simultaneous reproducibility of this method was 2.0-5.1%. The CV for day-to-day precision was 3.5-4.3%, and the recovery rates were 88-100%. A comparison of analysis of NA, A on this method with the HPLCfluorescence method (trihydoroxyindole method) shows that correlation for each are very high (correlation coefficients>0.9). The normal values obtained using this method (n=60) were (pg/ml): 160.1±70.5 (±SD) for NA, 30.1±15.2 for A, and 9.1±3.5 for DA. Loading tests in the standing position in patients with hypertension showed significant differences between NA, renin, and aldosteron values before loading and those after loading (p<0.05). Londing tests in other diseases also showed the usefulness of NA as a diagnostic index. This method is more sensitive than the conventional method and the limit concentration for detection (signal/noise=3) was considered to be 5pg/ml for DA. In addition, since no drug for deproteinization are used, free CA only can be measured without hydrolysis of conjugated CA. The handling and maintenance of the measurement system are also simple. This method seems to be appropriate for routine examinations of plasma Catecholamime levels.

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We developed a rapid enzymatic method forthe measurement of plasma branched chain amino acids based on the following principle. The branched chain amino acids are converted to a-ketoacids by a leucine dehydrogenase coupling the reduction of NAD. NADH converts 3-(4, 5-dimethy1-2-thiagolyl)-2, 5-diphenyl-2H tetrazolium bromide to formazan which is measured, with the catalyst of 1-methoxy-5-methylphenazium methylsulfate. The method allowed measurement of the branched chain amino acids in the visible range. In addition, application to various automated analysis system was possible. The impression of the reproducibility was 2% or less and the measurement was not affected by other amino acids. The obtained values correlated well with those of amino acid analysis by HPLC (y=0.967x+9.75, r=0.981), showing excellent precision of the method.

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Specific antibody concentration in human sera was determined by the end point method using double antibody solid phase radioimmunoassay (SP-RIA). The concentration of standard IgG at the end point was measured with sandwitch SP-RIA, and used as standards for determining antibody concentratlons in human sera. Error extents of the sensitivity of double antibody SP-RIA showed within 10-fold ranges, and its minimum detection levels gave the values from 1.42 to 4.25ng/ml. When the SP-RIA was applied to determine human antibody, 11 of 102 samples were judged as anti-FBS antibody positive samples. The positive distribution ranges revealed the values from 50.5 to 455.5ng/ml of anti-FBSantibody concentrations.

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A new enzymic method for blood levels of pyruvate kinase (PK) activity has been developed for the emergency clinical use. After preincubation of 20μl serum (or plasma) with 1 ml solution of enzyme and substrate at pH 7.4 for 3 min, PK activity was measured as increase of absorbance at 340 nm from 5 min to 10 min. Time-dependent increase of PK activity was observed from 6 min to 30 min, and a linear dilution curve was obtained by diluted human muscle PK and patient serum. intra- and inter-assay variations wereC.V.=3.0% and 2.4%, respectively. Analytical recovery of enzymic PK method was 93-107%. A significant correlation was noted between enzymic and colorimetric assay.
In 95 normal subjects, normal value of PK activity was 102±2.2 (mean±SEM) mIU/ml. Apparent increase of serum PK levels were observed in acute transmural myocardial infarction, with the mean peak value was 289±22.1 mIU/ml, which was 3 fold over than normal. Although serum levels of CK were elevated in subjects with hypothyroidism and post operation of chest-open surgery, serum levels of PK were not elevated.
These results indicate that the present assay kit for serum level of PK is clinically useful for the emergency diagnosis and follow up of myocardial infarction when measured simultaneously with CK.

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Assaying conditions of γ-Glutamyltransferase (γ-GTP EC.2.3.2.2) activity in serum were studied by using L-γ-glutamy1-3-hydroxymethy1-4-nitroanilide as substrate. This new substrate is readily soluble even in a neutral aqueous solution, and the substrate solution is stable at least for five months at 5°C. The measurment of γ-GTP activity was carried out under the conditions of pH 8.2, 67mmol/l glycylglycine and 1.5mmol/l substrate. γ-GTP activity in human serum was determined as almost identical value with that obtained under the conditions recomended by Scandinavian Society of Clinical Chemistry (S. S. C. C.) using L-γ-glutamyl-4-nitroanilide. Further, the values of γ-GTP activities obtained from pig or bovine kidney by proposed method also closely approximated to that measured by S. S. C. C. method. The results of determination by using Hitachi automatic analizer model 7050 were as follows; the dynamic range was 0-2,000U/l, the reproducibility of a sample (X=66U/l) presenied S. D. =0.63, C. V. =0.95% and a good correlation with two recomended methods that are S. S. C. C. and l. F. C. C. was demonstrated for serum γ-GTP activity (r=0.999).

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