裂解加热-荧光毛细管法快速测定血液红细胞内丙酮酸  

Rapid Determination of Pyruvic Acid in Erythrocytes by Lysing-heating Fluorescence Capillary Method

作　　者：周朗[1] 李乔婧[1] 李永生[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》2012年第11期1725-1729,共5页Chinese Journal of Analytical Chemistry

摘　　要：在常规红细胞处理方法中,用生理盐水对红/白细胞清洗分离会导致细胞内丙酮酸外渗,造成丙酮酸测定值不能反映细胞内丙酮酸的真实浓度。本研究设计了"氯化铵裂解液特异性裂解红细胞-离心分离白细胞/血小板-加热去除蛋白-丙酮酸酶荧光毛细管分析"方法。最优化的血样预处理条件是:以16000 r/min离心血液1 min,获得红细胞;裂解液裂解红细胞后在100℃下加热5 min。对比实验发现,本方法优于其它红细胞处理法。本研究中丙酮酸测定方法的线性范围为10～120 mmol/L;检出限为0.98 mmol/L;灵敏度为5.64 F L/mmol,高于文献方法60倍以上;测定红细胞内丙酮酸值的相对标准偏差小于2.8%(n=11),回收率在98.3%～104.1%之间。In the conventional method of erythrocytes treatment,there was the efflux of pyruvic acid in erythrocytes（PAE） during washing and separation of erythrocytes/leukocytes using the physiological saline.This phenomenon caused the deviation of the measured value of PAE from its real concentration. This research designed a method comprising the steps of employing the erythrocyte lysis buffer to specifically lyse the red blood cells,separating leukocytes and platelets by centrifugation,removing the protein by heating the hemolysate,and detecting PAE using the pyruvate-enzyme fluorescence capillary analysis.The optimized procedures for the blood pretreatment were as follows：at the centrifugal speed of 16000 r/min,the blood sample was centrifuged for 1 min to obtain erythrocytes, after the erythrocytes were lysed by the lysis buffer,the hemolysate was heated for 5 min at 100℃. Through the contrast experiment,it was found that this method was superior to other erythrocyte treatment method.The pyruvate determination method in this research had a good linearity in the range of 10—120 mmol/L,a detection limit of 0.98 mmol/L,and a sensitivity of 5.64 F L/mmol,it was 60 times higher than that of references method.This method offered a relative standard deviation of PAE less than 2.8%（n=11）,and a recovery range of 98.3%-104.1%.

关 键 词：酶荧光毛细管分析 丙酮酸 红细胞 裂解加热法 

分 类 号：R446.1[医药卫生—诊断学] O657.8[医药卫生—临床医学]