基于丙酮酸/还原型辅酶Ⅰ/乳酸脱氢酶/氧化型辅酶Ⅰ/乳酸正逆反应同时测定丙酮酸/乳酸的酶荧光毛细管分析  

Enzyme Fluorescence Capillary Analysis for Simultaneously Determining Trace-Level Pyruvate and Lactate Based on Forward and Reverse Reactions of Pyruvate/NADH/LDH/NAD^+/Lactate System

作　　者：李永生[1] 杨微[1] 李乔婧[1] 周朗[1] 高秀峰[2] 

机构地区：[1]四川大学化学工程学院,成都610065 [2]四川大学基础医学与法医学院,成都610065

出　　处：《分析化学》2011年第7期1058-1064,共7页Chinese Journal of Analytical Chemistry

基　　金：四川大学214振兴计划基金项目(No.0082204127098)资助

摘　　要：利用丙酮酸(PA)/还原型辅酶I(NADH)/乳酸脱氢酶(LDH)/氧化型辅酶I(NAD+)/乳酸(LA)荧光反应体系的正逆反应,建立了一种可直接用于临床检验、能同时测定血清中微量PA/LA的酶荧光毛细管分析法。本方法可在常规荧光光度计上,用普通玻璃毛细管同时实现了PA/LA的高灵敏分析,每次分析试剂和样品的用量仅9μL,而且方法重现性好,血清样品预处理操作简单。得到的最佳条件:60μmol/L NADH,100 U/L LDH,3 mmol/L NAD+,测定乳酸的pH值为9.0、丙酮酸的pH值为7.5,反应时间10 min;LA的线性范围是0.05～1.0 mmol/L;检出限为0.023 mmol/L;RSD<1.6%;PA的线性范围是4～80μmol/L;检出限为0.73μmol/L;RSD<1.3%;血清中PA和LA的添加回收率在94%～105%之间;测定结果与常规紫外分光光度法一致。A new enzyme fluorescence capillary analysis for simultaneous determination of trace-level pyruvate（PA） and lactate（LA） contents in micro-volume serum（P/L-LE-FCA） was proposed by means of forward and reverse reactions of pyruvate/reduced form of nicotinamide-adenine dinucleotid（NADH） /lactate dehydrogenase（LDH）/nicotinamide-adenine dinucleotid（NAD＋）/lactate system.This method＇s has realized high-sensitive determination of PA and LA using a common glass capillary and a capillary holder on conventional fluorophotometer,and dosage of reagent and sample was only 9μL at one-time test,moreover,its reproducibility was better and pretreatment procedure of the serum samples was very simple.The optimum experimental conditions were as follows： the concentrations of NAD＋ and LDH as well as NADH were 3 mmol/L,100 U/L and 60 μmol/L,respectively;the pH of buffer solution was 9.0 for LA test and 7.5 for PA test;the reaction time was 10 min.For determination of LA,its linear range was 0.05-1.0 mmol/L and detection limit was 0.023 mmol/L and RSD was 1.6%（n=11）;for the determination of PA its linear range was 4-80 μmol/L and detection limit was 0.73 μmol/L and RSD was 1.3%（n=11）.Recovery of the method was 94%-105%.Results determined by using P/L-LE-FCA and UV method were well coincident each other.The characteristics of our method are that it realized simultaneous determination of trace-level PA/LA at normal fluorophotometer,and its sample′s pretreatment is simple and timesaving,it has higher sensitivity and better reproducibility,and can be directly used for the measurement and research on the LA/PA content in trace sample in the clinical testing.

关 键 词：荧光毛细分析 丙酮酸 乳酸 乳酸脱氢酶 血清 

分 类 号：R446.1[医药卫生—诊断学]