高效液相色谱直接衍生化法测定血清游离脂肪酸组成

High-performance liquid chromatographic analysis of serum free fatty acid compositions by direct derivatization method

目的脂肪酸摄入和代谢在高血压的发病和进展中发挥作用,但血清游离脂肪酸(FFA)组成测定方法尚未统一,本研究探讨高效液相色谱直接衍生化法测定空腹血清游离脂肪酸组成的可靠性。方法社区筛选出原发性高血压患者103例,其中肥胖患者65例,非肥胖患者38例,和血压正常个体128例,其中肥胖个体70例,非肥胖者58例,年龄35～59岁,各组饮食习惯相近。采空腹静脉血离心取上清分成两个1.5ml,用75%的乙醇60℃下除去血清中与FFA结合的白蛋白,无需常规的分离步骤,应用2-硝基苯肼盐酸盐(2-NPH.HCl)作为衍生试剂,在1-乙基-3-(3-二甲基氨丙基碳化二亚胺)盐酸盐(1-EDC.HCl)的催化下,直接衍生血清中的FFA,形成不挥发的能在紫外-可见光区产生吸收的脂肪酸的肼盐衍生化物,再用磷酸缓冲液酸化增加各种脂肪酸的分离度,衍生化的脂肪酸用正己烷萃取法与其它的酰肼和干扰成分分离出来,氮气吹干后甲醇定容,应用C18反向色谱柱进样30min内洗脱分析测定。结果共测定血清中主要的9种脂肪酸,从C16:0到C22:6,未鉴定的峰少于总量的1%。脂肪酸组成以个体脂肪酸浓度表示,浓度单位μmol/L。应用C17:0作内标,各脂肪酸测定的线性范围为2.6～200.0μmol/L,回收率在87.8%～120.5%之间,重复性检验变异系数<10%,相关系数为0.973～0.999,批内RSD范围为2.78%～12.90%,批间RSD范围为为0.95%～15.4%。与血压正常个体比较,原发性高血压患者血清游离多不饱和脂肪酸(PUFA)水平降低(P=0.004),饱和脂肪酸(SFA)水平有增高趋势(P=0.06),P/S比值降低(P=0.000)。结论高效液相色谱直接衍生化法测定空腹血清FFA组成重复性、准确性良好,可作为血清游离脂肪酸组成常规检测的有用的工具。

Objective Fatty acids intake and metabolism may play a role in the pathogenesis of essential hypertension, but the methods of determining serum free fatty acid （FFA） compositions have not been unified. The aim of this study was to investigate the validity of high performante liquid chromatogramin by direct derivatization method. Methods Subjects aged between 35 and 59 years from communities including 103 essential hypertensives and 128 normotensives were selected. Fasting blood （ 12 hours） was collected, separated and dispensed into two 1.5 - ml aliquots. Serum FFA was dissolved in ethanol （75% , v/v ）. Without commonly used isolation stops, fatty acids in serum were directly derivatized by treatment with 2 - nitrophenylhydrazine hydrochloride in the presence of l - ethyl -3 - （3 - dimothylarsinopropyl） carbodiimide hydrochloride（ 1 -EDC · HCl）, which produces their non -volatile hydrazine derivatives. The dorivatized fatty acids were separated from other acid hydrazides and interfering components by a simple solvent extraction （which were extracted into n -hexane）, and were cluted isoeratically on a reversed - phase C18 colunm within 30 rain. Results A total of 9 serum fatty acids from C16：0 to C22：6 were determined. Using margaric acid （ C17：0） as internal standard, each fatty acid could be quantitated with linearity over the range of 2.6 - 200 μmol/L. Analytical recoveries ranged from 87. 8% to 120.51% and coefficients of variation were less than 10%. Compared with normotensives, hypertensives had lower levels of serum polyunsaturated fatty acids （PUFAs, P = 0. 004 ） ） , and polyunsaturated/saturated fatty acids ratio （P/S ratio, P = 0. 000 ） , which were in good agreement with those in other reports. Conclusion The analytical results showed good recovery and reproducibility using each internal standard. Due to its excellent selectivity and sensitivity, the present method can serve as a useful tool for routine determinations of FFA compositions in serum.