Serum lipids, apolipoproteins and red cell lipids were investigated in the patients with spur cells or target cells in their peripheral blood. The patients were divided into 4 groups by the levels above and below 15 percent of the both deformed cells. However, the patients with cholestasis were separated into another group, because they had special characteristics in their serum lipids and apolipoprteins.
1.Mean serum total cholesterol concentrations were lower in both patients with spur cell and with target cells than those in normal control, of which degrees were markedly in the groups with higher percentages of abnormal cells. By contrast, cholestatic patients had higher cholesterol level than normal control. Serum phospholipids showed the same tende ncies, but triglyceride was not affected in these patients except high triglyceride level in cholestasis. The cholesterol ester ratio and HDL cholesterol were reduced in all groups, especially in both higher groups and cholestasis.
2. Serum apo A- I and A- II protein concentrations decreased in all suffered subjects. Although apo C-III showed the significant low levels in both higher groups, apo C-II: in cholestatic patients was almost the same level to control. Consequently, apo C-II/C-BI ratio was reversed in cholestasis as contrast to normal control and other groups. Apo B proteins in suffered groups were identical to control except high concentration in cholestasis. Further remarkable increase was observed in apo E protein of cholestatic patients, though other groups had the same mean levels to control.
3.Cholesterol in erythrocytes increased in every groups together with the increase of mean corpuscle volume. But the increases of phospholipids were more pronaunced in target cell groups than in the spur cells. Therefore phospholids/cholesterol ratio decreased in spur cells but not in target cells. The correlation coefficient between this ratio of erythro-cyte and serum phospholipids/free cholesterol ratio was 0.574 except cholestatic group and that between the former and phospholipids/free cholesterol ratio in HDL was 0.734.
4.Increase of the percent phosphatidyl choline and decrease of sphyngomyeline were proven in all groups with abnormal cells. These tendencies were clearly in the higher groups, especially in cholestatic group.
5. The masses of sphyngomyeline in erythrocytes were little affected in the suffered groups but these of phosphatidyl choline increased markedly. The increases were more remarkable in the higher group of target cells and cholestatics than in other groups.
6.Choline esterase activities were shown to decrease in all suffered groups. More abnormalities were found in GOT and GPT tests of target cell groups than in spur cells, but no intimate relationship was suggested between transaminase activities and appearance of the abnormal cells. Serum total bilirubin and bile acid acid concentrations were extremly elevated in both severe groups.

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Glucuronidase and sulfatase which hydrolyzed various conjugates in urine were found to occur in the extracts of digestive juice from Ampullaria and to be useful rapid hydrolysis of steroid conjugates. Ampullaria hydrolases had a good thermal stability, at 60°C, were not inactivated for 3 hours in urine. More than 90% activity of the sulfatase activity was inhibited by Pi in urine, but β-glucuronidase activity was slightly inhibited by urine. Pi could be removed by treating with the magnesium and ammonium solution at pH 11.5. Incubation for 2hr at 60°C of the Pi free urine with 800 U β-glucuronidase and 400 Roy U sulfatase from Ampullaria per milliliter at pH 5.0 resulted in complete hydrolysis of 17-OS conjugates in the sample. This hydrolysis method showed a good recovery of dehydroepiandrosterone (DHEA) sulfate by gas-chromatography. A good correlation between this rapidly hydrolysis method and the long time incubation with Helix pomatia enzyme was obtained expect the value of DHEA.(y=2.806x-0.068 y: Ampullaria x: Helix pomatia) Normal range of DHEA on male was obtained 0.12-5.20 mg/day, this value was higher than that by the other hydrolysis methods.

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When various concentrations of an authentic NADH were added to a solution of 1-methoxy-5-methylphenazium methylsulfate in a sealed vessel equipped with an oxygen electrode, O₂ in the mixture was consumed in proportion to concentrations of NADH added.Conversely, it was possible to make a quantitative estimate of the amount of NADH by measuring O₂ consumption in this reaction system. In addition, H₂O₂ was also produced in the reaction system. The amount produced, determined colorimetrically using peroxidase with 4-aminoantipyrine and ADOS as chromogens, was also equivalent to that of NADH added. Although keeping the reaction solution, which contained a pigment formed, at room temperature led to a rapid discoloration, this discoloration was inhibited by addition of superoxide dismutase.
On the basis of the above results, LDH was assayed by measuring O₂ consumption or H₂O₂ production in the enzymatic reaction using lactate as substrate. The activity obtained by the present method, especially in the case of NADH measurement by means of O₂consumption, showed a good correlation with that obtained by the UV method, indicating that this is a valid new LDH assay method.

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We developed field methods without sample blank reactions to estimate the reference methods proposed by the Japan Society of Clinical Chemistry (JSCC methods) for the measurement of aspartate (AST) and alanine (ALT) aminotransferase activities in human sera.
In our methods, activities were measured at 30°C but there were no dicrepancies between the activites at 30°C and 37°C. Conversion factors from 37°C to 30°C were obtained as 1/1.49 and 1/1.45 for AST and ALT respectively.
AST and ALT activities by our methods were so well correlated to those of the JSCC methods that clinical evaluations of the JSCC methods were made possible by our methods. AST and ALT activities by our methods were also well correlated to those of the conventional Reitman-Frankel's and Karmen's methods in all the patient sera.
Conversion factor of AST/ALT ratios by the conventional methods to the present methods was about 0.7 in healthy subjects and also in the patients with various liver diseases, however, correlation between AST and ALT activities were different and specific in these groups.
Therefore, even in the sera including mitochondrial AST, AST and ALT activities and AST/ALT ratios by JSCC methods were easily converted to those of the conventional methods by the respective conversion factors.

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Using appropriate conditions for head space gas chromatography, we investigated the plasma components which facilitate decarboxylation of acetoacetate. The highest activity was found in the albumin fraction. Our results on various properties (effects of pH, type of Michaelis-Menten plot, Kcat and Km values) differed appreciably from those of earlier investigators.
Weak acetoacetate decarboxylation activity was observed in serum ultrafiltrate. The acetoacetate decarboxylation activity of several amino acids and related compounds was examined: all amino acids showed some activity, but acylated amino acids did not. The activity in serum ultrafiltrate appears to come almost entirely from the amino acids.
Because the acetoacetate decarboxylation properties of intact serum is almost all attributable to serum protein, for accurate acetone and acetoacetate determination, blood sample should be deproteinized before storage.

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We present a micromethod for determining theophylline and its three major metabolites (1 methyl uric acid, 1, 3, dimethyl uric acid, 3 methyl xanthine) in 25 μl serum. Serum sumple was deproteinized by acetonitrile, then concentrated and injected into high pressure liquid chromatograph (HPLC) equipped with ODS reversed phase column run with a mixture of 0.01M sodium acetate (adjusted pH to 5.0): acetonitrile/90: 10 as eluent at flow rate of 0.8 ml/min. Absorption was mas monitored at 275 nm. Theophylline and metabolites were well separated with above chromatographic conditions. Concentrations of three metabolites were about one tenth of theophylline. Accuracy of the method was verified by comparing concentration of theophyline measured by this method with those by enzyme immunoassay (EIA) and fluorescent polarization immunoassay (FPIA). The correlation correlation coefficient (C.C) between this method and EIA was 0.984. The C.C between this and FPIA was 0.987.

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Hyper-lactatemia and lactic acidosis are known to be an important clinical disorder. When blood levels of lactate/pyruvate ratio increased in lactic acidosis, it indicates the serious and poor prognosis of life, and the ratio reflects tissue hypoxia.
A rapid, specific, and simple enzyme electrode method for the determination of pyruvic acid was studied. The principle is as follows: The pyruvic acid is oxidized to acetylphosphate, carbon dioxide, and hydrogen peroxide with pyruvate oxidase in the presence of phosphate ion, thiamine pyrophosphate, and metal ion such as magnesium or manganese. The hydrogen peroxide generated by the enzymatic reaction is detectable using a polarographic electrode. The electrode current is directly proportional to concentration of pyruvic acid.
Pyruvate oxidase was immobilized onto the porous side of a cellulose acetate membrane with asymmetric structure which has selective permeability to hydrogen peroxide in various oxidative materials. Properties of immobilized enzyme membrane, enzyme activity, apparent Km, optimum pH, optimum temperature, pH stability, and temperature stability, were 0.03 U/cm², 9.4 mmol/L, pH 6.5, 37°C, pH 5.0-8.0, and below 50°C, respectively in the given conditions.
The pyruvate electrode was constructed by combination of a hydrogen peroxide electrode with the immobilized enzyme membrane. The pyruvate electrode responded linearly to pyruvic acid over the concentration 0-1.0 mmol/L within 30s on the flow system. When the enzyme electrode was applied to the determination of pyruvic acid in control serum, within-assay precision (CV), analytical recovery, and correlation coefficient between the electrode method and the colorimetric method were 1.1% with mean value of 0.28 mmol, /L, 88.0%, and r=0.930 (Y=1.048X+0.0002, X: colorimetric method), respectively. The dried immobilized enzyme membrane retained 96% of its initial activity after storage at 4°Cduring 15 months. However the pyruvate electrode was unstable to assay the concentration of pyruvate continuously under the conditions at 30°C, pH 6.0 in sodium acetate buffer solution. The apparent output decreased by about 20% after a day.

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The following problems stemed from changes of the molar absorption of 4-nitrophenol (4NP) were found in the assay of a-amylase [EC 3.2.1.1] using 4-NP-maltooligosaccharides as the substrate.
1. The absorbance of 4-NP around 400nm at pH 7.0 varied with temperature and the buffer used.
2. The addition of serum caused a shift to shorter wave length in addition to the variation of the absorbance aronud 400nm. Albumin (4S fraction) seemed to shift the absorbance peak greater than other protein fractions (μ-globulin or γ-globulin).
3. Molar absorptivity of 4-NP should be measured using reagent for α-amylase activity with normal serum.

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