Serum Cholesteryl Ester N-3 Fatty Acid Index Determined By High Performance Liquid Chromatography

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.