Kimura S, Iyama S, Yamaguchi Y, Hayashi S, Fushimi R, Amino N
Central Laboratory for Clinical Investigation, Osaka University Hospital, Japan.
Clin Chem. 1996 Aug;42(8 Pt 1):1202-5.
We established a simple and rapid kinetic assay for measurement of calcium in serum by using urea amidolyase (EC 3.5.1.45) from yeast species. The method is based on inhibition of the enzyme by calcium. In the assay, we eliminated endogenous ammonium ion by use of glutamate dehydrogenase (GLDH; EC 1.4.1.4); then in the presence of urea amidolyase, urea, ATP, bicarbonate, magnesium, and potassium ions, ammonium ion production was inversely proportional to calcium ion concentration in serum. The concentration of ammonium ion formed was determined by adding GLDH to produce NADP+ in the presence of 2-oxoglutarate and NADPH; we then monitored the change of absorbance at 340 nm. The within-run CVs of this method were 1.7-3.2% (n = 10) at 1.53-3.08 mmol/L, respectively. Day-to-day (total) CVs were 2.8-4.1%. Analytical recovery was 92-112%. The presence of other ions, ascorbic acid, reduced glutathione, bilirubin, hemoglobin, citrate, lipemic material, or human serum albumin did not affect this assay system. The correlation between values obtained with our method (y) and o-cresolphthalein complexone method (CPC) (x) was: y = 1.001x + 0.077 mmol/L (r = 0.949, Sy[symbol: see text]x = 0.079, n = 100); with the other enzymatic method (x) it was: y = 0.952x + 0.021 mmol/L (r = 0.955, Sy[symbol: see text]x = 0.074, n = 100). The SEs for each method were: 0.025 mmol/L, our method; 0.023 mmol/L, CPC method; and 0.025 mmol/L, the other enzymatic method.
我们利用酵母中的脲酰胺酶（EC 3.5.1.45）建立了一种简单快速的血清钙动力学检测方法。该方法基于钙对该酶的抑制作用。在检测中，我们利用谷氨酸脱氢酶（GLDH；EC 1.4.1.4）消除内源性铵离子；然后在脲酰胺酶、尿素、ATP、碳酸氢盐、镁离子和钾离子存在的情况下，铵离子的产生与血清中钙离子浓度呈反比。通过加入GLDH在2-氧代戊二酸和NADPH存在下产生NADP⁺来测定形成的铵离子浓度；然后我们监测340nm处吸光度的变化。该方法在1.53 - 3.08 mmol/L时批内CV分别为1.7 - 3.2%（n = 10）。日间（总计）CV为2.8 - 4.1%。分析回收率为92 - 112%。其他离子、抗坏血酸、还原型谷胱甘肽、胆红素、血红蛋白、柠檬酸盐、脂血物质或人血清白蛋白的存在不影响该检测系统。我们的方法（y）与邻甲酚酞络合酮法（CPC）（x）所得值之间的相关性为：y = 1.001x + 0.077 mmol/L（r = 0.949，Sy[x = 0.079，n = 100]）；与另一种酶法（x）的相关性为：y = 0.952x + 0.021 mmol/L（r = 0.955，Sy[x = 0.074，n = 100]）。每种方法的标准误分别为：我们的方法为0.025 mmol/L；CPC法为0.023 mmol/L；另一种酶法为0.025 mmol/L。
