The plasma metabolome of long COVID patients two years after infection

Cumulative evidence from the last three years supports the dysregulation of metabolic and immune markers due to SARS-CoV-2 infection¹⁴. A retrospective cohort study has demonstrated that COVID-19 patients have a significantly higher risk to develop subsequent autoimmune diseases such as rheumatoid arthritis, ankylosing spondylitis, systemic sclerosis, type I diabetes mellitus, among others¹⁵. In the present work, our aim was to evaluate the persistence of long-term metabolic alterations in long COVID-19 patients, as well as to measure immune markers that, when chronically produced, can trigger autoimmune diseases.

Since well-defined classification or diagnostic criteria are not available for the long COVID assessment, there is an urgent need for molecular methods able to stratify patients according to the severity of the symptoms they are experiencing. Quantitative and validated scales, such as HAM-A, HAM-D, MoCA and mMRC are considered gold standards for neurocognitive impairment and for dyspnea assessment. However, their practical utility could be limited for complex conditions such as long COVID where a broader range of self-reported symptoms with different severity and duration are present. It has been reported that some long COVID-19 patients complain about extreme cognitive disorders (self-reported symptoms) but without any objective alterations, while others do not report symptoms but exhibit severe cognitive disorders after 6 to 9 months following SARS-CoV-2 infection¹⁶. In our study, several symptoms were corroborated through objective measures, but with lower rates when using validated scales. Therefore, molecular markers are urgently needed for the correct classification of patients.

Our results revealed that 50% of analyzed plasma metabolites showed statistical differences between COVID-19 and long COVID-19 phases in patients with a more complicated evolution. One of the most dysregulated metabolites was glucose. Montefusco et al.¹⁷ reported glycemic abnormalities in recovered patients two months after the onset of disease. The hyperglycemic state has been reported to be even worse in hospitalized patients, pointing to a possible causal role of administered drug regimens, including remdesivir and corticosteroids. These drugs stimulate hepatic gluconeogenesis from amino acids released from muscles, which then inhibits glucose uptake¹⁸. Long COVID has been associated with new-onset insulin resistance which may contribute to the onset of depressive symptoms by enhancing overall neurotoxicity¹⁹.

A number of other metabolites were also found to be dysregulated. Increased plasma pyruvate levels could be both a consequence of glycolytic dysregulation and protein degradation. The increase in putrescine levels in the long COVID phase may be an indicator of increased protein degradation to help fuel pyruvate metabolism.

Taurine and spermidine were found significantly decreased in the long COVID phase, although a trend towards normalization was observed when compared with controls. Decreased levels of serum taurine have been observed in patients with Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS)²⁰. The depleted levels observed in long COVID-19 phase could explain at least in part the fatigue, since taurine has multiple roles in skeletal muscle, the central nervous system, and energy metabolism. Nevertheless, based on our results, we did not find any correlation between fatigue or myalgia and taurine concentrations in long COVID-19 patients. Holmes et al.²¹ found that taurine levels were increased in post-COVID-19 patients, suggesting hepatic injury, hepatotoxicity, or muscle damage. However, the cohort evaluated in the Holmes study had a three-month follow-up after the initial infection, which is much shorter than the follow-up used in this study.

Furthermore, we observed increased levels of kynurenine, and a trend towards normalization in tryptophan and the kynurenine/tryptophan ratio in long COVID-19 patients. This indicates that, although lower in magnitude, the inflammatory conditions attributable to the hyperactivation of this metabolic pathway are still present and may account for some persistent physiological symptoms in these patients. Increased levels of hippuric acid in the long COVID-19 phase could be associated with a residual intestinal dysbiosis. This metabolite has been found increased in patients with chronic kidney disease and several age-related conditions²². An increase in the plasma levels of hippuric acid may also be either the result of an increased fruits and vegetable intake.

Our study revealed increased levels of metabolites associated with collagen metabolism in long COVID patients. Among these metabolites, proline is particularly noteworthy due to its involvement in protein structure and function, as well as its role in maintaining cellular redox homeostasis through the generation of ATP and reactive oxygen species (ROS) during its catabolism. Proline can be synthesized from arginine through various enzymes, including arginase (both type I and type II), ornithine aminotransferase, and P5C reductase²³. The glutamate/P5C synthase pathway in the intestine is responsible for most of the proline synthesis in the body. The increased levels of proline may arise from arginine or glutamine pathways, potentially in response to hypoxia²⁴ or tissue damage. Elevated blood levels of hydroxyproline have been proposed as a biomarker for diseases characterized by fibrosis, indicating an increased demand for proline in collagen synthesis. Trans-hydroxyproline plays a crucial role in collagen synthesis and contributes to the thermodynamic stability of the triple-helical conformation of collagen and associated tissues²⁵. In our study, both class A and class B patients had partial lung recovery, as evidenced by persistent lung function alterations observed in the lung CT scans (Supplementary Fig. 3). However, the measured levels of trans-hydroxyproline did not show a significant correlation with these findings. Nonetheless, the potential presence of collagen vascular diseases cannot be ruled out, as they may contribute to a more widespread systemic dysfunction, sometimes associated with viral infections²⁶.

The increase in glutamine (and decrease in glutamate levels) indicates a partial reestablishment of critical processes that took place during the COVID-19 infection phase, such as severe immunometabolic dysregulation. During COVID-19 phase, a decrease in circulating levels of glutamine has been widely described^{27,28,29,30,31,32}. This depletion is associated with the consumption of glutamine to generate ATP and precursors (purines and pyrimidines) for the synthesis of macromolecules to assemble progeny viruses; to fuel TCA cycle (as in cancer cell metabolism); to regulate the function of immune cells for maximal cytokine production, lymphocyte function, and for the growth, plasma T cell differentiation, and antibody production by B lymphocytes; and to promote interorgan nitrogen exchange, ammonia detoxification, and pH homeostasis. Glutamine/glutamate pathway is therefore closely related to energy metabolism. Dysregulations in this axis have been previously associated with increased risk of type 2 diabetes and other inflammatory diseases^33,34. In the post-COVID phase, by the contrary, an increase in glutamine concentrations and a decrease in glutamate (higher glutamine/glutamate ratio) could be associated with long-term recovery, since glutamine demand for immune activation, nucleotide synthesis and ammonia detoxification has decreased. However, in long COVID patients experiencing multiple symptoms (neuropsychiatric and systemic), immune dysregulation persists, leading to a persistent imbalance in glutamine/glutamate metabolism, since glutamine may act as a signaling metabolite. Disruption within the glutamatergic pathway can lead to important neurological consequences, such as cognitive deficits^35,36. In fact, this exquisitely sensitive glutamine/glutamate homeostasis has been reported disturbed in schizophrenia³⁷ and frontotemporal dementia³⁸. The glutaminergic dysfunction could be associated with some psychiatric and neurologic symptoms like those reported in the present work.

Alterations in lipid metabolism are evident in most long COVID-19 patients. These patients exhibited significantly higher levels of carnitine and some short, medium, and long acylcarnitines. These alterations have been largely associated with altered fatty acid metabolism, dysfunctional mitochondria-dependent lipid catabolism, and immune processes or the lysis of white blood cells. Similar results have been reported by Guntur et al.³⁹, pointing to mitochondrial dysfunction, as was also recognized during COVID-19 acute phase. Besides, decreased levels of LysoPC 16:0, LysoPC 17:0, LysoPC 18:0, and LysoPC 20:4, were found with respect to negative controls. These reductions have been reported in other inflammatory conditions⁴⁰ and other septic processes⁴¹. Depleted levels of lysophosphatidylcholines and phospholipid ethers, as well as depleted levels of PCs, can impede mitochondrial respiration, as has been also demonstrated in ME/CFS⁴². In line with the lipid dysregulation demonstrated by the targeted metabolomic analysis, routine clinical laboratory tests exhibited elevated levels of total cholesterol, triglycerides, and VLDL, as well as normal levels of HDL and LDL. Xu et al.⁴³ found increased LDL, triglycerides, total cholesterol, and decreased HDL in survivors of COVID-19, based on a large observational study with participants from the US Department of Veterans Affairs database compared to controls who had never tested positive for COVID-19.

As positive findings for the metabolic state of long COVID patients, we found that 30 metabolites fell within normal levels. Phenylalanine, which has been widely associated with sepsis and COVID disease severity¹⁴ decreased to normal levels. Beta-hydroxybutyric acid and citric acid were also normalized, indicating partial recovery of the tricarboxylic acid cycle^14,44. Butyric acid and propionic acid, two short-chain fatty acids that were found to be altered during COVID-19 phase, also fell within normal levels in post-COVID-19 patients, probably indicating that the leaky gut phenomenon and gut dysbiosis detected during COVID infection could be partially reestablished⁴⁵. Spermidine was also normalized. The decrease in spermidine levels could reflect a trend for normalization in overall redox balance. Although excessive levels of spermidine (as those reported in COVID-19 patients) trigger the production of superoxide radicals, optimal concentrations mitigate oxidative stress and diminish overall ROS production⁴⁶.

In addition, sphingomyelins and long-chain monounsaturated and saturated LysoPCs were found to be within normal levels. We previously noted altered sphingolipids levels during COVID-19 infection¹⁴. Sphingolipids play a crucial role in the regulation of signal transduction pathways and in certain pathological conditions, such as inflammation-associated illnesses and innate immune response.

In a recent report, Holmes et al.²¹ found a high degree of interindividual variability in follow-up patients, reflecting the heterogeneity of post-COVID-19 patients and the fact that long COVID is a spectrum of disorders. Indeed, computationally modeling of the long COVID phenotype data based on electronic healthcare records found six distinct clusters, each with distinct profiles of phenotypic abnormalities⁴⁷. Since symptom classification is still highly subjective, we decided to arbitrarily classify long COVID patients as: class A (less than five symptoms, mainly neuropsychiatric disorders), and class B (more than five symptoms, with a broad spectrum of systemic disorders). In a recent article⁴⁸, the authors proposed a definition of PASC based on self-reported symptoms, identifying four distinct clusters with concomitant symptoms ranging from two to six, to address the heterogeneity of long COVID. The four PASC subgroups were identified as follows: cluster 1: loss of or change in smell or taste; cluster 2: post-exertional malaise and fatigue; cluster 3: brain fog, post-exertional malaise, and fatigue; cluster 4: fatigue, post-exertional malaise, dizziness, brain fog, gastrointestinal symptoms, and palpitations. Our own classification scale aligns with this study, both in terms of the number and types of symptoms included.

We believe that metabolic information may complement, and partially explain the phenotypic differences among long COVID-19 patients. Xu et al.⁴⁹ classified recovered patients based on abnormal pulmonary functions, finding increased levels of triacylglycerols, phosphatidylcholines, prostaglandin E2, arginine, and decreased levels of betaine and adenosine in patients with abnormal pulmonary function.

In our work, lactic acid levels were increased in patients with more than five symptoms and systemic disorders (class B patients). Ghali et al.⁵⁰ found that patients with ME/CFS exhibited elevated blood lactate at rest. Mitochondrial dysfunction, with increased blood lactate, low levels of ATP, and increased levels of oxidative stress markers have been associated to these alterations⁵¹, as well as relative deficiency of mitochondria type I fibers on muscle biopsies, and low intracellular pH during recovery phase^52,53,54. De Boer et al.⁵⁵ also reported altered lactate levels in long COVID patients, suggesting that long COVID patients have significant impairment in fat beta-oxidation and increased blood lactate accumulation even during low-intensity exercise. In contrast, Guntur et al.³⁹ reported low levels of lactic acid and pyruvate in long COVID patients. However, this study was conducted in non-hospitalized patients who had recovered from COVID-19 in March 2020.

Increased level of the lactate/pyruvate ratio in class B patients is another important indicator of mitochondrial dysfunction. The lactate/pyruvate ratio has been proposed as a marker for mitochondrial disorders since it indirectly reflects the NADH/NAD + redox state⁵⁶, lipid metabolism (fat oxidation), and ATP generation. In our study, both markers (lactate and the lactate/pyruvate ratio) were found positively correlated with fatigue, myalgia and arthralgias (Spearman correlation, R > 0.6, p < 0.05).

The increased ornithine/citrulline ratio level in class B patients reflects abnormal metabolic activity in the urea cycle. It is notable that Yamano et al.⁵⁷ reported a similarly increased ornithine/citrulline ratio in CFS patients. An adequate balance of citrulline and ornithine is vital for the clearance of ammonia via urea cycle⁵⁸. If ammonia accumulates intracellularly, the aerobic utilization of pyruvate to feed the TCA cycle is inhibited, resulting in lactate production, which further contributes to fatigue.

In addition, class B patients had decreased levels of arginine in comparison with the other subgroups. The reduced bioavailability of arginine to produce adequate levels of nitric oxide in endothelial cells and vascular tissues leads to the impairment of multiple physiological functions of skeletal muscles, including contractile functions, and muscle repair⁵⁹. Arginine is also a substrate for ornithine production by arginase. It is well known that under certain inflammatory conditions, arginase activity is increased⁵⁹, producing an excess of ornithine and an imbalance in the urea cycle.

Previous studies have pointed to the persistent immune dysregulation following COVID-19 infection⁶⁰. We found increased levels of monocytes in class B patients. Nuber-Champier et al.⁶¹found that monocyte percentage in the acute phase of the disease allowed them to distinguish between patients with anosognosia for memory deficits in the chronic phase (6–9 months after SARS-CoV-2 infection) and nosognosic patients.

We also measured IL-17 levels in post-COVID-19 patients since it is well known that this cytokine is persistently altered in several chronic inflammatory and autoimmune diseases⁶², and previous reports have indicated an increased risk of such diseases in COVID-19 patients¹⁵. IL-17 is a proinflammatory cytokine mainly produced by T helper type 17 cells, playing a vital role in the regulation of host immune response against SARS-CoV-2. IL-17-induced dysregulated immune responses have been shown to potentially cause hyperinflammatory COVID-19 disease⁶³. It has been reported that IL-17 downregulates protein phosphatase 6, resulting in increased arginase-1 expression in psoriatic keratinocytes⁶⁴. IL-17A has been found to be associated with neurological sequelae and pulmonary fibrosis in post-COVID-19 patients^65,66. Fluctuations in IL-17 have been associated with fatigue and fatigue severity in ME/CFS patients⁶⁷. Additionally, we measured leptin as it is believed to cause inflammatory fatigue. Leptin is produced mostly by adipose tissue and plays a role in regulating food intake, basal metabolism, and the β-oxidation of fatty acids. In metabolic diseases such as obesity, chronically elevated levels of leptin are observed, which can induce the production of proinflammatory molecules and impair immune self-tolerance, predisposing to develop conditions such as rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, and others⁶⁸. Increased levels of leptin have been associated with higher fatigue scores in people with CFS⁶⁹. In a study conducted by Stringer et al.⁶⁹, the authors demonstrated that daily fatigue severity was significantly correlated with daily serum leptin levels in women with CFS. However, in our study, we did not find any statistical differences in leptin levels, despite observing higher levels in class B patients. Based on our results, levels of leptin cannot be associated with fatigue, which suggest that the action of IL-17 on metabolic pathways may play a more significant role in this regard.

Metabolomics is not only useful in providing a snapshot of transient physiological or pathophysiological processes taking place in a living organism, but it has also proven to be a powerful tool for proposing and monitoring therapeutic interventions. In the case of long COVID, a common situation worldwide is that patients have reported an absence of adequate support and a poor recognition of their condition, initially attributed to psychiatric issues. People with long COVID have tried a vast range of self-prescribed medicines, supplements, remedies, and dietary changes to manage the disease and to overcome the effects it has on their quality of life and work capacity. Based on our findings, some interventions could be tested for treating long COVID patients: (1) supplementation of taurine (reducing musculoskeletal disorders); (2) supplementation of citrulline (enhancing ammonia clearance and reducing blood lactate, as well as increasing arginine bioavailability for adequate NO production); (3) supplementation of glutamine (primary source for neurotransmitters and immune function balancing); (4) supplementation of antioxidants such as N-acetylcysteine or NAD + (redox balance); (5) supplementation of arginine (targeting endothelial dysfunction in Long-COVID), as has been previously suggested by Tosato et al.⁷⁰. Similarities found in our results with the ME/CFS pathophysiology may pave the way to common therapeutic interventions for both diseases.

We need to acknowledge several limitations with this study. The small sample size was due to the limited number of patients who agreed to participate. While several objective measures of mood and cognition (HAM-A, HAM-D, MoCA mMRC) were used, the sample size did not allow for stratification of patients according to the different test scores obtained, and only self-reported symptoms were used for sub-group classification. Furthermore, we were unable to have a detailed tracking of treatments, medications or alternative therapies during the period evaluated. This limited our interpretation with regard to the impact of pharmacological interventions on the metabolome. Also, some compounds such as hexoses were measured by direct injection (DI); therefore, it was not possible to differentiate glucose (the most abundant sugar) from its epimers. We did not have access to Ct values from the electronic files of the patients indicating the initial viral load. All the patients were infected with the original strain, which was the predominant strain circulating in 2020. There are limited studies examining the association between the initial viral load, as determined by Ct values, and the various long COVID-19 effects⁷¹. A study conducted in Mexico found that patients with a low Ct experienced between 15 and 20 symptoms, while patients with a high Ct value experienced fewer than six symptoms. Asymptomatic patients with Ct values between 33–36 showed no or very few post-COVID-19 symptoms⁷². However, in larger studies conducted more recently⁷³, the focus has shifted towards viral persistence as a potential mechanism for long COVID, rather than solely considering the initial viral load.

Patients from two different hospitals participated in our study: one private hospital located in Chihuahua city, and one public hospital located in Zacatecas city. In general, private hospital patients have relatively high incomes while public hospital patients have lower incomes. Logistic regression models showed no effects of sex, age, comorbidities, vaccination status or severity during the acute phase in the metabolomic profile associated with long COVID. However, patients from the public hospital reported more systemic symptoms in general, while patients from the private hospital reported principally neuropsychiatric symptoms. A recent study found that patients diagnosed with a post-COVID-19 condition were more likely to be unemployed or on public health insurance, illustrating racial and social disparities in access to and experience with healthcare, at least in the USA⁷⁴. Whether the socioeconomic conditions and lifestyles, along with causes of biological origin influence the metabolic phenoreversion of patients recruited in our study, needs to be further investigated. This is particularly important in countries with significant health system disparities and significant differences in population life conditions.

At the moment of this study, most of the negative controls recruited in 2020 were tested positive for COVID-19 in subsequent waves in 2021 or 2022. Therefore, we could not compare the prevalence of symptoms in post-COVID-19 patients with a non-COVID-19 matched group. In a study conducted by Ballering et al.⁷⁵, of the 76 422 participants longitudinally surveyed before and after COVID-19, 4231 (5.5%) had COVID-19 and were compared with 8462 matched controls. The proportion of participants who had at least one core symptom of substantially increased severity to at least moderate was 21.4% in COVID-19 participants versus 8.7% in controls, suggesting that core symptoms were attributed to COVID-19 in 12.7% of participants. These numbers are at present increasing as more epidemiological data are reported worldwide.