We summarized mutations in human genes of lactate dehydrogenase (LDH) A (M) or B (H) subunits. In a collection of 19 analyzed cases, mutations were demonstrated in 13 (4 in A subunit and 9 in B subunit), including 3 transitions, 5 transversions and 5 deletions/duplications.
Four missense mutations causing LDH deficiency are located at a conserved sequence which are indispensable for the function of LDH molecule, such as the stability of P-axis, nicotinamide adenine dinucleotide (NAD)-binding and substratebinding, Two electrophoretic variants were identified to be due to missense mutations located at codon 6 or 314. These residues are not buried in the subunit molecules but located at the surface of protein. We used a computer model based upon Protein Data Bank to predict the location and function of point mutations of LDH.
Two nonsense mutations, two duplications (8 by and 4 bp) and two deletions (20 by and 1 bp) resulted in premature termination of LDH subunits. Because the premature termination induces serious perturbation and the instability of tertramer formation, the enzyme deficiency may be due principally to the more rapid in vivo denaturation degradation of the variant enzyme.
These mutations are different from each other except two mutations, which are 20 by deletion in exon 6 of LDH-A gene (5 families including 21 individuals) and a missense mutation in exon 4 of LDH-B gene (3 cases). It is concluded that there is considerable heterogeneity among genetic mutations of human LDH genes.

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Polyclonal and monoclonal antibodies to porcine elastase were produced and characterized. Production of monoclonal antibodies was performed by somatic cell fusion. Titers and cross reactivites of the antibodies were studied by enzyme-linked immunosorbent assay (ELISA). Each of these polyclonal (R90-03, R90-04) and monoclonal (MAb653F2) antibodies reacted in dose-dependent fashion with porcine elastase. The affinities, as estimated by cross-reactivities of polyclonal antibodies, were highest for porcine elastase and low for human elastase and trypsin. On the other hand, the affinity of the monoclonal antibody was highest for porcine elastase, intermediate for human elastase and lowest for trypsin. Using ELISA methods, with the antibody capture assay, the quantitative determination of porcine elastase was possible using polyclonal antibodies between the range from 0.8 to 80 ng/ml. The quantitative determination of human elastase by antibody capture assay was also possible with use of the monoclonal antibody between the range from 4 to 80 ng/ml. In addition, the presence of elastase in vascular wall smooth muscle cells was demonstrated and the secretion of smooth muscle cell-derived elastase in normal and arteriosclerotic conditions was studied with fluorescent antibody methods using one of the polyclonal antibodies (R90-04). Findings suggested that the secretion of elastase was markedly reduced in intimal smooth muscle cells in arteriosclerotic lesions. The antibodies produced and characterized in the present study may be useful for future pathophysiological investigation of elastase.

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Elastin peptides (α-elastin and elastase-solubilized elastin Peptide) were prenared from insoluble elastin extracted from normal human aorta. Rabbits were immunized with these elastin peptides. We obtained two antibodies, one each to a -elastin and elastase-solubilized elastin peptide. Using these antibodies, an enzyme immunoassay system for the measurement of elastin peptide was devised, and the competitive binding curve obtained with its use was found to be similar to that obtained for radioimmunoassay. Anti-a-elastin antibody recoanized larger and hydrophobic peptides, whereas anti-elastase-solubilized elastin peptide antibody (anti-elastin peptide antibody) recognized smaller and hydrophilic peptides. The enzymelinked immunosorbent assay (ELISA) using anti-α-elastin antibody was found to be superior to that using anti-elastin peptide antibody for measurement of urinary elastin peptides. The urinar excretion of elastin peptide recognized by anti-α-elastin antibody reflected decree of smokina: the content of elastin peptide in the urine of light smokers (39±22 ng/mg creatinine) was sianificantly less than that in the urine of heavy smokers (188±24 ng/mg creatinine). However, the level of elastin peptide in the urine of light smokers (8.30±0.9μg/mg creatinine) as measured using anti-elastin peptide antibody was not significantly different from that in the urine of heav smokers (7.42±0.95g/mg creatinine). The ELISA for the detection of elastin peptide in human urine using anti-α-elastin antibody described here may be useful for monitoring of elastin metabolism in patients with a variety of connective tissue abnormalities.

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Purification of a recombinant human thyroid-stimulating hormone (rhTSH) was carried out, which was produced in Chinese hamster ovary cells by co-expression of complementary deoxyribonucleic acid for human chorionic gonadotropin (hCG) α and hTSH β subunit as previously described. The cell culture medium was treated by successive chromatographies on S-Sepharose, DEAE-Sepharose, concanavalin-A Sepharose, TSK-G2000SW, and Bio-Gel P-30. Each separation step was monitored by immunoradiometric assay for hTSH. A unique property of rhTSH was observed in the size exclusion chromatography using TSKG2000SW with its flow grossly retarded in the gel. But at the final step by Bio-Gel P-30 column, a single peak of rhTSH with 95% purity was obtained at a position corresponding to the molecular size of pituitary derived authentic hTSH. The overall recovery rate of hTSH immunoreactivity was 10%.
On Western blotting, bands of both the purified rhTSH and a pituitary-derived reference hTSH migrated to the position twice of their molecular weight, showing their tendency of aggregation under a non-reducing condition.
The purification procedures for rhTSH reported here will be applicable to a larger scale production because of its relative simplicity and practical yield.

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In order to establish a speedy and convenient assay of serum CA 19-9, we developed a latex photometric immunoassay of the antigen. Latex reagent was prepared by physical binding of F (ab) 2 fragment of murine monoclonal antibody (1116 NS 19-9) onto latex particles. Latex reagent showed dose-dependent agglutination after being mixed with CA 19-9 antigen. The latex assay required only 10 minutes and could be automated by using conventional clinical chemistry analyzer, COBAS MIRA. Intensity of signal of the reaction could be adjusted by modifying the concentration of reaction accelerating polymer, polyvinyl pyrrolidone K90 (PVP K90). When we used the assay buffer containing 2 g/l of PVP K90, minimum detection dosage was 2 units/ml and measuring range was up to 400 units/ml; they were similar to those of radioimmunoassav (RIA) and enzyme immunoassay (EIA). Within and between assay precisions were both about 5% in coefficient of variation CVI at normal ranae and about 1-2% in CV at increased level. Correlation versus commercially available EIA was y (Latex assay) =1.08x (EIA) +3.41, r=0.981 (n=101). Prozone phenomenon could be detected by utilizing mechanism of the instrument. These results indicate that this assay can be applied to daily analysis in clinical laboratories as a speedy and convenient substitute of RIA and.EIA.

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We described the formation of bilirubin from recombination of intermediates formed in the bilirubin diazo coupling reaction. Two types of intermediates formed in the diazo coupling reaction of bilirubin changed to other yellow compounds, whose absorption spectrum was similar to that of bilirubin, during storage after extraction from the reaction mixture. The yellow substance was separated into three yellow compounds by thin layer chromatography. The RI values of these three compounds were similar to those of bilirubin IIIα, IXα and XIIIα. The relative proportions of formation of these three yellow compounds appeared to be 1: 2.6: 2. These substances reacted with the diazonum salt to form an azo dye and formed a green product with the same absorption spectrum as that of biliverdin upon oxidation with a nitrous acid solution. These findings suggested that three isomers, bilirubin IIIα, IXα and XIIIα, are formed by recombination of endovinyl and exovinyl dipyrrole intermediates.

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A fluorometric method for determining urinary estriol which is the principal estrogen in pregnancy has been developed. The method includes enzymatic hydrolysis of estrogen conjugates with β-glucuronidase from Ampullaria, extraction of free estrogen with dichloromethane followed by fluorometric analysis using a modified Kober color method.
Hydrolysis of estriol 16 β-glucuronide (E3 16G) was completed by an incubation with 1000 Fishman units of Ampullaria β-glucuronidase at 60°C for 5min. The calibration curve for E3 16G was linear in the wide range of 0.5-80mg/l. Reproducibility of within-run assay was CV 2.0-8.4% (n=10). Results obtained by this method were closely correlated with those by the other Kober reaction method and enzyme immunoassay. This method is applicable to the determination of estrogen in pregnancy urine, and useful for evaluation of feto-placental function.

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A new method for measuring bilirubin using vanadic acid as oxidizing agent was studied. A solution of a cation surfactant, cetyltrimethylammonium bromide (CTAB) as the reaction accelerator and that of a reducing agent, hydroxylamine as the reaction inhibitor in citrate or tartarate buffer, pH 3 (the first reagent) were used for measuring the concentration of total bilirubin and direct bilirubin, respectively. A solution of vanadic acid (4 mmol/l) containing ethylenediamine tetraacetic acid (EDTA) or hydroxyethan-di-phosphonic acid, pH 7 was used as the second reagent. The bilirubin concentrations were calculated by a two point method measuring absorbances at a specific wavelength before and after oxidation reaction. Under these experimental conditions, the oxidation reaction was accomplished within 2 to 3 minutes.
This new method had fundamental abilities for the determination, such as an appropriate dynamic range, a high reproducibility and the good correlation with the diazo method. Furthermore, the results were affected very little by coexisting substances in serum.
In addition, such liquid type reagents are very convenient without being prepared for every determination and will contribute to the correction of the day-to-day differences and save works in the laboratories.

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In order to assess the extent of damage to renal tubular peroxisome granules, we developed a method of measuring the urinary concentration of D-amino acid oxidase, an enzyme well known to be present in these granules. A fundamental study was conducted using this method and its clinical application was also assessed.
The standard curve, reproducibility and recovery rate of the assay were all satisfactory. There was no diurnal variation of urinary excretion or any variation between different ages, and the standard urinary D-amino acid oxidase level was <10.0μg/g creatinine.
Measurement of urinary D-amino acid oxidase levels in diabetic patients and patients with renal damage using this method revealed that this enzyme was a more sensitive marker than urinary microalbumin or other parameters, and it was concluded to be useful for detecting early kidney damage.

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