When the activity of a pure LDH preparation was measured in a reaction system consisting of NAD, lactate, an electron carrier and 2-(p-lodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium Chloride (INT) a tetrazolium salt, and estimated from the amount of formazan formed, the activity values gained were much lower than those determined by the UV-method. On the other hand, when the activity of the pure LDH was determined in the colorimetric assay system, to which either Triton X-100 or heated human serum was added, formazan formation was markedly increased and the activity value estimated from its amount was consistent with that determined by the UV-method. This action of Triton X-100 and heated human serum on the formazan formation was investigated using 9-dimethylaminobenzo-α-phenazoxonium chloride (Meldola's Blue, MB), phenazine methosulfate (PMS) and 1-methoxy-5-methylphenazinium methylsulfate (1-M-PMS) as electron carriers. It was found that when INT was omitted from the colorimetirc LDH assay system with or without either Triton X-100 or heated human serum, a large amount of hydrogen peroxide (H₂O₂) was produced. This finding indicates that in the formazan formation based on the reduction of INT by NADH via electron carrier, the electron carrier reduced by NADH reacted not only with INT, but also easily with molecular oxygen (O₂). It is assumed, therefore, that Triton X-100and heated human serum enhance the reactivity of redu ced electron carriers with tetrazolium salts by changing the redox potentials of the tetrazolium salts, resulting in a marked increase in formazan formation.

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A simple and sensitive method for direct determination of whole-blood iron without deproteinization is described. Whole-blood iron was split and oxidized by the treatment of diluted blood with periodic acid solution. Ascorbic acid solution was then added to this solution to reduce the oxidized iron (Fe³⁺) to Fe²⁺. 2-Nitoroso-5-(N-propyl-N-sulfopropylamino) phenol solution added to the reacted mixture formed a complex with Fe2+ which exhibited a peak absorption at 745nm by addition of ammonium acetate solution. The method was suited to the determination of whole-blood iron at concentrations of 1-100mg/dl. The C. V. in within-run precision was 1.83% with the recovery of 101.7%. A close correlation was found between this method and other employed methods.

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A new sensitive and simple method for color-developing determination of plasma total bilirubin is described. This method is based on oxidative reaction involving formaldehyde. In the presence of hydrogen peroxide, peroxidase in horseradish shows a peroxidase activity and catalyzes the formation of a deep greenish blue dye with an absorption peak at 615 nm, caused by the oxidative reaction with bilirubin and formaldehyde. The proposed method using 5% solution of Triton X-100 in 0.1 M phosphate buffer (pH 7.0) eliminated the interferences derived from blood components. The method was suited to the measurement of bilirubin at concentrations of 0.5-40.0 mg/dl. The C. V. in within-run precision was 1.41% with the recovery of 105.3%. A close correlation was found between this and other employed methods.

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Changes in the levels of γ-glutamyltranspeptidase (GGT) and alkaline phosphatase (AP) were measured in control subjects and in patients with renal and non-renal diseases. The ion retardation chromatographic method was employed for estimating the GGT activity. Both GGT and AP levels were increased in renal disorders associated with nephrotic syndrome. In acute nephritis there was a slight increase or normal levels in both enzyme activities. In renal failure of different etiologies the GGT levels were either normal or decreased whereas in 34% cases the AP activity was increased, Among non-renal diseases, in leukemia there was a significant increase in the levels of GGT in all cases investigated. On the other hand only in about half of the cases, the AP level was found to be increased.

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A systematic study on the changes in levels of γ-glutamyltranspeptidase (GGT), protein and creatinine in amniotic fluid has been made. GGT activity in amiotic fluid showed a gradual decline with advancing pregnancy. Highest enzymatic activity was observed during 12-16 weeks of gestation. The correlation between GGT activity and protein content was found to be significant statistically. On the other hand a significant negative correlation between GGT activity and creatinine concentration was also observed in amniotic fluid. The GGT activity in amniotic fluid at term was comparable to the enzyme level in the maternal serum. The GGT activity in the umbilical-cord serum was found to be 10 fold higher than that of serum and amniotic fluid at term. No variation in the levels of serum GGT during different stages of pregnancy was noticed.

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Phenobarbital was detected by an enzyme-linked immunosorbent assay (ELISA) using an avidin-biotin-peroxidase complex (ABC) technique. The antigen, p-succinamidopheno-barbital conjugated to bovine serum albumin, was adsorbed to wells of a microtiter plate made of polystylene. Rabbit anti-phenobarbital antiserum, samples, biotinylated anti-rabbit IgG antibody, and an ABC reagent were then added to the wells. The absorbance produced by the interaction of the peroxidase activity with a chromogenic substrate was measured at 492 nm.
Less than 1 ng of phenobarbital could be detected by this procedure, and the color development was linearly inhibited up to the addition of 20 ng of phenobarbital. Good correlation between this ELISA and the corresponding radioimmunoassay (RIA) was obtained (r=0.95).

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Mouse monoclonal antibodies against human IgG, IgA and IgM were produced. Monoclonal antibodies were used singly or combinedly. Automated direct turbidimetric procedures, which do not require sample dilution, have been developed for determination of immunoglobulins, IgG, IgA and IgM in serum using a commercial automated chemical analyzer. Three immunoglobulins are measured simultaneously with other routine chemical parameters such as sugars, lipids and enzymes in a single serum sample. Values determined by the new direct automated procedures correlate well with values obtained by single radial immunodiffusion. The within and between assay precisions are lower than 3%. The new method offers great potential for clinical managements of patients.

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A method for the determination of bile acids in normal serum specimen was investigated using a high-performance liquid chromatograph equipped with an automated pretreatment system (Serumout-25, Sekisui Chemical Co.). The pretreatment system could serve for purification and concentration of bile acids in serum within a few minutes. So, it would be possible to determine their contents in normal serum by the system. The presence of 6-12 kinds of bile acids (ursodeoxycholic acid, cholic acid, glycoursodeoxycholic acid, glycocholic acid chenodeoxycholic acid, deoxycholic acid, glycochenodeoxycholic acid, glycodeoxycholic acid, taurochenodeoxycholic acid, taurodeoxycholic acid, lithocholic acid, glycolithocholic acid) was demonstrated in serum specimens from healthy individuals. In these cases, chenodeoxycholic acid and glycochenodeoxycholic acid were always detected, but tauroursodeoxycholic acid and taurolitholic acid were not. This method has proved to be free from the interference of drugs and other constituents in serum, and therefore, is suitable for the reliable determination of bile acids in biological samples.

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An accurate enzymatic procedure to for the determination of blood lactate is explored. The hydrogen peroxide is produced from lactate which is oxidized by lactate oxidase extract from Pediococcus sp. It is oxidatively coupled with 4-aminoantipyrin and N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine sodium salt (TOOS) by peroxidase to yield a chromogen with an absorption maximum at 550 nm.
As the procedure preparing protein-free supernatant, Somogyi method was employed, but with a exception that the order of adding the barium hydroxide soiution and zinc sulfate solution is reversed.
The method requires only 25 μl of ear-lap blood or venous blood and a 15 min. incubation. The present method offers a measurement of high concentration of lactate up to 10 m mol/l blood.
The results obtained by this method show good correlation with those obtained by the LDH-Hydrazin UV method and the LDH-GPT UV method (correlation coefficient is 0.99).
The method has a highly specific that requires a smaller quantity of sample and a shorter time than those previously reported, and has excellent precision.

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A method for the determination of urinary sulfate conjugates of 17-oxosteroids (17OS-sulfates), which can directly determine 17 OS-sulfates without hydrolysis of the sulfate conjugates, is described. The four major urinary 17OS-sulfates are effectively extracted (about 100% recovery) with benzene in the presence of benzyltributylammonium chloride (BTBA). The four chromogens produced by the reaction of 17OS-sulfates with m-dinitrobenzene reagent with BTBA added are quantitatively extracted with diethylether without occurrence of cloudy appearance. The chromogens can then be measured at 515 nm without interferences of contaminants. The proposed method shows a sufficient recovery and a good reproducibility. The values of 17OS-sulfates in urine samples that were determined by Kanbegawa's method after the solvolysis by Barstein and Lieberman's method showed good correlation with the values of 17OS-sulfates obtained by the proposed method.

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A two-dimensional electrophoretic method for the determination of the isoenzyme specifictiy of LDH-binding immunoglobulin in human serum is described. The five isoenzymes of LDH for reference are separated by electrophoresis on a cellulose acetate membrane. The test serum, whose enzyme activity has been destroyed by acid, is then applied as a line over the unstained zymogram, and the second electrophoresis is run in the vertical direction to the first. The interaction between immunoglobulin in the serum and the reference isoenzymes is detected as diffuse trailing, spliting or retardation of bands in the second electrophoresis. lgA-bound LDH isoenzymes are trapped in the precipitin line if immunoelectrosyneresis with anti-lgA antibody is used for the second electrophoresis. The isoenzyme specifities of LDH-binding immunoglobulin have been determined in 8 sera out of 9. The method can be applied also to alkaline phosphatase-binding immunoglobulins.

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A simple, accurate and micro-scale method for the simultaneous determination of procainamide (PA) and N-acetylprocainamide (NAPA) in breast milk and serum using high-performance liquid chromatography is described. Only 50μl of specimen is required. PA and NAPA in the specimen are purified by solid-phase extraction method using CLIN-ELUT and then liquid-liquid back extraction method using dilute phosphoric acid. An aliquot of the acidic aqueous solution is injected directly into the chromatograph. within-run and day-to-day variation for PA at 4μg/ml was below 2.0% and that for NAPA at 12μg/ml was below 1.5%. This method and fluorescence polarization immunoassay have shown a good correlation.
The concentrations of both PA and NAPA in the breast milk were 4.3-6.4 times higher than the maternal serum values. The trace amount of both PA and NAPA was found in serum of an infant fed with milk from mother under PA therapy.

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