一种基于微孔板的酶法，用于同时测定血清中的苯丙氨酸和酪氨酸。

A microplate-based enzymatic assay for the simultaneous determination of phenylalanine and tyrosine in serum.

作者信息

Wibrand Flemming

机构信息

John F. Kennedy Institute, Gl. Landevej 7, 2600 Glostrup, Denmark.

出版信息

Clin Chim Acta. 2004 Sep;347(1-2):89-96. doi: 10.1016/j.cccn.2004.04.012.

DOI:10.1016/j.cccn.2004.04.012

PMID:15313145

Abstract

BACKGROUND

Several methods have been reported for the quantitation of phenylalanine (Phe) or tyrosine (Tyr) in blood, but the simultaneous determination of Phe and Tyr usually requires sophisticated equipment. Here we describe a simple and reliable microplate assay for measurement of Phe and Tyr in serum of patients with phenylketonuria (PKU).

METHODS

Serum was deproteinised and incubated with phenylalanine ammonia-lyase at 37 degrees C for 2 h. Phe and Tyr were converted to trans-cinnamate and trans-coumarate, respectively, which were determined UV spectrophotometrically.

RESULTS

The assay demonstrated linearity up to 3200 micromol/l Phe and 640 micromol/l Tyr. The sensitivity of the assay was 15 micromol/l for Phe and 5 micromol/l for Tyr. Analytical recovery was 105% and 97% for Phe and Tyr, respectively. The between-run coefficient of variation (CV) varied with concentration but was 4.7-18.8% for Phe and 4.2-8.8% for Tyr. The within-run CV was 2.7% for Phe and 6.5% for Tyr. Results obtained with the assay correlated well with those of an ion exchange chromatographic method.

CONCLUSIONS

This novel assay is sensitive, accurate, precise and suitable for routine monitoring of PKU patients.

摘要

背景

已有多种方法用于定量血液中的苯丙氨酸（Phe）或酪氨酸（Tyr），但同时测定Phe和Tyr通常需要精密设备。在此，我们描述一种简单可靠的微孔板分析法，用于测量苯丙酮尿症（PKU）患者血清中的Phe和Tyr。

方法

血清经去蛋白处理后，与苯丙氨酸解氨酶在37℃孵育2小时。Phe和Tyr分别转化为反式肉桂酸和反式香豆酸，通过紫外分光光度法进行测定。

结果

该分析方法在Phe浓度高达3200微摩尔/升和Tyr浓度高达640微摩尔/升时呈线性。该分析方法对Phe的灵敏度为15微摩尔/升，对Tyr的灵敏度为5微摩尔/升。Phe和Tyr的分析回收率分别为105%和97%。批间变异系数（CV）随浓度变化，但Phe为4.7 - 18.8%，Tyr为4.2 - 8.8%。批内CV对Phe为2.7%，对Tyr为6.5%。该分析方法获得的结果与离子交换色谱法的结果相关性良好。