第1報 四塩化炭素中毒時に於ける血液酵素の研究
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By zone electrophoresis of plasma and hemolyzed erythrocyte solution, distribution of phosphomonoesterase, peptidase and cholinesterase in protein fractions was tested in normal rabbits and rabbits suffering fiom CCl_4 poisoning. Potato starch as described by Kunkel was employed as the supperting medium. Experiments were performed at about 10℃, using a voltage of 150V. which furnished a current of 7ma. and gave adequate separation of enzymes in 20 hours. At the experiment with plasma, M/20 veronal mixture of pH 8.6 was used and origin was set at 15〜16cm. from the cathodic end of the cell. To certify the position of separated plasma proteins, a filter paper was piessed on the surface of the starch block and this moistened paper was then stained; with bromophenolblue procedure. The starch block was sliced into albumin, α_1-globulin, α_2-globulin, β-globulin and γ-globulin segments. Each segment was extracted with equal volume of saline solution for estimation of enzymes. Electrophoresis of hemolyzed erytherocyte solution was carried out at pH7.0 with M/20 veronal buffer, and origin was set at 15〜16cm. from the anodic end of the cell. After 20 hours, the starch block was cut into segments of 3cm. wide and each segment was extracted with equal volume of saline solution. The substrates used were as follows : p-nitro-phenylphosphate for phosphomonoesterase (determination of liferated p-nitrophenoF), leucyl-m-aminobenzoate or glycyl-m-aminobenzoate for peptidase (determination of aminobenzoate by diazo methoe) and acetylcholine-HCl for cholinesterase (determination of remaining ester by hydroxylamate method). In the case of normal plasma, the maximal activity of alkaline phosphomonoesterase was observed in the segment of arand α_2-globnlin; peptidase was distributed equally in the β-globulin and albumin segments, and that of cholinesterase was found in the γ-globulin segment. Of the enzymes in hemolyzed erythocyte solution, acid phosphomonoesterase migrated toward the cathode, though its ztrne was far behind that of hemoglobin, and cholinesterase remained near the origin, while peptidase moved toword the anode. CCl_4 poisoning caused no change in the activities of erythrocyte enzymes above mentioned, while in the plasma the activities of those were increased. The pattern of migration of plasma enzymes remained unaltered, except that the increase of peptidase was visible in β-globulin fraction. The appearance of β-gloucosidase and arginase was observed. Measurement of the. former was based on its activity to hydrolyze p-nitrophenyl-β-glucoside, While in the case of the latter was cairied out by enzyme the remaining arginine was estimated by modification of Sakaguchi reaction. β-Glucosidase wsa found in the γ-globulin fraction, while the distribution of arginase was wide, showing the peak at the point of origin, extended on the anode side so far as to α-globulin fraction, but on the cathode side only slightly beyond the origin. Arginase electrophoretically separated could be activated by Mn^<++>. Excretion of β-glucosidase in urine was not observed. The increase of phosphomonoesterase and cholinesterase and the appearance of β-glucosidase and arginase in CCl_4 intoxication was already known. The increase of peptidase was certified by the present experiment. The result should be attributed to the migration of enzyme protein from the liver into blood. (2) Quantitative Determination of Serum Arginine and Glycocyamine The change in the amount of blood and urine arginine as the result of the appearance of arginase in blood was studied in rabbits suffering from CCl_4 poisoning. For measurment of arginine and glycocyamine in serum, serum was deproteinized by trichloroacetic acid, since minute amounts of high molecular substance which gives positive Sakaguchi reaction could not be precipitated by sulfosalicylic acid-oxine solution of the modified Sakaguchi's method. The filtrate was adjusted to pH 9 and to it arginase solution was added, prepared from hog liver, and before and after arginase action, the Sakaguchi reaction modified by Watanabe was carried out and the extinction was determined. The liver arginase solution specifically hydrolyzed arginine. The difference of extinction indicates the amount of arginine. From the extinction attained after the addition of arginase the concentration of glycocyamine was calculated. For estimation of arginine and glycocyamine in urine urea and other Sakaguchi reaction interfering substances were removed by elution with sodium hydroxide of the guanidino compounds absorbed on Dowex 50 (×4, mesh 100〜200). Differential determination of arginine and glycocyamine was carried out also by application of arginase. Paper chromatography was carried out to as certain whether the substances in serum arid urine which gave a positive Sakaguehi color reaction were not other than arginine and glycocyamine. For chromatography, serum was deproteinized with perchloric acid, excess perchloric acid in the filtrate was removed as potasium base, and the solution was concentrated in desiccator. The urine which had been made free from interfering substances by ion exchange resin procedure was also concentrated for application to chromatography. When the paper was sprayed with the reagents of the modified Sakaguehi reaction, only the spots of arginine and glycocyamine were revealed on the paper chromatogram. Arginine levels in normal labbits serum ranged from 1.88mg% to 5.46mg%, with an average of 2.96mg%, and serum glycocyamine levels were from 1.11mg% to 1.76mg%, with an average of 1.30mg %. In rabbits suffering from CCl_4 poisoning serum arginine levels were lower than in normal rabbits, andrangedfrom 0.19mg% to 0.35mg %, with an average of 0.25mg %, while serum glycocyamine levels were within normal ranges, and from 0.95mg% to 1.74mg% with an average of 1.31mg%. The serum of CCl_4 poisoning showed, when allowed to stand at a neutral pH at 37℃, a gradual decrease of arginine with time. When arginine was extra added it, could be hydrolyzed, while neither arginic acid, agmatine nor glycocyamine were attacked. In the urine of.toxicated rabbits arginase was not detected. The ratio of the amount of arginine to that of glycocyamine was 1:2 in the urine of normalrabbits, while it was 1:7 in the case of toxication. Exceretion of arginine seems to be decreased in intoxication. Therefor, 'the decrease of serum arginine might be due to its hydrolysis in blood by arginase caused to appear in it by CCl_4 poisoning. Arginnie in the serum from patients with cancer was 1.78mg% in average of three cases and showed a tendency to be below the normal level of 2.05 mg. %, but glycocyamine was 1.15mg% and within normal ranges. Arginase was not detected in the serum and urine of the patients.
- 1958-11-28
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