Plasma acylcarnitine levels increase with healthy aging

Abstract

Acylcarnitines transport fatty acids into mitochondria and are essential for β-oxidation and energy metabolism. Decreased mitochondrial activity results in increased plasma acylcarnitines, and increased acylcarnitines activate proinflammatory signaling and associate with age-related disease. Changes in acylcarnitines associated with healthy aging, however, are not well characterized. In the present study, we examined the associations of plasma acylcarnitines with age (range: 20-90) in 163 healthy, non-diseased individuals from the predictive medicine research cohort (NCT00336570) and tested for gender-specific differences. The results show that long-chain and very long-chain acylcarnitines increased with age, while many odd-chain acylcarnitines decreased with age. Gender-specific differences were observed for several acylcarnitines, e.g., eicosadienoylcarnitine varied with age in males, and hydroxystearoylcarnitine varied in females. Metabolome-wide association study (MWAS) of age-associated acylcarnitines with all untargeted metabolic features showed little overlap between genders. These results show that plasma concentrations of acylcarnitines vary with age and gender in individuals selected for criteria of health. Whether these variations reflect mitochondrial dysfunction with aging, mitochondrial reprogramming in response to chronic environmental exposures, early pre-disease change, or an adaptive response to healthy aging, is unclear. The results highlight a potential utility for untargeted metabolomics research to elucidate gender-specific mechanisms of aging and age-related disease.

Keywords: aging; carnitine; lipid metabolism; mitochondria.

Conflict of interest statement

CONFLICTS OF INTEREST: The authors declare no conflicts and interest.

Figures

Figure 1

Background characteristics of 163 healthy adults of the predictive medicine cohort. (A) Mean values with standard deviation (SD) of gender, race and clinical measures are shown. (B) Age distribution of the subset. Stacked bars are shown with men in dark gray and women in light gray. Mean age was 43.5 years, and ages ranged from 20 to 90 years.

Figure 2

Metabolome-Wide Association Study (MWAS) of plasma metabolites correlated with age. (A) Type 1 Manhattan plot showing -log10p for correlation of each metabolite plotted by m/z (mass-to-charge ratio) and type 2 Manhattan plot showing -log10p for correlation of each metabolite plotted by chromatographic retention time (RT) in seconds, as separated the C18 column. Plots are shown with significance (n = 1505, p = 0.05) and false discovery rate (n= 140, FDR = 0.2) thresholds by dashed lines, and the detailed information of metabolic features is provided in Supplementary Table 1. (B) Plot of acylcarnitine correlation strength and direction (Spearman ρ) by –Log10 p. Acylcarnitines with p < 0.05 are labeled by the chain length, saturation and modification of the acyl group (see Table 1 for details). For acylcarnitines detected on both C18 and anion exchange columns, only the C18 data is represented in the plot. The plot is shown with significance (n = 26, p = 0.05) and false discovery rate (n= 4, FDR = 0.2) thresholds by dashed lines In all plots, significant negative correlations are shown in blue, and significant positive correlations are shown in red.

Figure 3

Heat map of one-way hierarchical clustering analysis (HCA) of the 26 acylcarnitines significantly associating with age. Along the x-axis, individuals are organized by age, with youngest on the left. The y-axis is comprised of the one-way HCA of acylcarnitines. Each column represents an individuals’ metabolic profile of the 35 acylcarnitines. Degree of deviation of acylcarnitine concentration below the mean of the study population are indicated by saturation of blue coloration, and degree of deviation of acylcarnitine concentration above the mean of the study population are indicated by saturation of red coloration. Short-chain and medium-chain acylcarnitines are labeled in gray, and long-chain and very-long-chain acylcarnitines are highlighted by labeling in black. For acylcarnitines detected on both C18 and anion exchange columns, only the C18 data was included in the HCA. The lower major acylcarnitine cluster is labeled by its subgroups, G1-3.

Figure 4

Highest correlations of acylcarnitines with age in human plasma. Log2 transformed intensity values for ions, identified by mass-to-charge ratio (m/z) and retention time (RT) for individual plasma samples are plotted against individual ages. Confidence intervals (95%) are shown in gray. (A) Eicosenoylcarnitine (C20:1), (B) eicosadienoylcarnitine (C20:2), (C) arachidylcarnitine (C20:0) and (D) decadienoylcarnitine (C10:2) were significant at FDR = 0.2. (E) Hydroxystearoylcarnitine (C18OH) and (F) decatrienoylcarnitine (C10:3) were significant at p < 0.05.

Figure 5

Identification of acylcarnitines by MS/MS. Experimental MS/MS fragmentations of (A) valerylcarnitine, (B) hydroxyvalerylcarnitine, (C) octenoylcarnitine and (D) octanoylcarnitine are juxtaposed below a library MS/MS fragmentation of methylbutyroylcarnitine. Diagnostic fragments common between library and experimental fragmentations are labeled. Pertinent MS/MS peaks are labeled for mass-to-charge ratio (m/z) by broken line. Additionally, distinctive fragments equivalent in mass difference to that of the mass difference between the represented acylcarnitine and methylbutyroylcarnitine are labeled with proposed fragment structure displayed. MS/MS peaks and matching proposed fragment structure are labeled by dotted line.

Figure 6

Association of top 6 age-associated acylcarnitines with metabolome. (A) xMWAS network of top 6 age-associated acylcarnitines as associated with metabolome within females. Cluster 1 (orange) has features predominantly associated with hydroxystearoylcarnitine (C18OH). Cluster 2 (green) is comprised of features associated most closely with arachidylcarnitine (C20:0). Cluster 3 (yellow) contains features clustered around decadienoylcarnitine (C10:2) and decatrienoylcarnitine (C10:3). Cluster 4 (dark blue) has features mainly associated with eicosenoylcarnitine (C20:1). Cluster 5 (light blue) has features mainly associated with eicosadienoylcarnitine (C20:2). See Supplementary Table 6 for detailed annotation of metabolites included in the female network. (B) xMWAS network of top 6 age-associated acylcarnitines as associated with metabolome within males. Clusters 1-3 form around the same acylcarnitines as their respective acylcarnitines in the female subset. Cluster 4 (blue) has features mainly associated with both C20:1 and C20:2. See Supplementary Table 7 for detailed annotation of metabolites included in the male network. Positive associations are shown in red, while negative associations are shown with blue lines.