血清肌酐二步酶法检测新技术研究

目的探讨二步酶法测定肌酐的实验方法。方法以肌酐酰氨基水解酶为试剂Ⅱ,以N-(2-羟基-3-磺丙基)-3,5-二甲氧基苯胺钠盐(HDAOS)和4-氨基安替比林(4-AAP)色原物作为试剂Ⅰ,建立二步酶法测定血清肌酐。通过内空白法消除内源性肌酸和改变光谱吸收方法消除脂血、溶血、黄疸干扰。采用二步酶法测定36例患者血清肌酐,并与高效液相色谱(HPLC)法和一步酶法比较;同时测定200名健康体检者血清肌酐,以建立参考范围。结果患者组肌酐二步酶法结果(84.2±26.6)μmol/L]明显低于一步酶法(116.6±29.6)μmol/L,t=32.12,P<0.01];与HPLC法测定结果(83.9±26.8)μmol/L]差异无统计学意义(t=0.541 6,P>0.05),且呈良好相关性(Y二步酶法=1.042XHPLC法-0.182 1,r2=0.982 4)。二步酶法测定血清肌酐的线性范围达4 500μmol/L,平均回收率为100.8%,批内和批间变异系数(CV)分别为2.91%～4.20%和3.20%～4.60%,应用二步酶法每天测定室内质控定值血清低(78.0μmol/L)、中(206.0μmol/L)、高(900.0μmol/L)],共测定180 d,其日间总CV分别为4.46%、5.27%、7.24%。二步酶法试剂至少能稳定180 d。健康人群血清肌酐的参考范围男性为56～132μmol/L,女性为41～109μmol/L。结论以肌酐酰氨基水解酶为试剂Ⅱ和以HDAOS、4-AAP为色原物(试剂Ⅰ)的二步酶法可以消除肌酸和脂血、溶血、黄疸血的干扰,可用于临床肌酐常规测定。

Investigation on the new two-step enzymatic method for the detection of serum creatinine

Objective To investigate the two-step enzymatic method for the detection of serum creatinine.Methods The two-step enzymatic method for the detection of serum creatinine was established using creatinine amidohydrolase as the reagent Ⅱ and N-（2-hydroxy-3-sulfopropyl）-3,5-dimethoxyaniline sodium salt（HDAOS）and 4-mino-antipyrine（4-AAP） as the reagent Ⅰ.The inner blank method was used to eliminate endogenous creatine,and altered spectral absorption method was used to eliminate the interference of lipemia,hemolysis and jaundice.The levels of serum creatinine in 36 patients were determined by the two-step enzymatic method.The results were compared with those of high performance liquid chromatography（HPLC） and single-reagent method.The serum creatinine levels of 200 healthy subjects were determined and performed as reference range.Results There was significant difference between two-step enzymatic methodcreatinine：（84.2±26.6）μmol/L] and single-reagent method creatinine：（116.6±29.6）μmol/L,t=32.12,P0.01],whereas there was no statistical significance between two-step enzymatic method and HPLC creatinine：（83.9±26.8）μmol/L,t=0.541 6,P0.05],and showed good correlation（Ytwo-step enzymatic method =1.042XHPLC-0.182 1,r2=0.982 4）.The liner range of the two-step enzymatic method for the detection of serum creatinine was up to 4 500 μmol/L,the average rate of recovery was 100.8%,and the within-run coefficient of variation（CV） and between-run CV were 2.91%-4.20% and 3.20%-4.60%.The sera low level： 78.0 μmol/L,middle level： 206.0 μmol/L and high level： 900.0 μmol/L] were determined by the two-step enzymatic method for 180 d.The interday CV were 4.46%,5.27% and 7.24%.The reagents of the two-step enzymatic method could be stable at least for 180 d.The reference range of serum creatinine in healthy males was 56-132 μmol/L,and that in healthy females was 41-109 μmol/L.Conclusions The two-step enzymatic method based on creatinine amidohydrolase as reagent Ⅱ and based on HDAOS and 4-AAP as reagent Ⅰ could eliminate the interference of creatine,lipemia,hemolysis and jaundice,and it can be applied as routine test.