Metabolism of Citrate and Other Carboxylic Acids in Erythrocytes As a Function of Oxygen Saturation and Refrigerated Storage

Abstract

State-of-the-art proteomics technologies have recently helped to elucidate the unanticipated complexity of red blood cell metabolism. One recent example is citrate metabolism, which is catalyzed by cytosolic isoforms of Krebs cycle enzymes that are present and active in mature erythrocytes and was determined using quantitative metabolic flux analysis. In previous studies, we reported significant increases in glycolytic fluxes in red blood cells exposed to hypoxia in vitro or in vivo, an observation relevant to transfusion medicine owing to the potential benefits associated with hypoxic storage of packed red blood cells. Here, using a combination of steady state and quantitative tracing metabolomics experiments with ¹³C_1,2,3-glucose, ¹³C₆-citrate, ¹³C₅¹⁵N₂-glutamine, and ¹³C₁-aspartate via ultra-high performance liquid chromatography coupled on line with mass spectrometry, we observed that hypoxia in vivo and in vitro promotes consumption of citrate and other carboxylates. These metabolic reactions are theoretically explained by the activity of cytosolic malate dehydrogenase 1 and isocitrate dehydrogenase 1 (abundantly represented in the red blood cell proteome), though moonlighting functions of additional enzymes cannot be ruled out. These observations enhance understanding of red blood cell metabolic responses to hypoxia, which could be relevant to understand systemic physiological and pathological responses to high altitude, ischemia, hemorrhage, sepsis, pulmonary hypertension, or hemoglobinopathies. Results from this study will also inform the design and testing of novel additive solutions that optimize red blood cell storage under oxygen-controlled conditions.

Keywords: flux analysis; hypoxia; mass spectrometry; metabolomics; tracing experiments.

Figure 1

Acclimatization to high-altitude hypoxia decreases steady-state levels of carboxylic acids in human red blood cells. Twenty-one healthy volunteers (12 male and 9 female) were flown from sea level (Oregon) to Bolivia (>5,260 m) for up to 16 days (A), within the framework of the AltitudeOmics study (28, 29). While all of them successfully acclimatized to high-altitude hypoxia (28, 29), red blood cell (RBC) levels (B) of citrate, alpha-ketoglutarate, hydroxyglutarate, and succinate decrease from sea level to high altitude, proportionally to the duration of stay at over 5,000 m. Transient decrease and progressive increases in fumarate and malate were observed, paralleled by increases in the pyruvate/lactate ratios and reduced/oxidized glutathione (GSH/GSSG) ratios, suggestive of a progressively more reducing environment in the cytosol of RBCs from individuals acclimatizing to high-altitude hypoxia. *p < 0.05; **p < 0.01; ***p < 0.001 (repeated measures ANOVA). All data points on x axis were tested [n for each data point is reported in panel (C)].

Figure 2

Packed red blood cell (RBC) storage under controlled hemoglobin oxygen saturation conditions recapitulates high-altitude hypoxia-induced decreases in citrate and accumulation of fumarate/malate. RBCs were stored under normoxic conditions (untreated–SO₂ = 47 ± 21, mean ± SD—solid blue line), hyperoxia (SO₂ > 95%—solid purple line), or four hypoxic conditions (SO₂ = 20, 10, 5, or <3%—solid purple, green, orange, and red lines, respectively). Supernatant citrate was significantly lower than controls (p < 0.05) in SO₂ < 3% hypoxic RBCs at all tested time points. Fumarate was significantly higher than controls (p < 0.05) at storage day 7 and 14, while malate at day 14 onward in all hypoxic RBCs when compared to controls and hyperoxic counterparts. Dotted lines indicate ranges (same color-code—lighter tone). All data points on x axis were tested (n = 4).

Figure 3

Glucose tracing experiments indicate hypoxia-induced increases in carboxylic acids deriving from glucose. Cytosolic isoforms of Krebs cycle enzymes are present in mature red blood cells (RBCs) and can theoretically catalyze the reactions graphed here, reactions that could contribute to RBC reducing equivalent homeostasis. Heavy fumarate and malate accumulation in hypoxic RBCs was significantly higher (p < 0.05) than controls at all tested storage days after day 7. Hyperoxic RBCs had significantly (p < 0.05) lower heavy fumarate than control RBCs only at storage day 42. All data points on x axis were tested (n = 4).

Figure 4

Isotopologue distribution of heavy carbon atoms from heavy citrate, glutamine, glucose, and aspartate indicate a complex rewiring of red blood cell carboxylic acid metabolism in response to hypoxia, as summarized in the panels to the right. Bars indicate median (± SD)% accumulation of heavy isotopologues vs the total levels of the compound, as measured in three independent experiments per each condition (normoxia vs 24 h hypoxia—blue and red bars, respectively). Arrows in the panels to the right indicate metabolic rewiring in normoxia and hypoxia and color-code are consistent with the colors used to identify stable isotope tracers indicated in the four panels to the left. *p < 0.05; **p < 0.01; ***p < 0.001 (T-test to normoxic control).

Figure 5

Relative contribution of metabolic substrates (citrate, glutamine, glucoose, aspartate, other) to the generation of citrate, malate, lactate, glutamate, alpha-ketoglutarate, and 5-oxoproline under normoxic or hypoxic conditions (24 h). Mean ± SD are shown from three independent experiments per condition. Other here indicates either endogenous levels of the metabolite or derivation from other sources than the stable isotope tracers used here. Significant increases in glucose-derived lactate and glutamine-derived glutamate, but not ketoglutarate were observed under hypoxic conditions. Citrate and glucose-derived 5-oxoproline increased significantly (p < 0.05) under hypoxic conditions.