Plasma and serum lipidomics of healthy white adults shows characteristic profiles by subjects' gender and age

Abstract

Blood is a commonly used biofluid for biomarker discovery. Although blood lipid metabolites are considered to be potential biomarker candidates, their fundamental properties are not well characterized. We aimed to (1) investigate the matrix type (serum vs. plasma) that may be preferable for lipid biomarker exploration, (2) elucidate age- and gender-associated differences in lipid metabolite levels, and (3) examine the stability of lipid metabolites in matrix samples subjected to repeated freeze-thaw cycles. Using liquid chromatography-mass spectrometry, we performed lipidomic analyses for fasting plasma and serum samples for four groups (15 subjects/group) of young and elderly (25-34 and 55-64 years old, respectively) males and females and for an additional aliquot of samples from young males, which were subjected to repeated freeze-thaw cycles. Lysophosphatidylcholine and diacylglycerol levels were higher in serum than in plasma samples, suggesting that the clotting process influences serum lipid metabolite levels. Gender-associated differences highlighted that the levels of many sphingomyelin species were significantly higher in females than in males, irrespective of age and matrix (plasma and serum). Age-associated differences were more prominent in females than in males, and in both matrices, levels of many triacylglycerols were significantly higher in elderly females than in young females. Plasma and serum levels of most lipid metabolites were reduced by freeze-thawing. Our results indicate that plasma is an optimal matrix for exploring lipid biomarkers because it represents the original properties of an individual's blood sample. In addition, the levels of some blood lipid species of healthy adults showed gender- and age-associated differences; thus, this should be considered during biomarker exploration and its application in diagnostics. Our fundamental findings on sample selection and handling procedures for measuring blood lipid metabolites is important for ensuring the quality of biomarkers identified and its qualification process.

Figure 1. Differences in lipid metabolite levels in human blood samples between plasma and serum.

Lipid metabolites with marked fold changes (serum/plasma >1.5 or <0.67) and p<0.01 values between plasma and serum in any of the four subject groups are plotted for each group. Statistical significance was determined by Wilcoxon matched-pairs signed-rank test (*p<0.05, **p<0.01, ***p<0.001). Some metabolites (^{†, ††}) with same exact mass were eluted with different retention time (Table S1) and therefore seem to be different molecular species of DG. LysoPC, lysophosphatidylcholine; DG, diacylglycerol; TG, triacylglycerol.

Figure 2. Levels of representative lipid metabolites with significant matrix-associated differences.

(A) 18∶0 lysoPC, (B) 36∶3 DG, (C) 5,6-diHEtrE, (D) 8-HETE. The graph shows medians and interquartile ranges. Statistical significance was determined by Wilcoxon matched-pairs signed-rank test (*p<0.05, **p<0.01, ***p<0.001). YM, young male; YF, young female; EM, elderly male; EF, elderly female. LysoPC, lysophosphatidylcholine; DG, diacylglycerol; diHEtrE, dihydroxyeicosatrienoic acid; HETE, hydroxyeicosatetraenoic acid.

Figure 3. Differences in lipid metabolite levels in human blood samples between males and females.

Lipid metabolites with marked fold changes (female/male >1.5 or <0.67) and p<0.01 values between males and females in either plasma or serum are plotted for young (A) and elderly (B) age groups. Statistical significance was determined by Mann-Whitney U-test (*p<0.05, **p<0.01, ***p<0.001). SM, sphingomyelin; Cer, ceramide; PC, phosphatidylcholine; PE, phosphatidylethanolamine; ChE, cholesteryl ester; TG, triacylglycerol; HETE; hydroxyeicosatetraenoic acid; diHETE, dihydroxyeicosatetraenoic acid.

Figure 4. Levels of representative lipid metabolites with significant gender- and age-associated difference.

(A) d18∶1–18∶0 SM, (B) 18∶0–22∶6 PC, (C) 18-HETE, (D) 5-HEPE. The graph represents medians and interquartile ranges. Statistical significance was determined by Mann-Whitney U-test (*p<0.05, **p<0.01, ***p<0.001). YM, young male; YF, young female; EM, elderly male; EF, elderly female. SM, sphingomyelin; PC, phosphatidylcholine; HETE, hydroxyeicosatetraenoic acid; HEPE, hydroxyeicosapentaenoic acid.

Figure 5. Differences in lipid metabolite levels in human blood samples between young and elderly age groups.

Lipid metabolites with marked fold changes (elderly/young subjects ratio >1.5 or <0.67) and p<0.01 values between young and elderly age groups in either plasma or serum are plotted for male (A) and female (B) groups. Statistical significance was determined by Mann-Whitney U-test (*p<0.05, **p<0.01, ***p<0.001). PC, phosphatidylcholine; Cer, ceramide; ChE, cholesteryl ester; TG, triacylglycerol; HEPE, hydroxyeicosapentaenoic acid; lysoPC, lysophosphatidylcholine; HETE, hydroxyeicosatetraenoic acid.

Figure 6. Effects of repeated freeze-thawing on lipid metabolite stability in plasma and serum of young males.

Lipid metabolite levels after 10 freeze-thaw cycles were compared with those after two freeze-thaw cycles for plasma and serum samples. (A) 18∶0–18∶2 PC, (B) arachidonic acid, (C) Ch, (D) 20∶4 ChE. The graph shows medians and interquartile ranges. Statistical significance was determined by Wilcoxon matched-pairs signed-rank test (**p<0.01, ***p<0.001). PC, phosphatidylcholine; Ch, cholesterol; ChE, cholesteryl ester.