Linnet K
Department of Clinical Biochemistry, Rigshospitalet, Risskov, Denmark.
Clin Biochem. 1994 Jun;27(3):177-82. doi: 10.1016/0009-9120(94)90052-3.
Currently used isotope-dilution mass spectrometry methods for serum total cholesterol are performed without control in each sample for completeness of hydrolysis of cholesterol esters. In order to monitor this step in the analysis, we developed a method based on both high-pressure liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC/MS). 13C3-cholesterol and cholesteryl [1-14C] oleate were added to serum that was saponified and extracted into hexane. The extract was subjected to HPLC with collection of the fractions corresponding to cholesterol and cholesteryl oleate. The radio-activity of the latter was counted in order to estimate the nonhydrolysed fraction that on average amounted to less than 0.1% for commonly used ester hydrolysis procedures. The cholesterol fraction was subjected to GC/MS for quantitation using the traditional isotope-dilution principle. Evaluation of accuracy by assaying sera from the National Institute of Standards and Technology showed a deviation from the target value of < 1% with a coefficient of variation of < or = 1.0%. In conclusion, the present reference method for serum total cholesterol assures results based on complete hydrolysis of the cholesterol ester fraction.
目前使用的血清总胆固醇同位素稀释质谱法在分析过程中，没有对每个样本中胆固醇酯水解的完整性进行控制。为了监测分析中的这一步骤，我们开发了一种基于高压液相色谱（HPLC）和气相色谱/质谱（GC/MS）的方法。将13C3-胆固醇和胆固醇[1-14C]油酸酯添加到经皂化并萃取到己烷中的血清中。提取物进行HPLC分析，收集对应于胆固醇和胆固醇油酸酯的馏分。对后者的放射性进行计数，以估计未水解部分，对于常用的酯水解程序，该部分平均含量不到0.1%。胆固醇馏分采用传统的同位素稀释原理进行GC/MS定量分析。通过分析美国国家标准与技术研究院的血清来评估准确性，结果显示与目标值的偏差<1%，变异系数<或 = 1.0%。总之，目前的血清总胆固醇参考方法确保了基于胆固醇酯部分完全水解的结果。
