高效液相色谱测定血清胆固醇酯n-3脂肪酸指数
摘要
目的：
建立一种简便、快速、精密的高效液相色谱(HPLC)测定血清胆固醇酯n-3脂肪酸指数(CE n-3指数)的方法,用于多不饱和脂肪酸(PUFA)摄入人群营养筛查,并研究CE n-3指数与心血管疾病危险因素的关系。
方法：
采集血液样品,凝血后离心分离血清,用氢氧化钠(NaOH)乙醇溶液水解血清甘油三酯(TG),乙酸终止水解反应,正己烷提取胆固醇和胆固醇酯(CE), HPLC分离脂质,紫外(205 nm)检测,计算n-3脂肪酸CE在所有CE中的百分比(CEn-3指数)。胆固醇二十碳五烯酸酯(20：5-CE)和二十二碳六烯酸酯(22：6-CE)用液相色谱串联质谱(LC/MS/MS)鉴定,各CE峰面积校正因子用测定相应组分胆固醇含量的方法确定,归一化法定量,各CE的百分含量用校正的CE摩尔百分比表示。征集106名健康北京居民,测量身高、体重、血压,用上述方法测定血清标本CE n-3指数,描述此范围的人群分布特征,用HPLC法测定FERHDL,用酶法测定血清总胆固醇(TC)和TG,用超速离心HPLC法测定HDL-C、HDL亚类胆固醇(HDL2-C和HDL3-C)、低密度脂蛋白(LDL)胆固醇(LDL-C)、LDL亚类胆固醇(LDLa-C和LDLb-C)和脂蛋白(a)胆固醇[Lp(a)-C],分析CE n-3指数与其他指标的关系。
结果：
1.HPLC测定CE n-3指数的色谱条件为：采用Nova-Pak C18色谱柱(5μm,3.9mmx 150 mm);流动相为乙腈-异丙醇-正己烷=55-50-15；紫外205nm检测；流速为1.2mL/min;进样50uL；色谱分析在6分钟内完成；且血清中n-3 CE与其他CE分离良好。
2.影响CE n-3脂肪酸指数测定的TG用4mol/LNaOH的乙醇溶液水解30秒,可被去除,CE n-3指数预计比实际水平低约3%,对测定结果影响较小,出于实用考虑,可以作为去除TG的方法。
3.比较两种提取溶剂对样品中各脂质的提取效率,结果显示一致,表明溶剂可以充分提取各脂质组分,在一定程度上可以保证测定结果的准确性。
4.测定血清CE n-3指数的批内和总变异系数分别为0.36%-0.86%和0.65%～1.17%,远优于红细胞膜n-3指数的分析重现性(CV=4-7%)。
5.测定106例北京居民血清CE n-3指数,全体指数略呈正偏态分布(偏度值=1.25,峰度值=1.70),中位数为0.98%(0.37%～2.40%)。n-3指数对数基本呈正态分布(偏度值=0.24,峰度值=0.27),均值为0.0037%,标准差为0.15%,95%分布区间为(-0.0248%,0.0322%)。性别组n-3指数分布与全体类似,女性和男性中位数分别为1.08%(0.60%～2.40%)和0.95%(0.37%-2.11%),女性明显高于男性(t=2.694,P=0.008)。相关分析结果显示,CE n-3指数与收缩压、TG和FERHDL显著正相关(P<0.05或0.01);CE n-6/n-3与身高、TC、TG、LDL-C、LDLa-C、FERHDL和CE n-3指数显著负相关(P<0.05或0.01)；PUFA/MUFA与HDLC、HDL2-C、HDL3-C、LDL-C和LDLa-C显著正相关(P<0.05或0.01)；与年龄、体重、BMI、收缩压、TG和CE n-6/n-3显著负相关(P<0.05或0.01)。
结论：
1.建立了HPLC测定血清CE n-3指数的方法,该法简便、快速、精密、成本低,有望在营养监测和心血管病危险分析中发挥作用。
2.测定了106名北京居民CE n-3指数,给出了此人群CE n-3指数的参考值及分布特征,并分析了该指标与一般体征指标及常规血脂指标的关系。
Objective To develop an HPLC method for the measurement of n-3 fatty acid index of serum cholesteryl esters(CE n-3 index),and to investigate the correlation of CE n-3 index with diatery intake of n-3 polyunsaturated fatty acid and cardiovascular disease risk factors.
Methods Blood samples were collected after an overnight fast, and the sera were separated. Serum triglycerides were hydrolyzed with ethanolic sodium hydroxide,and the reaction was terminated with acetic acid. Cholesteryl esters (CEs) were extracted with hexane. The extracted CEs were analyzed by reversed phase HPLC with a UV detection at 205 nm. Cholesteryl eicosapentaenoate and docosahexaenoate (major n-3 fatty acid cholesteryl esters) were identified by liquid chromatography-tandem mass spectrometry. Cholesterol levels in each CE fraction was measured. Peak areas of CEs were corrected for cholesterol and CE n-3 index was calculated using the corrected peak area and expressed as the percentage of n-3 fatty acid CEs in total CEs. The indices of n-6/n-3, polyunsaturated fatty acid/monounsaturated fatty acid(PUFA/MUFA), unsaturated fatty acid/satuated fatty acid(LVS), polyunsaturated fatty acid/satuated fatty acid(P/S) and linoleic acid/oleic acid(L/O) were calculated respectively.106 Beijing residents were recruited, and Blood samples were collected. Fractional esterification rate of high density lipoprotein cholesterol (FERHDL) was measured by HPLC method. HDL-C, HDL2-C, HDL3-C, LDL-C, LDLa-C, LDLb-C, Lp(a)-C were measured by ultracentrifugation-HPLC method, and the correlation between CE n-3 index and these parameters were analyzed.
Results Triglycerides, which interfere with the determination of n-3 fatty acid index, were hydrolyzed with ethanolic sodium hydroxide (4mol/L) in 30 seconds. The HPLC analysis was finished in 6 minutes. There was a good separation between n-3 CE and other CEs. The chromatography, within-run, between-run and total CVs for CE n-3 index measurements were 0.25%-0.46%,0.36%-0.86%,0.00%-0.76 and 0.65%-1.17%, respectively. CE n-3 index of 106 samples appeared approximate positively skewed distribution(skewness=1.25, kurtosis=1.70). The median of n-3 indices was 0.98% (0.37%-2.40%). The logarithm of n-3 index appeared normal distribution, and the average is 0.0037% with standard deviations of 0.15%. The distribution of n-3 indices of gender groups was similar to that of the total. The medians of females and males were 1.08%(0.60%-2.40%) and 0.95%(0.37%-2.11%), respectively, and the former was significantly higher than the latter (t=2.694, P= 0.008). CE n-3 index was positively correlated with systolic blood pressure, TG and FERHDL.
Conclusion A new method for the measurement of n-3 index of serum cholesteryl esters by HPLC has been established. The sample size is low,0.02ml is needed for the determination of CE n-3 index. The method is simple, precise and easy to operate, and can be used in predicting cardiovascular diseases risks and in monitoring dietary intake of n-3 fatty acids.
建立一种简便、快速、精密的高效液相色谱(HPLC)测定血清胆固醇酯n-3脂肪酸指数(CE n-3指数)的方法,用于多不饱和脂肪酸(PUFA)摄入人群营养筛查,并研究CE n-3指数与心血管疾病危险因素的关系。
方法：
采集血液样品,凝血后离心分离血清,用氢氧化钠(NaOH)乙醇溶液水解血清甘油三酯(TG),乙酸终止水解反应,正己烷提取胆固醇和胆固醇酯(CE), HPLC分离脂质,紫外(205 nm)检测,计算n-3脂肪酸CE在所有CE中的百分比(CEn-3指数)。胆固醇二十碳五烯酸酯(20：5-CE)和二十二碳六烯酸酯(22：6-CE)用液相色谱串联质谱(LC/MS/MS)鉴定,各CE峰面积校正因子用测定相应组分胆固醇含量的方法确定,归一化法定量,各CE的百分含量用校正的CE摩尔百分比表示。征集106名健康北京居民,测量身高、体重、血压,用上述方法测定血清标本CE n-3指数,描述此范围的人群分布特征,用HPLC法测定FERHDL,用酶法测定血清总胆固醇(TC)和TG,用超速离心HPLC法测定HDL-C、HDL亚类胆固醇(HDL2-C和HDL3-C)、低密度脂蛋白(LDL)胆固醇(LDL-C)、LDL亚类胆固醇(LDLa-C和LDLb-C)和脂蛋白(a)胆固醇[Lp(a)-C],分析CE n-3指数与其他指标的关系。
结果：
1.HPLC测定CE n-3指数的色谱条件为：采用Nova-Pak C18色谱柱(5μm,3.9mmx 150 mm);流动相为乙腈-异丙醇-正己烷=55-50-15；紫外205nm检测；流速为1.2mL/min;进样50uL；色谱分析在6分钟内完成；且血清中n-3 CE与其他CE分离良好。
2.影响CE n-3脂肪酸指数测定的TG用4mol/LNaOH的乙醇溶液水解30秒,可被去除,CE n-3指数预计比实际水平低约3%,对测定结果影响较小,出于实用考虑,可以作为去除TG的方法。
3.比较两种提取溶剂对样品中各脂质的提取效率,结果显示一致,表明溶剂可以充分提取各脂质组分,在一定程度上可以保证测定结果的准确性。
4.测定血清CE n-3指数的批内和总变异系数分别为0.36%-0.86%和0.65%～1.17%,远优于红细胞膜n-3指数的分析重现性(CV=4-7%)。
5.测定106例北京居民血清CE n-3指数,全体指数略呈正偏态分布(偏度值=1.25,峰度值=1.70),中位数为0.98%(0.37%～2.40%)。n-3指数对数基本呈正态分布(偏度值=0.24,峰度值=0.27),均值为0.0037%,标准差为0.15%,95%分布区间为(-0.0248%,0.0322%)。性别组n-3指数分布与全体类似,女性和男性中位数分别为1.08%(0.60%～2.40%)和0.95%(0.37%-2.11%),女性明显高于男性(t=2.694,P=0.008)。相关分析结果显示,CE n-3指数与收缩压、TG和FERHDL显著正相关(P<0.05或0.01);CE n-6/n-3与身高、TC、TG、LDL-C、LDLa-C、FERHDL和CE n-3指数显著负相关(P<0.05或0.01)；PUFA/MUFA与HDLC、HDL2-C、HDL3-C、LDL-C和LDLa-C显著正相关(P<0.05或0.01)；与年龄、体重、BMI、收缩压、TG和CE n-6/n-3显著负相关(P<0.05或0.01)。
结论：
1.建立了HPLC测定血清CE n-3指数的方法,该法简便、快速、精密、成本低,有望在营养监测和心血管病危险分析中发挥作用。
2.测定了106名北京居民CE n-3指数,给出了此人群CE n-3指数的参考值及分布特征,并分析了该指标与一般体征指标及常规血脂指标的关系。
Objective To develop an HPLC method for the measurement of n-3 fatty acid index of serum cholesteryl esters(CE n-3 index),and to investigate the correlation of CE n-3 index with diatery intake of n-3 polyunsaturated fatty acid and cardiovascular disease risk factors.
Methods Blood samples were collected after an overnight fast, and the sera were separated. Serum triglycerides were hydrolyzed with ethanolic sodium hydroxide,and the reaction was terminated with acetic acid. Cholesteryl esters (CEs) were extracted with hexane. The extracted CEs were analyzed by reversed phase HPLC with a UV detection at 205 nm. Cholesteryl eicosapentaenoate and docosahexaenoate (major n-3 fatty acid cholesteryl esters) were identified by liquid chromatography-tandem mass spectrometry. Cholesterol levels in each CE fraction was measured. Peak areas of CEs were corrected for cholesterol and CE n-3 index was calculated using the corrected peak area and expressed as the percentage of n-3 fatty acid CEs in total CEs. The indices of n-6/n-3, polyunsaturated fatty acid/monounsaturated fatty acid(PUFA/MUFA), unsaturated fatty acid/satuated fatty acid(LVS), polyunsaturated fatty acid/satuated fatty acid(P/S) and linoleic acid/oleic acid(L/O) were calculated respectively.106 Beijing residents were recruited, and Blood samples were collected. Fractional esterification rate of high density lipoprotein cholesterol (FERHDL) was measured by HPLC method. HDL-C, HDL2-C, HDL3-C, LDL-C, LDLa-C, LDLb-C, Lp(a)-C were measured by ultracentrifugation-HPLC method, and the correlation between CE n-3 index and these parameters were analyzed.
Results Triglycerides, which interfere with the determination of n-3 fatty acid index, were hydrolyzed with ethanolic sodium hydroxide (4mol/L) in 30 seconds. The HPLC analysis was finished in 6 minutes. There was a good separation between n-3 CE and other CEs. The chromatography, within-run, between-run and total CVs for CE n-3 index measurements were 0.25%-0.46%,0.36%-0.86%,0.00%-0.76 and 0.65%-1.17%, respectively. CE n-3 index of 106 samples appeared approximate positively skewed distribution(skewness=1.25, kurtosis=1.70). The median of n-3 indices was 0.98% (0.37%-2.40%). The logarithm of n-3 index appeared normal distribution, and the average is 0.0037% with standard deviations of 0.15%. The distribution of n-3 indices of gender groups was similar to that of the total. The medians of females and males were 1.08%(0.60%-2.40%) and 0.95%(0.37%-2.11%), respectively, and the former was significantly higher than the latter (t=2.694, P= 0.008). CE n-3 index was positively correlated with systolic blood pressure, TG and FERHDL.
Conclusion A new method for the measurement of n-3 index of serum cholesteryl esters by HPLC has been established. The sample size is low,0.02ml is needed for the determination of CE n-3 index. The method is simple, precise and easy to operate, and can be used in predicting cardiovascular diseases risks and in monitoring dietary intake of n-3 fatty acids.
引文
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