Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an enveloped positive-sense RNA betacoronavirus that appeared in Wuhan (China) in December 2019 and is the cause of coronavirus disease 2019 (COVID-19). COVID-19 has a variable clinical presentation from asymptomatic to milder symptoms or, far more seriously, as a severe and often deadly respiratory disease (Chen et al., 2020). The initial symptoms of COVID-19 mainly include fever, cough, myalgia, or fatigue and some patients develop gastrointestinal problems, such as diarrhoea and vomiting. In the later stages of the disease may occur dyspnea, hypoxia, confusion, or chest pain and gradually progress into complications such as acute respiratory distress syndrome (ARDS), which may result in lung injury, decreased oxygen saturation, and widespread tissue damage, thus, causing a multiple organ system failure, unfavourable prognosis, and even death (Hu et al., 2021, 2020).

The main cause of COVID-19 disease severity and death in patients is the extreme inflammatory response caused when the damaged cells trigger lung inflammation, largely mediated by proinflammatory macrophages and granulocytes, along with severe lymphopenia, neutrophil activation, thrombosis, and massive mononuclear cell infiltration in multiple organs (Merad and Martin, 2020). Accordingly, severe or fatal cases of COVID-19 disease are associated with elevated levels of interleukin-6 (IL-6), IL-7 and reduced levels of cytokines related to T-cell antiviral activity, such as IL-2, IL-12 or interferon (IFN)-γ (Vigón et al., 2021), as well as other laboratory abnormalities that include elevated levels of urea, creatinine, tissue markers (adipose tissue, kidney and muscle), ferritin, C-reactive protein (CRP), lower counts of lymphocytes (<1,000/µL), and platelets (<100 × 10⁹/L), as well as reduced levels of albumin (Goyal et al., 2020; Malik et al., 2021).

For a long time, no evidence-based or clinically demonstrated strategy was shown for the treatment of COVID-19 (Mishra and Tripathi, 2021). Different drugs, designed for treating other diseases, have been used to limit the adverse effects of viral pathogenesis, such as antivirals (e.g., remdesivir), IL-6 inhibitors (e.g., tocilizumab), Janus kinase (JAK) inhibitors (e.g., baricitinib), or corticosteroids (e.g., dexamethasone, methylprednisolone). The excessive immune response and inflammation in severe COVID-19 patients have prompted many clinical studies to use a combination of different drugs (Esposito et al., 2020). As a consequence, FDA recently authorized the simultaneous administration of nirmatrelvir and ritonavir for the treatment of mild-to-moderate COVID-19 in children and adults, as well as oral molnupiravir for adults, but this authorization has only been issued in case of emergency for those individuals with high risk to progress to severe disease (Couzin-Frankel, 2021; Parums, 2022).

Risk factors such as older age, cardiovascular disease, diabetes mellitus, chronic respiratory disease, arterial hypertension, and cancer are associated with a higher probability to develop a severe form of COVID-19 (Huang et al., 2020; Jordan et al., 2020; Yang et al., 2020). In addition, there is now sufficient evidence to indicate that pre-existing conditions such as vitamin D deficiency may also predispose to SARS-CoV-2 infection and increase COVID-19 severity (Bergman, 2021; Rhodes et al., 2021; Seal et al., 2022). Vitamin D is widely known for its role in calcium and phosphate balance but supports also key functions in many organs, including the brain, muscle and the immune system (Carlberg and Muñoz, 2022; Holick, 2022). Its immunomodulatory role promotes the development of an anti-inflammatory and antiviral environment and consequently, its deficiency seems to contribute to airway and gastrointestinal infectious illnesses (Ilie et al., 2020; Watkins, 2020). It has been suggested that vitamin D₃ supplementation may be used prophylactically as an affordable and safe treatment strategy that could be added to existing COVID-19 protocols. However, there is still no clear evidence that vitamin D₃ supplementation can prevent the severity and/or mortality of COVID-19.

Accordingly, the main objective of this study was to evaluate the safety, tolerability and effectiveness of vitamin D₃ supplementation to reduce the duration and severity of COVID-19 in hospitalized individuals who were recruited for a randomized pilot clinical trial to receive the moderate dose of 2000 International Units (IU)/daily of cholecalciferol or a higher dose of 10,000 IU/daily for 14 days, in combination with the standard drug therapeutic regimen.

Vitamin D is a fat-soluble secosteroid hormone with a well-defined spectrum of immunomodulatory, anti-inflammatory, and antioxidant actions (Ebadi and Montano-Loza, 2020). Its deficiency is considered a public health issue that has been identified in over half of the population worldwide in people of all age groups (Rubin, 2021). Vitamin D deficiency has been associated not only with non-communicable diseases but also with increased susceptibility to infectious diseases; especially, upper respiratory tract infections (RTI) (Cannell et al., 2006; Ginde et al., 2009) and higher risk of community-acquired pneumonia (Zdrenghea et al., 2017; Zhou et al., 2019). Similarly, there is now sufficient evidence to support that vitamin D deficiency may predispose to SARS-CoV-2 infection and increase COVID-19 severity and mortality (Rhodes et al., 2021; Seal et al., 2022; Wang et al., 2022) due to several mechanisms: first, vitamin D reduces lung permeability by modulating the renin-angiotensin system (RAS) pathway and angiotensin converting enzyme 2 (ACE2) expression; second, it promotes the production of antimicrobial peptides in the epithelium of the respiratory tract, thereby reducing the possibility of virus infection and COVID-19 symptoms; third, vitamin D favours an anti-inflammatory environment, mostly by reducing the concentration of pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α and IFNγ and by increasing the expression of anti-inflammatory cytokines by macrophages; and finally, vitamin D stabilizes the physical barriers by contributing to the maintenance of functional tight junctions, gap junctions and adherence junctions (Pedersen et al., 2009; Baeke et al., 2010). On the other hand, COVID-19 is characterized by the dysregulation of the inflammatory response, especially the RAS pathway. Consequently, the degree of immune overactivation has been associated with a poorer prognosis (Hu et al., 2021). Accordingly, the use of anti-inflammatory drugs during COVID-19 has been proved to be beneficial, as occurs with corticosteroids (Recovery Collaborative Group et al., 2021).

Vitamin D deficiency has been linked to geographical differences in ARDS and COVID-19 mortality (Marik et al., 2020). Therefore, vitamin D₃ supplementation could be used prophylactically to reduce the severity of COVID-19, especially in those regions where hypovitaminosis D is frequent (Panarese and Shahini, 2020). Despite Spain is a country with an adequate amount of daily sunlight, the overall vitamin D intake is lower than the recommended levels of 10 µg/day and consequently, the prevalence of deficient 25(OH)D levels among the Spanish population is high (González-Rodríguez et al., 2013; Olza et al., 2017). Indeed, 78.8% of the participants recruited for our study showed basal 25(OH)D serum levels <20 ng/ml (50 nmol/L). A similar study that was performed in adults who were hospitalized for mild to moderate COVID-19 and were supplemented with a 5,000 IU/day dose of vitamin D₃ during the same time (14 days), serum 25(OH)D levels raised to sufficiency (≥30 ng/ml) (Sabico et al., 2021). However, these individuals were younger than our cohort and showed median baseline serum 25(OH)D levels that were substantially higher. Accordingly, we decided to provide a higher dose of cholecalciferol (10,000 IU) that was administered daily due to the protective effects are expected to be higher when the individuals received daily or weekly doses versus high dose boluses (Martineau et al., 2019). After the daily supplementation of 10,000 IU cholecalciferol for 14 days, 93% of the participants achieved an increase of serum 25(OH)D levels ≥20 ng/ml versus 32% of the individuals who received the 2000 IU/day dose. Although there was a significant 1.5-fold increase of serum 25(OH)D levels that were close to sufficiency in those participants who received the higher dose of cholecalciferol (29 ng/ml; IQR: 25–34) in comparison to those participants that received the moderate dose (17 ng/ml; IQR: 14–23), the primary outcome of a serum 25(OH)D concentration ≥30 ng/ml after 14 days of treatment was not achieved. This could be due to 25 [OH]D is a negative component of the acute phase response (Waldron et al., 2013; Silva and Furlanetto, 2015) and it is usually reduced during acute inflammatory procedures such as COVID-19 (Antonelli and Kushner, 2021). In fact, the deficiency of vitamin D at hospital admission in individuals with COVID-19 has been appointed as a potential prognostic marker of disease severity that would indicate the progression to the inflammatory form of the disease (Chiodini et al., 2021; Seal et al., 2022). Accordingly, the achievement of the primary endpoint of >30 ng/ml of 25 [OH]D in individuals with COVID-19 may be impaired due to the inflammatory process, even with the higher dose of 10,000 IU/day. However, we determined that the administration of the higher dose of 10,000 IU/day was safe and well tolerated by all the participants in the study as we did not observe episodes of hypercalcemia, hypercalciuria, or hyperphosphatemia that are considered to be the initial signs of vitamin D intoxication, or any other related adverse events.

Previous data from other intervention trials showed a preventive effect of vitamin D₃ supplementation in acute RTIs that was increased in patients with vitamin D deficiency (Martineau et al., 2017; Jolliffe et al., 2021). However, in individuals hospitalized with COVID-19, the clinical effects of the use of vitamin D₃ supplementation have yielded mixed results. A Spanish population-based study concluded that patients on cholecalciferol treatment had a lower risk of SARS-CoV2 infection, lower risk of severe COVID-19 and lower COVID-19 mortality than unsupplemented 25(OH)D-deficient patients (Oristrell et al., 2022), and a case-series study in COVID-19 patients who received 50,000 IU/day for 5 days showed a reduction in the inflammatory markers and time for recovery in comparison to those who received 1,000 IU/day (Ohaegbulam et al., 2020). Similarly, a previous RCT in Spain demonstrated that the administration of a high dose (0.532 mg) of calcifediol (25(OH)D₃) significantly reduced the need for ICU admission (Entrenas Castillo et al., 2020), and a placebo-controlled, double-blinded clinical trial reported that in 106 hospitalized patients, those who received vitamin D had a lower trend for hospitalization, ICU duration, need for ventilator assistance and mortality (Maghbooli et al., 2021). On the contrary, data from previous studies and a systematic review evaluating vitamin D₃ supplementation found low to moderate certainty evidence that a single high dose of vitamin D₃ is effective to reduce mortality, LOS, ICU admissions, and D-dimer or CRP levels (da Rocha et al., 2021; Murai et al., 2021). Our results did not report a reduction in the LOS or the ICU admissions when the administration of a high dose of vitamin D₃ for 14 days was compared with the administration of a moderate dose. However, the oxygen requirements measured by FiO2 were significantly reduced in those patients assigned to the high dose group, thereby confirming previous reports (Ohaegbulam et al., 2020). Interestingly, those participants with ARDS from our cohort who received the higher dose of vitamin D₃ showed a significant reduction in the LOS of 23.5 days on average in comparison with those patients who received the moderate dose. Despite the small number of patients who accelerated their recovery, these results achieved statistical significance and they were in accordance with previous studies that support that a higher vitamin D₃ status is associated with a significant reduction in infectivity, morbidity and mortality from SARS-CoV-2 infection (Seal et al., 2022; Wang et al., 2022) and also that treatment with vitamin D₃ during COVID-19 may also shorten the hospital stay and decrease mortality (Gönen et al., 2021). Moreover, according to previous observations in animal models (Yang et al., 2016), the higher dose of vitamin D₃ in patients with ARDS would increase the expression levels of ACE2, inducing moderate lung changes that might contribute to reduce the symptoms of this syndrome. In our study, the potential positive effect of vitamin D₃ could be related to the early administration just after hospitalization, as the accelerated improvement of 25(OH)D₃ levels may develop an anti-inflammatory environment, thereby helping to reduce the length of stay, ICU admission, and mortality in COVID-19 patients (Holick, 2022). Finally, the biochemical inflammatory parameters related to poor prognosis improved mainly during the first week, regardless of the dose of vitamin D administered, as we did not observe significant changes in the serum levels of LDH, ferritin, CRP, or D-dimer serum levels between the two groups. Although the platelet count showed a modest but significant difference in those patients who were supplemented with the higher dose, these changes may not be clinically meaningful.

On the other hand, a high prevalence of vitamin D deficiency has been described in obese and OW patients (Pereira-Santos et al., 2015), likely caused by a dilution into the greater volumes of fat, serum, liver, and muscle that are present in obese individuals (Vranić et al., 2019), although other mechanisms, such as the sequestration of vitamin D by the adipose tissue may not be completely discarded (Wortsman et al., 2000). Due to risk factors such as excess visceral fat or high BMI have been associated with a higher probability to develop more severe forms of COVID-19 (Zhou et al., 2019), a possible link may exists between COVID-19 severity, vitamin D deficiency and BMI. In our study, significant increases in serum 25(OH)D levels were observed among obese and OW participants who received the highest dose in comparison with those who received the moderate dose, while the serum levels in normal weight participants were not different between the groups. However, most of our participants were either OW or obese, as these factors are related to a higher probability to develop severe COVID-19. Therefore, we may not rule out that this imbalance in the BMI between the participants might be affecting the results. Accordingly, the effectiveness of vitamin D₃ supplementation in these individuals with high BMI to positively influence the clinical recovery from COVID-19 needs to be further analyzed by interventional studies.

This study presents two potential limitations. First, our study was not designed as a double-blinded RCT due to the Pharmacy services of the different hospitals needed to change the commercial medication into non-commercial syringes to be supplied to the participants that had been assigned to each treatment group, which made necessary that both the Pharmacy service and the Researchers knew the treatment group to which the participant had been assigned. Therefore, we cannot rule out that the single-blinded RCT design might have biased the assessment of more subjective parameters (e.g chest X-ray) in favour of those participants who received the high dose. On the other hand, the beneficial effect of vitamin D₃ supplementation over the reduction of the LOS in individuals who developed ARDS have been described in a small fraction of the participants (10/85), and although these results reached statistical significance, they need to be considered with caution until confirmation in a larger cohort.

In conclusion, the supplementation of a high dose of 10,000 IU/day of cholecalciferol for 14 days in hospitalized patients with COVID-19 was well tolerated and did not cause significant adverse events in comparison with the moderate dose of 2000 IU/day. The higher dose was more effective than the moderate dose to induce a significant increase of 25(OH)D levels in serum, especially in OW and obese individuals, although the participants did not achieve the target level (≥30 ng/ml). Moreover, the results obtained in our cohort supported that a higher dose of vitamin D₃ supplementation in association with the standard clinical care may be effective to improve the oxygen requirements during hospitalization by COVID-19 and to reduce the LOS in those individuals who developed ARDS. Although this study supports the notion that vitamin D₃ supplementation during COVID-19 may be safe, tolerable and beneficial, more randomized clinical trials are needed to determine the true efficacy of vitamin D supplementation as part of the therapeutic arsenal used against COVID-19 disease.

David Alonso-Menchén (Hospital Universitario Príncipe de Asturias), Adriana Álvarez Jusdado (Hospital Universitario Severo Ochoa), Daniel Barranco Maroto (Hospital Universitario Severo Ochoa), Ytsel Bello Pastor (Hospital Universitario Severo Ochoa), Cristina Córdoba Chicote (Hospital Universitario Severo Ochoa), Guillermo Cuevas Tascón (Hospital Universitario Infanta Leonor), Víctor Díez Viñas (Hospital Universitario Infanta Leonor), Inés Dorado Manzanares (Hospital Universitario Severo Ochoa), Ismael Escobar Rodríguez (Hospital Universitario Infanta Leonor), Eva Fonseca Aizpuri (Hospital Universitario de Cabueñes), Rosa María Galán Zuheros (Hospital Universitario Severo Ochoa), Concepción García Lacalle (Hospital Universitario Severo Ochoa), Mario García Peña (Hospital Universitario Severo Ochoa), Sebastiano Garotti (Hospital Universitario Severo Ochoa), Marta Gómez Sanz (Hospital Universitario Severo Ochoa), Tamara Gómez Zuil (Hospital Universitario Severo Ochoa), María González Pozuelo (Hospital Universitario Infanta Leonor), Cristina Helguera Amezua (Hospital Universitario de Cabueñes), María del Carmen Herrero Alonso (Hospital Universitario Fundación Alcorcón), Javier Marcos Arias (Hospital Universitario Fundación Alcorcón), Emilio José Martínez Martín (Hospital Universitario Fundación Alcorcón), Javier Montoya Adarraga (Hospital Universitario Infanta Leonor), Patricia Moreira Escrich (Hospital Universitario Severo Ochoa), María Jesús Moro Álvarez (Hospital Universitario Infanta Leonor), Paula Novo González (Hospital Universitario Severo Ochoa), Raquel Paz Peño (Hospital Universitario Severo Ochoa), Alejandro Pérez Cachaldora (Hospital Universitario Severo Ochoa), Miriam Rodríguez Fernández (Hospital Universitario Severo Ochoa), María del Carmen Romero Pérez (Hospital Universitario Severo Ochoa), José Sanz-Moreno (Hospital Universitario Príncipe de Asturias), Mercedes Toral Leiva (Hospital Universitario Severo Ochoa), Nuria Toro Moreno (Hospital Universitario Severo Ochoa), Rafael Torres Perea (Hospital Universitario Severo Ochoa), María Velasco Arribas (Hospital Universitario Fundación Alcorcón), Gema Vico Díaz-Parreño (Hospital Universitario Severo Ochoa), Ana Villanueva Fernández-Ardavín (Hospital Universitario Fundación Alcorcón), Rocío Minaya Carrero (Hospital Universitario Severo Ochoa), Monserrat Rodríguez Miguel (Hospital Universitario Severo Ochoa).