胆固醇、磷脂和脂肪酸代谢在心血管疾病和炎-癌转化的发病中起着重要作用,在生物体液样本中这3条代谢通路脂质成分的理化性质和浓度水平差别很大,同时定量颇具挑战。基于此,以体积比65∶35的甲醇-二氯甲烷混合液为提取溶剂超声提取血清样本后,采用超高效液相色谱-串联质谱法(UHPLC-MS/MS)同时测定3条代谢通路中145种脂类成分的含量。145种脂类成分的测定可在40 min内完成,测定下限为0.03~50.0 μg·L^-1。以内标磷脂酰甘油(15:0)[PG(15:0)]、1-十七碳酰-甘油-3-磷酰胆碱[LysoPC(17:0)]、7α-羟基-4-胆固醇-3-酮-D7、¹³C-甘氨胆酸、d₈-花生四烯酸为代表研究对象,评估方法的基质效应、精密度和准确度。结果显示,基质效应因子值为76.7%~115%,说明基质效应较弱。加标回收试验中目标物的回收率为71.3%~110%,测定值的相对标准偏差(n=5)均小于10%。方法用于测定30例健康人和30例乙型肝炎(HBV)患者血清中145种脂类成分的含量,并将筛选后的29种差异代谢物进行热图分析和相关性分析。结果表明,与健康人血清相比,HBV患者血清中磷脂(PL)和结合态的胆汁酸(BA)含量均是上升的,溶血磷脂(LPL)和脂肪酸(FA)的含量均是下降的。PL与BA表达呈显著正相关,与LPL呈显著负相关。受试者特征曲线分析(ROC)结果显示溶血磷脂酸(16:0)[LysoPA(16:0)]的ROC分析时曲线下面积(AUC)值等于1.000,具有作为HBV诊断生物标志物的前景。

Cholesterol, phospholipid and fatty acid metabolism played an important role in occurrence of atherosclerosis and inflammation-cancer transformation. Quantitative analysis of many lipids in the three metabolism pathways in a single run was an enormous challenge, due to the huge differences in physicochemical properties and concentration levels of pre-selected metabolites in biofluid samples. Based on this, the serum sample was extracted ultrasonically in the mixture of methanol and dichloromethane at a volume ratio of 65∶35, and then 145 lipid components in three metabolic pathways were determined simultaneously by ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) within 40 min, with lower limits of determination in the range of 0.03-50.0 μg·L^-1. Internal standards of phosphatidylglycerol(15:0)[PG(15:0)], 1-heptadecayl-glycerol-3-phosphorylcholine[LysoPC(17:0)], 7α-hydroxy-4-cholesterol-3-one-D7, ¹³C-glycylcholic acid and d₈-arachidonic acid were the representative research objects to evaluate the matrix effect, precision and accuracy of this method. As shown by the results, the values of matrix effect factor were found in the range of 76.7%-115%, indicating that the matrix effect was weak. In the spiked recovery test, the recovery of the target was in the range of 71.3%-110%, with RSDs (n=5) of the determined values less than 10%. This method was applied for determination of 145 lipid components in the serum from 30 healthy people and 30 hepatitis B (HBV) patients, and the 29 differential metabolites screened were analyzed by thermography and correlation analysis. It was shown that the levels of phospholipid (PL) and conjugated bile acid (BA) were elevated, but the levels of lysophospholipid (LPL) and fatty acid (FA) were decreased in serum of HBV patients compared with healthy people. According to correlation analysis test of differential metabolites, there was a significant positive correlation between BA and PL expression, and there was a significant negative correlation between LPL and PL expression. Moreover, as shown by the analytical results from receiver operating characteristic (ROC) curve, the area under the ROC curve (AUC) value of LysoPA(16:0) was 1.000, which meaned that LysoPA(16:0) was a very potential biomarker for HBV diagnosis.