Gray M C, Plant A L, Nicholson J M, May W E
Department of Chemistry, Howard University, Washington, District of Columbia 20059.
Anal Biochem. 1995 Jan 1;224(1):286-92. doi: 10.1006/abio.1995.1042.
An enzymatic assay using fluorometric detection for cholesterol determination in serum is described. Results were compared to a conventional enzymatic colorimetric procedure and to the definitive method, which is based on isotope dilution mass spectrometry. Fluorescence detection enhances sensitivity over current colorimetric methods by approximately two orders of magnitude, and the assay response is linear over three orders of magnitude of cholesterol concentration. The reaction is performed in a single step and can be performed with small sample (1 microliter) and reaction (200 microliters) volumes. The fluorescence intensity is stable after a 30-min sample incubation at room temperature. The sensitivity of this fluorescence assay makes it possible to measure subnanomoles of cholesterol, allowing accurate measurement of total cholesterol in 1 microliter of serum or less. This level of sensitivity will also allow measurement of cholesterol in various isolated lipoprotein fractions.
本文描述了一种采用荧光检测法测定血清中胆固醇的酶法测定方法。将结果与传统的酶比色法以及基于同位素稀释质谱法的权威方法进行了比较。与当前的比色法相比，荧光检测法的灵敏度提高了约两个数量级，并且该测定方法在三个数量级的胆固醇浓度范围内呈线性响应。该反应一步完成，且可以使用少量样本（1微升）和少量反应体积（200微升）进行。在室温下对样本孵育30分钟后，荧光强度保持稳定。这种荧光测定法的灵敏度使得测量亚纳摩尔级别的胆固醇成为可能，从而能够准确测量1微升或更少血清中的总胆固醇。这种灵敏度水平还将允许对各种分离的脂蛋白组分中的胆固醇进行测量。
