A causal relationship between plasma levels of Cer and the onset of Covid-19 respiratory distress symptoms is claimed. Cer involvement have been highlighted in reports of lung diseases including: acute lung injury, cystic fibrosis, emphysema, lung infections, and asthma (13,16). These reports show pulmonary manifestation to be the main symptoms of Cer mediated toxicity. That Cer’s can regulate major aspects of lung endothelial cell function and are involved in the pathogenesis of several conditions associated with pulmonary vascular dysfunction (16). These pulmonary manifestation symptoms are common with the symptoms also seen in patients with Covid-19 associated respiratory distress (17). The reported high levels of plasma Cer concentration in the obese, and asthmatic individuals is consistent with the observed sensitivity of this group to Covid-19 infection supporting its biological relevance (25).
In this study, we show that in addition to the described biological relevance, association, and concentration dependency, of specific subclasses of Cer in plasma of Covid-19 infected individuals, with respiratory distress symptoms are observed. These findings provide the support for existence of a causal type relationship.
Covid-19 infection causes profound changes in the metabolome of infected individuals, including plasma lipids levels. In this study, changes in plasma levels of 283 lipids covering 8 lipid classes, including PS, PE, PG, Cer, HexCer, Hex2Cer, Hex3Cer has been identified to be associated with Covid-19 infection. Figure 3a, 3b, shows the levels of subclasses of the above classes, as represented by peak area, to be markedly increased in the plasma of Covid-19 infected individuals, describing an association between the identified subclasses of lipids to the Covid-19 infected plasma.
Figure 5a, shows changes in concentration of Cer as a class, in terms of fold induction, the total levels of Cer are increased in plasma of Covid-19 infected individuals with mild symptoms by more than 250-folds or to 500 µmol/l (Fig. 5b). In cases of Covid-19 infected individuals with severe respiratory distress symptoms this total Cer level is increased by over 450-folds or to 720 µmol/l. Demonstrating concentration dependency, the difference between a Covid-19 infection with mild symptoms to an infection with severe respiratory distress response at the lipid class level to be an increase of 220 µmol/l plasma in total Cer class concentration. The predominant Cer subclasses (Fig. 6a and 6b) detected to be associated with these plasma levels in respiratory distress Covid-19 infection are Cer(d18:1/16:0) at 3.63 µmol/l, and Cer(d18:1/24:1) at 95.63 µmol/l. The concentrations of which are 2.79 umol/l (30% less) and 39.57 umol/l (142% less) respectively in plasma of Covid-19 infected individuals with no symptoms of distress.
The Cer(d18:1/16:0), Cer(d18:1/24:1) subclasses were reported to be involved in a cardiac mortality study and to be associated with increased risk of cardiac death outcomes, in patients with stable coronary artery disease (18). In this study we report that Covid-19 infection causes a significantly greater mortality risk with a 10 and a 30-fold increase in plasma levels of these subclasses over the reported cardiac mortality predictions level.
Causality assessment, leads us to believe that monitoring and therapeutic aim of reducing Cer(d18:1/16:0), and Cer(d18:1/24:1) levels in the plasma of patients is required for enhancing survival from Covid-19 respiratory distress. A reduction in plasma levels of the above Cer subclasses (bringing down the observed plasma levels to the level seen associated with mild symptoms) can be an aim to aid in the recovery process of the severe symptoms associated with Covid-19 infection.
Cer are generated by de novo synthesis, salvage of sphingosine, and breakdown of complex sphingolipids, including sphingomyelin. In which the necessary substrates are: monohexocylceramide, sphingomyelin, and dihydroceramide (20). An increase in all three of these substrates is observed in plasma of Covid-19 infected individuals (Fig. 3a, Figs. 6a, and 6b).
To effectively modulate toxic effects of Cer, in the context of Covid-19 infection, is to reduce its circulating concentrations. This can be achieved by inhibiting its biosynthesis or by chemical modification of the circulating Cer in the plasma. The Cer de novo synthesis pathway includes a series of enzymes that produce Cer from the starting components serine and palmitoyl CoA. Researchers seeking to pharmacologically inhibit Cer synthesis in vivo have generally used myriocin, which inhibits serine palmitoyltransferase, the rate limiting initial step in the biosynthesis of all sphingolipids (20).
Ceramide synthase (CerS) catalyzes the acylation of the amino group of sphingosine, sphinganine, and other sphingoid bases using acyl CoA esters. CerSs consists of six enzymes (CerS1–6), with each isoform synthesizing a subset of Cer with partially distinct acyl chain lengths. CerS1 forms Cer18, CerS2 forms Cer24, and CerS6 forms Cer16 ceramide (21). Since, Cer(d18:1/16:0), and Cer(d18:1/24:1) are the predominant subclasses seen in the plasma samples of Covid-19 infected group, focusing on modulation of CerS2 and CerS6 seems to be the logical therapeutic approach. Inhibitors of Cers2 enzyme include the fumonisins, the related AAL-toxin, and australifungins (22).
Reduction of circulating concentration of Cer in the plasma can be achieved by causing chemical modifications with introduction of enzyme or agonist to at least one ceramide-modifying enzymes such as Glucosylceramide synthase, Ceramidase (SMase), Ceramide kinase, and Sphingomyelin synthase. Both acid and neutral SMase inhibitors, as well as inhibitors of the de novo pathway of Cer synthesis, effectively inhibited Cer-induced apoptosis in the lung in various acute or chronic injury models in vivo, as recently reviewed by Uhlig and Gulbins (22). Carpinteiro et al. reported that by pharmacological and genetic inhibition of acid SMase enzyme they can prevent infection of cells with Covid-19, VSV and PP.VSV viruses (21).
Indirect examples of regulators or circulating Cer levels have also been described to include TNF-alpha inhibitors, TLR-4 inhibitors, adiponectin, FGF21, apoptosis inhibitors, and mitophagy inhibitors (23, 24).
Future studies should address more effectively, the limitations of this study, the third criterion for causality which requires that alternative explanations for the observed relationship between two variables to be ruled out (non-spuriousness, or “not false.”). To better address these criteria one would need to directly monitor the effect of lowering plasma concentrations of Cer, treatment with Cer inhibitors, on the Covid-19 mediated respiratory distress. Furthermore, our investigations have focused on the detected subclasses of Cer isolated through a none comprehensive metabolomics analysis paradigm, a more comprehensive approach may lead to identification of more sensitive subclasses of these lipids.
In summary, uncovering causal relationships is an important first step towards understanding disease and predicting the course of future treatments. In this study a causal relationship between plasma Cer plasma concentration and respiratory distress symptoms in Covid-19 patients is presented. Specific subclasses of Cer has been identified that can be used for monitoring Covid-19 infection severity and progression. The causality relationship also defines that modulating ceramide synthesis pathways, and its salvage and its regulatory mechanisms, to be a validated approach towards enhancing survival from Covid-19 respiratory distress.