Top-down lipidomics of low density lipoprotein reveal altered lipid profiles in advanced chronic kidney disease

Abstract

This study compared the molecular lipidomic profile of LDL in patients with nondiabetic advanced renal disease and no evidence of CVD to that of age-matched controls, with the hypothesis that it would reveal proatherogenic lipid alterations. LDL was isolated from 10 normocholesterolemic patients with stage 4/5 renal disease and 10 controls, and lipids were analyzed by accurate mass LC/MS. Top-down lipidomics analysis and manual examination of the data identified 352 lipid species, and automated comparative analysis demonstrated alterations in lipid profile in disease. The total lipid and cholesterol content was unchanged, but levels of triacylglycerides and N-acyltaurines were significantly increased, while phosphatidylcholines, plasmenyl ethanolamines, sulfatides, ceramides, and cholesterol sulfate were significantly decreased in chronic kidney disease (CKD) patients. Chemometric analysis of individual lipid species showed very good discrimination of control and disease sample despite the small cohorts and identified individual unsaturated phospholipids and triglycerides mainly responsible for the discrimination. These findings illustrate the point that although the clinical biochemistry parameters may not appear abnormal, there may be important underlying lipidomic changes that contribute to disease pathology. The lipidomic profile of CKD LDL offers potential for new biomarkers and novel insights into lipid metabolism and cardiovascular risk in this disease.

Keywords: N-acyltaurine; cholesterol; cholesterol sulfate; dyslipidemias; inflammation; mass spectrometry; partial least squares discriminant analysis; phospholipids.

Fig. 1.

Typical normal-phase LC/MS chromatograms of normolipidemic LDL lipid extract (Control, top) and LDL extract from CKD patients (Disease, bottom) in positive (+ve, left) and negative (−ve, right) ion mode. Lipid extracts were prepared according to the Folch method, and chromatograms were normalized to relative intensity (%). Labeled peaks are triacylglycerides and cholesteryl esters (TAG + CE), phosphatidylinositols (PI), phosphatidylethanolamines (PE), PCs (PC), and SMs (SM). The insets depict a zoomed region for the elution of cholesterol sulfate (CS), N-acyltaurines (NAT), ceramides (Cer), and fatty acids (FA) in negative ion mode.

Fig. 2.

Box plots showing changes in major lipids in the disease group (n = 10) against the age-matched control group (n = 10). Samples were analyzed in triplicate (n = 3), and statistical analysis was carried out using the Mann-Whitney test to estimate the P values. Differences were considered statistically significant at P < 0.05. Plots are total lipids detected (A), TAGs (B), total PC (C), ratio of pPE to total PE (D), total LPC and LPE (E), and ratio of LPC to LPE (F).

Fig. 3.

Box plots of minor lipids that showed significant change in disease. Samples from the disease group (n = 10) and the age-matched control group (n = 10) were analyzed in triplicate, and statistical analysis was carried out using the Mann-Whitney test to estimate the P values. Differences were considered statistically significant at P < 0.05. The plots are Cer’s (A), NATs (B), CS (C), and STs (D).

Fig. 4.

PLSDA scores plot for merged data (lipids detected in positive and negative ion mode). Numbers adjacent to the symbols are patient sample codes.

Fig. 5.

Boxplots of two individual lipid species that were altered in positive (PC) and negative (PE) ion mode. Samples from the CKD group (n = 10) and age-matched control group (n = 10) were analyzed in triplicate, and statistical analysis was carried out using the Mann-Whitney test to estimate the P values. Differences were considered statistically significant at P < 0.05. Plots are pPC 40:7 (A) and pPE 38:7 (B), where the numbers (C:n) correspond to the total number of carbon atoms:number of double bonds present in the acyl and alkyl chains (including the vinyl ether bond).