Metabolome Changes during In Vivo Red Cell Aging Reveal Disruption of Key Metabolic Pathways

Abstract

Understanding the mechanisms for cellular aging is a fundamental question in biology. Normal red blood cells (RBCs) survive for approximately 100 days, and their survival is likely limited by functional decline secondary to cumulative damage to cell constituents, which may be reflected in altered metabolic capabilities. To investigate metabolic changes during in vivo RBC aging, labeled cell populations were purified at intervals and assessed for abundance of metabolic intermediates using mass spectrometry. A total of 167 metabolites were profiled and quantified from cell populations of defined ages. Older RBCs maintained ATP and redox charge states at the cost of altered activity of enzymatic pathways. Time-dependent changes were identified in metabolites related to maintenance of the redox state and membrane structure. These findings illuminate the differential metabolic pathway usage associated with normal cellular aging and identify potential biomarkers to determine average RBC age and rates of RBC turnover from a single blood sample.

Keywords: Medical Biochemistry; Metabolomics.

Graphical abstract

Figure 1

Red Cell Survival, Cell Separation, and Experimental Design (A) Results of a representative RBC survival experiment in male C57Bl6/J mice using the in vivo biotin labeling approach. Care was taken to collect very small volumes of blood (1–5 μL) at each time point to avoid skewing the results with random cell loss due to phlebotomy. Under these conditions, RBC half-life is approximately 23 days, with maximal RBC lifespan 58 days. The survival curve closely approximates a linear function, with the loss of ∼2.4% of RBC each day. Arrows indicate the timing of harvest for separate cohorts used for metabolomic analyses. Data are represented as +/− SEM. (B) Results of a representative magnetic bead separation of RBC at the 35-day time point. Unfractionated cells (left), biotin(−), and biotin(+) are shown. Bead separations routinely yielded high-purity populations in both biotin(−) and biotin(+) fractions. Complete purification results are presented in Table S1. (C) Schematic view of the age distribution of the RBC populations used in metabolomic analyses. Without evidence for significant random destruction of RBCs, the best approximation of age distribution is a conveyor belt, where RBCs enter the circulation and remain until removal at or near the maximal lifespan.

Figure 2

Overview of Metabolomic Analysis (A) Principal component analysis was used as an overall method to assess data quality and compare sample groups. At each time point, biologic replicate samples cluster together by age (biotin + or biotin -). Biotin(+) samples at different time points are more similar to each other than biotin(−) samples, with cells ≤8 days being the most divergent. (B) Distribution of metabolites according to compound class, with lipids and amino acids representing two-thirds of all compounds identified. (C) Heatmap summarizing all metabolite data by compound class over the time course of this study. The predominant pattern of metabolite change across all compound classes is a decline with increasing RBC age. Data presented are from five biologic replicates at day 8 and six replicates for days 15 and 35.

Figure 3

Changes in Metabolite Production and Consumption in the Red Cell Metabolic Network RBC metabolic map highlighting systemic metabolic shifts during cellular senescence. Reaction arrow thickness and color coding correspond to rMAR magnitude changes (day 8 versus day 35; all ratios greater than two are shaded red and black is 0). Three subsystems of metabolism are highlighted, sugar metabolism for energy (orange), amino acid/glutathione metabolism for redox homeostasis (green), and fatty acid metabolism for membrane/structural maintenance (pink). High rMAR reactions (thick, red arrows, rMAR ≥2) reflect bottlenecks with accumulation of enzyme substrates, identifying potential “age-limited” enzymes (the corresponding metabolites are highlighted in blue text). Metabolic subsystems are labeled in black font text. See Figure S2 for a high-resolution, detailed labeled version.

Figure 4

Change in Metabolites in Glycolytic Pathway (A) Schematic of the glycolytic pathway. Metabolites with statistically significant changes in concentration are accompanied by a block arrow showing the direction of change over the course of RBC aging. Box plots for each metabolite are shown comparing metabolite concentration in cells ≤8 days versus cells ≥35 days. In addition to changes in the glycolytic pathway, we also found reduced concentration of sedoheptulose 7-P, a downstream metabolite in the pentose phosphate pathway. (B) Hexokinase activity was compared in RBC hemolysates prepared from cells of the indicated ages. Hexokinase activity is highest in the youngest RBC fraction and shows a modest progressive decline in activity with increasing RBC age. (C) ATP levels were determined in the same cell fractions as used above. ATP concentration is consistently higher in the young cell fractions, without evidence of a further decline as a function of RBC age. (Data presented in (A) were derived from five or six biologic replicates; data in (B and C) were derived from four biologic replicates, summarized as mean values +/− SEM. Asterisks indicate significance at ∗ <0.05, ∗∗ <0.01, and ∗∗∗ <0.001, respectively, assessed with paired t tests).

Figure 5

Glutathione Synthesis and Utilization as a Function of RBC Age (A) Schematic metabolic pathways including GSH synthesis, utilization, and regeneration are depicted along with a parallel pathway for ophthalmate synthesis indicating common precursors. Several intermediates in the glutathione synthetic pathway are present at significantly lower concentration in old RBC (bold downward arrows). Despite reduced precursor concentrations, GSH level was modestly increased in older cells (∗p ≤ 0.05) and GSSG level did not change with RBC age. (B) Box plots comparing concentrations of specific metabolites from young RBCs (≤8 days) and old RBCs (≥35 days). The combination of increased GSH level, unchanged GSSG level, and reduced cys-GSH level is consistent with reduced GSH cycling in older cells. The increased concentration of ophthalmate indicates that the GSH synthetic machinery is intact but that cysteine is lacking for GSH synthesis. (Data were derived from five or six biologic replicates, summarized as mean values +/− SEM. Asterisks indicate significance at ∗ <0.05 and ∗∗∗ <0.001, respectively, assessed with paired t tests and Welch's two-sample t test for non-paired sample groups).

Figure 6

Activity of GSH-Utilizing Enzymes, Ophthalmate Concentration, and Effect of Ophthalmate on Activity (A–D): Enzymatic activity of (A) glutathione-S-transferase (GST), (B) glutathione reductase (GReductase), (C) glutaredoxin (GRedoxin), and (D) glutathione peroxidase (GPx) was compared in RBC hemolysates prepared from young versus old RBCs harvested 8, 15, or 35 days after biotin labeling. GRedoxin and GST both had significantly reduced activity in the old RBC fractions and showed a progressive loss of activity with increasing RBC age. GReductase activity was higher in young RBCs, without progressive loss of activity with increasing RBC age. In contrast, GPx activity was preserved with RBC aging, and there was no differential activity observed when comparing young versus old RBCs. (E) Relative ophthalmate concentration data from untargeted analysis (showing five or six biologic replicates for each time point). (F) Absolute ophthalmate concentration derived from independent experiment with targeted metabolomic approach using deuterated ophthalmate spike (showing three or four biologic replicates per time point). The rise in ophthalmate concentration with increasing RBC age was confirmed in this independent experiment. (G) Enzymatic activity assays (as in A–D) were repeated using normal murine RBC hemolysate in the presence of increasing concentrations of ophthalmate. GRedoxin shows a dose-dependent reduction in activity with increasing concentrations of ophthalmate. (Inhibition data are derived from testing hemolysates from two animals, using triplicate wells for each sample at each concentration tested. Results are representative of two independent experiments, summarized as mean values +/− SEM. Asterisks indicate significance at ∗ <0.05, ∗∗ <0.01, and ∗∗∗ <0.001, respectively, assessed with paired t tests and Welch's two-sample t test for non-paired sample groups).

Figure 7

Changes in Glycerophosphocholine and Related Metabolites During RBC Aging (A) Schematic of phospholipid metabolism showing the relationship between GPC, choline, and GPC-containing lysophospholipids. The decreased concentration of several lysophospholipids with increasing RBC age may indicate lipid breakdown resulting in increased GPC levels. (B) Relative metabolite concentrations for choline and GPC are divergent over the course of RBC aging. The concentration of GPC is below the level of detection in the youngest RBC and rises progressively with increasing cell age. Choline levels remain low in older RBC, despite progressive increase in GPC. This may indicate a block in conversion of GPC to choline in older cells. (C) Sialic acid (N-acetylneuraminate), a sugar residue found in glycoproteins, shows a progressive decline with increasing RBC age. Horizontal bars reflect the mean measurements for each corresponding condition.

Figure 8

Ergothioneine Concentration During RBC Aging Ergothioneine, a xenobiotic with putative antioxidant properties, is highest in the youngest RBCs and shows a progressive decline with increasing RBC age. Horizontal bars reflect the mean measurements for each corresponding condition.