高效液相色谱法荧光测定肝脏δ-氨基乙酰丙酸合酶活性

Fluorimetric determination of hepatic delta-aminolevulinic acid synthase activity by high-performance liquid chromatography.

作者信息

Tomokuni K, Hirai Y, Ichiba M

机构信息

Department of Community Health Science, Saga Medical School, Japan.

出版信息

J Chromatogr. 1991 Jun 14;567(1):65-70. doi: 10.1016/0378-4347(91)80310-9.

DOI:10.1016/0378-4347(91)80310-9

PMID:1918261

Abstract

A fluorimetric method for measuring the activity of delta-aminolevulinic acid synthase (ALAS) in the liver of mice has been developed. The liver homogenate was used as the enzyme source. The final concentration of glycine (substrate) used for the assay was 100 mM. The delta-aminolevulinic acid (ALA) formed during incubation was converted into a highly fluorescent derivative by condensation with acetylactone and formaldehyde (application of the Hantzsch reaction). This derivative was completely separated from other fluorescent substances in the reaction medium, and it was determined using a high-performance liquid chromatograph equipped with a fluorescence monitor (370/460 nm). The activity of ALAS was expressed as nmol ALA formed per gram liver per hour.

摘要

已开发出一种用于测量小鼠肝脏中δ-氨基乙酰丙酸合酶（ALAS）活性的荧光测定方法。肝脏匀浆用作酶源。用于测定的甘氨酸（底物）的终浓度为100 mM。孵育过程中形成的δ-氨基乙酰丙酸（ALA）通过与乙酰丙酮和甲醛缩合（应用汉茨希反应）转化为高荧光衍生物。该衍生物与反应介质中的其他荧光物质完全分离，并使用配备荧光监测器（370/460 nm）的高效液相色谱仪进行测定。ALAS的活性以每克肝脏每小时形成的nmol ALA表示。