测定白细胞和成纤维细胞中胱氨酸的新方法。

New method for determining cystine in leukocytes and fibroblasts.

作者信息

de Graaf-Hess A, Trijbels F, Blom H

机构信息

Laboratory of Paediatrics and Neurology, University Hospital Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.

出版信息

Clin Chem. 1999 Dec;45(12):2224-8.

PMID:10585356

Abstract

BACKGROUND

Cystinosis is a rare inborn error of cystine transport, leading to accumulation of cystine in the lysosomes. To diagnose cystinosis and monitor treatment with cysteamine, adequate measurements of cystine concentrations in leukocytes and cultured fibroblasts are required.

METHODS

Cells were sonicated in the presence of excess N-ethylmaleimide to prevent oxidation of cysteine to cystine and disulfide exchange reactions of cystine with available sulfhydryl moieties. Cystine was measured as cysteine after reduction with sodium borohydride and derivatization with monobromobimane, followed by separation with automated HPLC and fluorescence detection.

RESULTS

The assay was linear to 200 micromol/L cysteine. Within-run and day-to-day (total) imprecision (CV) was <5%, and the detection limit was 0.3 micromol/L. Added cysteine, up to 200 micromol/L, was completely removed, and recovery of added cystine was 69-86%. Cystine was stable for at least 2 months in leukocytes frozen in liquid nitrogen and stored at -80 degrees C CONCLUSIONS: Oxidation of cysteine to cystine and disulfide exchange reactions of cystine with sulfhydryl moieties are prevented by N-ethylmaleimide. The detection limit for the determination of cystine is adequate to measure cystine in leukocytes and cultured fibroblasts for diagnosis of cystinosis and monitoring treatment with cysteamine.

摘要

背景

胱氨酸病是一种罕见的胱氨酸转运先天性代谢缺陷病，可导致胱氨酸在溶酶体中蓄积。为诊断胱氨酸病并监测半胱胺治疗效果，需要对白细胞和培养的成纤维细胞中的胱氨酸浓度进行准确测量。

方法

在过量N-乙基马来酰亚胺存在的情况下对细胞进行超声处理，以防止半胱氨酸氧化为胱氨酸以及胱氨酸与可用巯基部分发生二硫键交换反应。用硼氢化钠还原并与单溴双马来酰胺衍生化后，将胱氨酸作为半胱氨酸进行测量，随后通过自动高效液相色谱和荧光检测进行分离。

结果

该测定法在200微摩尔/升半胱氨酸范围内呈线性。批内和日间（总计）不精密度（CV）<5%，检测限为0.3微摩尔/升。添加至200微摩尔/升的半胱氨酸被完全去除，添加的胱氨酸回收率为69-86%。胱氨酸在液氮中冷冻并储存在-80℃的白细胞中至少稳定2个月。结论：N-乙基马来酰亚胺可防止半胱氨酸氧化为胱氨酸以及胱氨酸与巯基部分发生二硫键交换反应。胱氨酸测定的检测限足以测量白细胞和培养的成纤维细胞中的胱氨酸，用于胱氨酸病的诊断和半胱胺治疗的监测。