血液尿素微量測定法
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概要
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The Principle of this method is hydrolysis of urea by urease, diffusion of produced ammonia into dilute sulfuric acid and colorimetric estimation of ammonia by the previously reported phenol-antiformine procedure. Preparation of urease solution according to Folin and Youngberg was slightly modified as follows. About 1 g. of permutit in a flask is washed once with 2 per cent acetic acid, then twice with water ; 1 g. of jack bean or soya or soya bean meal and 20 ml. of M/50 phosphate buffer (pH 6.8) are added to it. The mixture is shaken gently for 15 minutes, then centrifuged and the supernatant is used as urease solution. One ml. of such solution can split 1 micromole urea at 37℃ in 10 minutes. In the outer chamber of Conway's unit 0.1 ml. of blood is mixed with 1 ml. of the buffered urease solution, and into the inner chamber of the unit 1 ml. of N/100 sulfuric acid is:pipetted. Then the unit is closed and left for 10 minutes at 37℃. Then 1 ml. of saturated potassium carbonate is added to the outer chamber and the closed unit is left in that condition for one hour at 37℃ in order to distillate the produced ammonia into the sulfuric acid in the inner chamber. The acid solution is then transferred by a 0.2 ml. capillary pipette from the inner chamber to a test tube 'graduated at 10 ml., where after the inner chamber is washed thrice every time with 0.5 ml. of water, the washings being transferred successively by the capillary pipette to the test tube. As a blank 0.1 ml. of water is treated simultaneously in the same manner. The solution in the test tubes is now nearly 2.5 ml.. The indophenol reagents are added to them as reported previously and after 20 minutes the blue solution is filled with water to the mark. To measure the extinction coefficient of indophenol blue out of known ammonia quantity, add 1.5 ml. of water, then the reagents to 1 ml. of M/1000 ammonium sulfate for standard and to 1 ml. of water as its blank respectively and dilute the solutions to the mark with water (Photometry by Pulfrich's apparatus.). The calculation of urea content in the tested blood is as follows. Blood urea mg. per 1.00 ml.=E. of test blood-E. of its blank/E. of standard-E. of its blank×60 mg. Similarly 0.1 ml. of serum may be used in the determination of urea, as its concentration is the same in blood and serum.
- 千葉大学の論文
- 1953-09-28