A new study by researchers from University of Otago-New Zealand, University of California-Davis-USA, University of Alabama at Birmingham-USA, University of Queensland-Australia and Birmingham VA Medical Center-USA has found that that vitamin B9 or Folic Acid supplementation can actually increase risk of SARS-CoV-2 infection by 1.5 times and mortality by 2.6 times compared to those that do not take these supplements.
The study findings were published on a preprint server and are currently being peer reviewed. https://www.medrxiv.org/content/10.1101/2022.02.10.22270804v1
Folate, a B-vitamin, carries out critical roles in the transfer of one-carbon units in intermediary metabolism. Folates exist in various forms depending on the one-carbon substituent attached to the parent molecule and are involved in numerous reactions, including the synthesis of methionine from homocysteine and are also utilized in purine and pyrimidine metabolism for DNA and RNA synthesis.
The oxidized form, folic acid, is added to fortified foods in the USA and over 80 other countries, including, recently, the UK1 to prevent neural tube defect pregnancies and is used in dietary supplements to prevent or treat folate deficiency.2
Additionally, folic acid supplementation of up to 5mg daily is often advised during pregnancy and in women of childbearing age and for other medical conditions (sickle cell anemia) 3 and during treatment with certain anticonvulsants 4.
Methotrexate, a structural analogue of folate has potent antifolate activity and is in widespread use as an antineoplastic agent and as a first-line disease-modifying antirheumatic drug (DMARD) treatment for rheumatoid arthritis (RA).5
The COVID-19 Global Rheumatology Alliance physician-reported registry has evaluated factors related to death from COVID-19 in individuals with rheumatic diseases.8 Compared with those receiving methotrexate monotherapy, use of rituximab (OR 4.0 [95% CI 2.3 ; 7.0]), sulfasalazine (3.6 [1.7 ; 7.8]), azathioprine, cyclophosphamide, cyclosporine, mycophenolate, and tacrolimus (2.2 [1.4 – 3.4]) or no DMARD (2.1 [1.5 – 3.0]) all had higher risks of death from COVID-19.
In an in vitro system using African green monkey kidney cells infected with SARS-CoV-2 intracellular glucose and folate were depleted, and this perturbation was sensitive to folate inhibitors such as methotrexate. 9
It is therefore plausible that methotrexate therapy for RA could have a beneficial effect on COVID-19 outcomes given its antifolate activity. However, since folic acid is routinely included with methotrexate to prevent methotrexate-related toxicity, such putative beneficial effect of methotrexate on viral proliferation and hence on COVID-19 outcomes may be negated by folic acid supplementation.
The aim of this study was to determine whether the use of methotrexate and folic acid prescription, together or individually, were associated with a lowered or increased risk, respectively, for COVID-19 diagnosis or mortality in a large population based-cohort.
In this population-based analysis, we report 1.5-fold increased risk for COVID-19 diagnosis and 2.6-fold increased risk for COVID-19-related death among those who had been prescribed folic acid supplementation. The prescription of methotrexate was not associated with an increased risk of diagnosis of COVID-19 and we were not able to make an estimate for COVID-19-related death in the small sample of those prescribed methotrexate only.
Notably, those prescribed methotrexate and folic acid did not have an increased risk for COVID-19 diagnosis or associated death, indicating that methotrexate might attenuate an increased risk for COVID-19 diagnosis and death conferred by folic acid.
In the context of SARS-Cov-2 infection it is established that hijacking of cellular metabolic pathways is important for viral replication.17 Zhang et al described that SARS-CoV-2 remodels host folate and one-carbon metabolism at the post-transcriptional level to support de novo purine synthesis, bypassing viral shutoff of host translation. 9
This suggests that viral replication could be sensitive to folate inhibitors, such as methotrexate. Intracellular glucose and folate are depleted in SARS-CoV-2-infected cells, and viral replication is exquisitely sensitive in vitro to inhibitors of folate and one carbon metabolism, notably methotrexate.9
Stegmman et al, based on cell culture experiments, reported that methotrexate alone or in combination with remdesivir limits the replication of SARS-CoV-2.18 With the caveat that our study is observational epidemiology and causality cannot be inferred, our study does support the possibility that external folate supply facilitates the production of large amounts of virus, contributing to clinical infection and mortality.
Our study also supports the notion that SARS-CoV-2 replication is enhanced by folate supply by our finding that co-prescription of an antifolate (methotrexate) can ameliorate the possibly adverse effect of supplementation with folic acid on COVID-19 outcomes.
There is also evidence that inadequate folate status may be harmful in the context of host resistance to infection with SARS-CoV-2. In addition to the well-recognized complication of anemia, folate deficiency has other detrimental health effects, including suppression of immune function.19
Additional support for the concept that adequate folate status is important in COVID-19 outcomes is provided by the observation that folate deficiency was associated with poorer outcomes in a cohort of COVID-19 patients.20 (It is important to note that it is possible that in the study by Itelman et al,20 if increased folate levels were causal of COVID-19 diagnosis and poor outcomes, that the association with lower folate levels could have been caused by selection (collider) bias.21) Vitamin B12 deficiency has also been proposed as a factor related to poor COVID-19 outcomes, presumed to be through the induction of functional folate deficiency.22
A drug-protein structure interaction analysis raises the possibility that folate blocks the 3CL hydrolase enzyme, which may affect viral entry and replication.23 It is therefore possible that both inadequate and excessive amounts of folate may be detrimental to host resistance to SARS-CoV-2 infection and that there may be an optimal range of physiological folate status related to host-resistance to COVID-19 infection and severity.
Data from the COVID-19 Global Rheumatology Alliance describes that a number of immunomodulatory drugs used in rheumatology are associated with an increased risk of infection and death compared with methotrexate. 8 Being on no DMARD therapy was associated with an increased risk of death with COVID-19 (OR 2.11 [1.48 ; 3.01]), which could be interpreted as either a protective effect of methotrexate or an increased risk for death associated with poor rheumatic disease control.
The authors of the study additionally noted that people not on DMARD therapy had increased use of glucocorticoids meaning that confounding by indication cannot be ruled out as an explanation.24 Methotrexate was also associated with lower odds for death when compared with sulfasalazine, other immunosuppressants and rituximab. In no case was methotrexate associated with an increased risk for death.
The COVID-19 Global Rheumatology Alliance study did not explore the effect of folic acid supplementation in the setting of methotrexate, although it is it is highly likely that almost all patients on methotrexate also were receiving folic acid supplementation.
Considering the widespread use of folic acid supplements and proposals to abandon entirely tolerable upper intake levels for folic acid 25 it would be prudent to monitor the effect of increased folic acid intake at a population level on COVID-19 morbidity and mortality, particularly at the upper end of folic acid intake.
A number of limitations of our analysis are important to note. One, given the small size of the methotrexate-only group and that there were no deaths related to COVID-19 in this group we could not test a beneficial effect on mortality of methotrexate in isolation. It is uncommon to find patients with methotrexate prescribed without supplemental folic acid as this is the standard of care.
Second, over the time period of this study (March 2020 – November 2021), COVID-19 outcomes (i.e. death) will have been influenced by the development of clinical treatments including antiviral drugs and monoclonal antibodies, changes to public health measures and the appearance of new COVID-19 strains 16.
We were unable to account for these factors in the population-based analysis however we attempted to account for this in the analysis within the COVID-19-positive group by including a time variable (Table S4). Third, findings are not necessarily generalizable outside of the middle-aged (>45 years of age) white European cohort that dominates the UK Biobank.
Fourth, the full extent of SARS-CoV-2 infection is not known in the UK population due to incomplete testing rates early in the pandemic. Fifth, prescription data were single script from General Practitioners only, and it was not possible to ascertain compliance or whether participants were taking the prescribed medication during the COVID-19 pandemic although we attempted to account for this by only using prescription information from 2019 and 2020.
Sixth, although we included RA in Model 2, we were unable to account for any potential effect of disease activity in RA in people prescribed folic acid. Disease activity negatively impacts death from COVID-19 outcomes.8 Finally, while it is a strength of our study that mandatory fortification of the UK diet with folic acid had not been introduced during the period of our study and thus did not confound our analysis, we were unable to account for the lower-dose over the counter folic acid supplementation available in the UK (400 micrograms being the most common formulation for over the counter tablets) because there were no self-report data on the use of folic acid supplementation.
In conclusion, and despite the limitations of our study enumerated above, our data support the hypothesis that increased folate resulting from folic acid prescription could contribute to a higher probability of contracting clinically-detectable infection with SARS-CoV2 and to an increase in the risk of death following the infection.
The study population was drawn from the >45 year old segment of the UK population and is predominantly of white European ethnicity, therefore our findings have reduced generalizability to younger people, to other ethnic groups and to other countries. Nevertheless, our findings justify future studies on the influence of folic acid supplementation on COVID-19 outcomes, particularly in pregnant women and people on anticonvulsants requiring supplementary folic acid.
As a final comment, we point out that attention is currently being directed toward establishing whether excessive intake of folate, particularly in the form of folic acid, may have undesirable and potentially deleterious effects.26 The possibility that susceptibility to COVID-19 infection and its serious and even fatal complications may be affected by folic acid intake and folate status should be thoroughly investigated.