We identified altered levels of fatty acids, amino-acids, Krebs cycle intermediaries, and α-HA in serum samples from mild and severe COVID-19 patients. The analysis of these metabolic profiles revealed a relationship between an altered metabolism of BCAA, glutamate, glutamine, and Warburg effect with the severity of the disease. In particular, hypoxemia in COVID-19 patients could impair redox, energetic, and immune responses 1. In this context, the term metabolic flexibility refers to the transcriptomic, proteomic and metabolic changes that need to be adapted to particular pathological conditions 10. Globally, hypoxia favors anaerobic glycolysis (over beta oxidation, pentose phosphate pathway and cellular respiration) as a source of ATP to fulfill the energetic requirements of several critical processes such as RNA expression, and protein and lipid synthesis 11.We detected modified levels of three intermediaries of the Krebs cycle. Citrate serum levels fell in severe COVID-19 patients and positively correlated with lower oxygen saturation levels. On the other hand, α-ketoglutarate and malate transiently increased in mild COVID-19 patients, showing a positive correlation with lactate and anion gap markers, both indicators of metabolic acidosis. Although it is unclear whether the extent of these changes were caused by the hypoxic conditions, the analysis of the differential metabolic signatures exhibited by mild and severe COVID-19 patients suggests that citric cycle and Warburg effect play a significant role for discriminating both groups. In normoxia, glucose conversion to acetyl Co-A and glutamine to α-ketoglutarate are the major sources of mitochondrial carbon intermediaries for the Krebs cycle 12. However, in hypoxia, the flux of acetyl-coA is inhibited and glutamine-derived α-ketoglutarate can be reductively carboxylated to isocitrate by isocitrate dehydrogenase as a way to replenish mitochondrial NAD levels and increase citrate pools in the Krebs cycle 12,13. This process has been extensively studied in cancer-derived hypoxia 14. Thus, citrate may diffuse to cytosol where it could be further processed by the enzyme ATP citrate lyase to Acetyl-Co-A and oxaloacetate 15. The cytosolic activity of malate dehydrogenase also might support an enhanced glycolysis through the conversion of oxaloacetate to malate, regenerating NAD 6. In addition to the potential inhibition of the Krebs cycle, ATP citrate lyase and malate dehydrogenase activities could also explain the altered levels of citrate and malate in mild COVID-19 patients.
Glutamine levels decrease in severe and mild COVID-19 patients and this change negatively correlated with lactate dehydrogenase (LDH), C reactive protein (CRP), and PO2 levels, and positively with PCO2; these markers have been associated with lung damage and altered oxygen homeostasis in COVID-19 patients 16,17. In vitro experiments also have demonstrated that hypoxia increases glutamine transport into the cells through HIF2-alpha upregulation of SLC1A5 genes 18. These interconnected processes might potentially link glutamine hypoxic catabolism with NAD recycling and malate synthesis. Nonetheless, further studies are needed to more precisely assess whether glutamine is linked to reductive carboxylation in hypoxic patients.
We observed reduced levels of stearic and decanoic acid in severe and mild COVID-19 patients. These changes showed a positive correlation with oxygen saturation levels. This latter observation is more likely associated with lipid accumulation in tissues or up-regulation of its biosynthetic pathways instead of enhanced β-oxidation, a process inhibited during hypoxia 19. Recently, Bensaad et al20 showed that HIF1-alpha regulates fatty acid uptake and storage in vitro, an effect that may convey beneficial properties on energy and reactive oxygen species (ROS) homeostasis during normalization of oxygen levels 20.
The enrichment pathway analysis identified an altered glutathione metabolism in our cohort of COVID-19 patients. The decrease of cysteine and methionine levels are in agreement with glutathione synthesis, an important antioxidant that becomes depleted by the increment of ROS in hypoxia and several infections 21. The lowering of cysteine negatively correlated with CRP and LDH, and positively with reduced SO2 levels. This could involve a physiological response aimed to restore glutathione levels during SARS-Cov-2 infection; elevated levels of this antioxidant seem essential for a better prognosis in COVID-19 22.
On the other hand, three different α-HA of amino acid origin (α-hydroxyisovaleric acid, α-hydroxybutyric, and 2,3 dihydroxybutanoic acid) were significantly increased in samples from mild and severe COVID-19 patients and positively correlated with CRP and LDH and negatively with SO2 and serum albumin. Reduced levels of this protein have been associated with increased mortality in severe hypoxic hepatitis and COVID-19 23–25. Similar to glutathione, albumin has an antioxidant activity over ROS control in ischemic and hypoxic liver 26. The association between increased α-HA and lower albumin levels may be related to the decline in protein synthesis and modification of amino acid metabolism due to hypoxia 27,28. In this regard, α-hydroxyisovaleric acid is a product of valine catabolism and is used as a marker of maple syrup urine disease (MSUD), a clinical condition derived from inactivating mutations in branched-chain α-keto acid dehydrogenase complex (BCKDH), an enzyme essential for BCAA catabolism 29. BCAA that are not required for protein synthesis are deaminated to α-KA (producing glutamate from α-ketoglutarate) by branched chain aminotransferases (BCAT) and funneled to mitochondrial BCKDH, where they are further metabolized in the Krebs cycle 30. However, in hypoxic conditions, the halt of respiratory chain promotes an increase in NADH/NAD ratio, inactivating BCKDH complex and boosting α-KA levels 31. Subsequently, cellular peroxidases oxidizes the resultant α-KA to α-HA in a NADH dependent manner 32,33. As in MSUD, α-KA and α-HA are increased in patients with respiratory diseases that disturb oxygen homeostasis, and this increment is probably related to BCKDH inhibition and α-keto oxidase activity 31,34,35.
Additionally, we identified modified serum valine, leucine, isoleucine, 2-keto-3-methylvaleric acid, and 3 hydroxyisovaleric acid levels. These changes suggest a modified BCAA metabolism in COVID-19 patients. Noteworthy, we also identified a trend towards restoration of the levels of these amino acids in patients with severe COVID-19 when compared to those with mild disease. In fact, 4-hydroxyproline, a marker of amino acid mobilization from skeletal muscle to liver, displayed a trend towards higher levels in severe COVID-19 patients, suggesting that in severe disease, there is an increased requirement of tissue BCAA in order to replenish NAD.
Threonine levels decreased in severe and mild COVID-19 patients; this decrement could be potentially related to an enhanced catabolism as we also identified increased levels of α-hydroxybutyric and 2,3 dihydroxybutanoic acids, both oxidized metabolites of threonine conversion to α-HA. α-hydroxybutyric acid is synthetized by the activity of lactate dehydrogenase over α-ketobutyrate, a product of methionine/threonine catabolism and cysteine anabolism 36. The increase of this metabolite correlated positively with higher levels of serum LDH. In this context, given that α-ketobutyrate is a substrate of BCKDH, the increase of its corresponding α-HA could be related with the potential inactivity of the mitochondrial enzymatic machinery 37,38. On the other hand, 2,3 dihydroxybutanoic acid is a product of threonine catabolism probably generated by the activity of cytosolic transaminases and oxidases, a conversion that may potentially restore NAD during hypoxia 39. All the enzymes involved in α-KA oxidation rely on NADH (or NADPH) as a co-substrate and their activities are important to maintain the energetic and redox states in health and disease. However, the mechanisms involved in BCAA and threonine intermediaries in hypoxia are still incompletely understood 40.
A mechanism that links HIF1-alpha with the increase in BCAA transport and aminotransferase activity in hypoxic glioma cells was recently described 41. In this cell context, the in vitro accumulation of BCAA in cerebral cortex of normoxic rats promotes glucose internalization and, interestingly, induces lower levels of CO2 release. These data suggest the presence of aerobic or anaerobic glycolysis regulated by the BCAA levels despite the presence of normoxia 42. Interestingly, BCAT exhibits a CXXC motif that presumably acts as redox sensor and predisposes its aminotransferase activity by the cellular redox state 43.
Although the effects of BCAA metabolism in hypoxia are still poorly understood, an evolutive advantage cannot be ruled out. In this regard, cells from the fungus Aspergillus nidulans increase the synthesis of BCAA as a mechanism to regenerate NAD and NADP during hypoxia 44. Interestingly, the in vitro addition of BCAA, such as valine and leucine, rises the survival rate of mice infected with Klebsiella Pneumoniae 45. Our present data suggest that hypoxia promotes the metabolic funneling of BCAA and threonine to α-KA synthesis and then to α-HA as a compensatory mechanism to replenish NAD levels in COVID-19 patients (Fig. 4).
Fig.4. Proposed model for the serum metabolic changes observed in COVID-19 patients. Red arrows represent metabolic flux inhibited during hypoxia. Blue arrows represent increased flux in hypoxia. ME Malic enzyme. The graphs were constructed with the normalized values from control subjects (C), mild (M), and severe (S) patients. Asterisks indicate statistical significance according to the Mann Whitney and Dunn tests. p*<0.05 **<0.01, ***<0.001, ****<0.0001
In support to this hypothesis, we observed decreased serum levels of BCAA and threonine in COVID 19 patients, an effect that has been previously identified in several studies of chronic obstructive pulmonary disease 40. It has been shown that BCAA mobilization from skeletal muscle to liver is essential for human adaptability to lower oxygen levels 46, which is in agreement with our findings. Recent data highlight the relevance of BCAA administration as an strategy to attenuate protein muscle loss in COVID-19 critical patients 47.
An scenario with high amino transferase activity requires a constant flux of α-ketoglutarate, a nitrogen acceptor, which could be achieved through an enhanced glutamine catabolism 12. In this regard, α-KA synthesis mediated by aminotransferases generates glutamate as a byproduct. Accordingly, we observed an increase in glutamic acid levels, which correlated positively with anion gap values in severe COVID-19. Glutamate is an important neurotransmitter that, if unbalanced, promotes several neurological abnormalities 48,49. In fact, patients with hypoxia may exhibit impaired activity of glutamate and several other neurotransmitters involved in homeostasis of the central nervous system, which may explain some of the neurological features found in COVID-19 patients 50.
On the other hand, the appearance of new onset or worsening of pre-existing diabetes has been identified in COVID-19 patients 51. α-hydroxybutyric acid is an early marker of insulin resistance in the non-diabetic population 52,53. Additionally, high levels of 2,3 dihydroxybutanoic acid and α-hydroxybutyric acid were detected in type I diabetes patients 39. Gall et al54 proposes that individuals with an augmented fatty acid metabolism display NADH/NAD imbalance resembling hypoxic conditions that unleash a change in BCAA metabolic fate, predisposing to diabetes development. Although there are still missing paths connecting BCAA metabolism with diabetes, a plausible explanation for the presence of insulin resistance in COVID-19 patients may be partly connected to BCKDH state 55.