Evaluating the Relationship Between Disease Severity and Serum Levels of Zinc, Calcium and Vitamin D in COVID-19 Patients

SARS-CoV-2 is spreading rapidly worldwide these days so that it has infected people in many countries. It is a zoonotic virus and the cause of COVID-19 infectious pneumonia. The World Health Organization (WHO) declared it a pandemic on January 30, 2020. Given that no standard treatment has been found for the new coronavirus so far, the present study seeks a way to reduce the incidence and severity of the disease along with health protocols. Some of the factors possibly effective in getting less infected by the SARS-CoV-2 are|taking medication supplements such as zinc, calcium, and vitamin D. Materials and methods This cross-sectional study was conducted from May 13 to May 30, 2020, on 93 COVID-19 patients admitted to Khatam Al-Anbia Hospital in Shushtar in southwestern Iran. Some patients' laboratory and clinical of were collected and analyzed using the Chi-squared test, the independent t-test, the Kruskal-Wallis test, and the Spearman rank-order correlation coecient by IBM SPSS Statistics 18.0 software. The severity of the disease (40%) of 37 patients was severe in pulmonary involvement. Serum levels of vitamin D and zinc were lower than the average in all patients. Still, the severity of COVID-19 in patients was not signicantly different from their zinc serum levels (P = 0.216). Serum vitamin D was not signicantly different (P = 0.102). The severity of COVID-19 in patients was signicantly different according to serum calcium levels (P = 0.005). The lower the calcium level, the more severe the disease. the in and no study has published on the appropriate these in taking economically viable calcium-rich food dairy, is


Introduction
The SARS-CoV-2 is spreading rapidly worldwide, so that it has infected many countries. It is a zoonotic virus and the causative agent of COVID-19 pneumonia, which the WHO identi ed as a pandemic (1). The SARS-CoV-2 causes much lower mortality in patients than SARSr-CoV and MERSr-CoV, but has a signi cant potential for infection and transmission (2).
The WHO declared COVID-19 to be an epidemic because it is still spreading rapidly worldwide (3).
Currently, the COVID-19 pandemic is one of Iran's most important health issues and the world's (4). The SARS-CoV-2 can be transmitted from human to human, and no effective treatment has been found so far.
The most effective measures are prevention and control, nding suspicious patients and close contact, con rming patients and virus carriers, and preventing transmission through isolation, disinfection, and personal protection (2). Given that standard treatment for the COVID- 19 has not yet been found (5), the present study seeks to reduce this disease's incidence and severity along with health protocols. A factor that may be effective in this way is taking supplements such as vitamin D, calcium, and zinc.
One of these elements is zinc, which is of particular importance due to the global epidemic of COVID-19.
Zinc is considered a potential supportive treatment in treating COVID-19 infection due to its immunomodulatory effect and direct antiviral effect (6).
Zinc ion has been shown to directly inhibit SARS-CoV-1 RNA-dependent RNA polymerase activity from suppressing viral genomic RNA replication and preventing viral particle proliferation as a mechanism of action (7). Not only does it reduce the number of lymphocytes, but it also impairs the function of T and B lymphocytes (8, 9). Earlier studies demonstrated that a decreased zinc level favors this interaction of ACE2 with SARS-CoV-2 spike protein and likewise that an increased zinc level inhibits ACE2 expression resulting in reduced viral interaction (10).
Calcium is one of the most abundant minerals in the body, which makes up 39% of the body's total salts.
Calcium plays a vital role in the immune system. It is very effective in nerve conduction, blood clotting, heart rate regulation, secretion of hormones and enzymes, and muscle contraction (11,12). In addition to playing an essential role in strengthening bones and teeth, as a messenger, calcium also plays a signi cant role in cellular signaling pathways throughout the body (13,14). Another element is vitamin D, which dates back hundreds of years to the link between vitamin D de ciency and infectious diseases.
Nineteenth-century studies found that sunlight was bene cial for patients suffering from tuberculosis (15).
Vitamin D plays an in uential role in autoimmune diseases, cardiovascular disease, allergic and respiratory disorders (16, 17)This vitamin has immune regulating properties. Acting on the immune system inhibits the production of in ammatory cytokines and stimulates the production of antimicrobial peptides. This peptide plays an essential role in the body's defense against respiratory pathogens, as it both strengthens the immune response and clears bacteria from defense barriers and immune cells (18).Vitamin D de ciency also increases the risk of viral infections such as RSV-induced bronchiolitis and seasonal in uenza (19). Numerous studies have shown an association between vitamin D and respiratory asthma symptoms. This connection is probably through the effect of vitamin D on immune system regulation (20).
This vitamin also plays an essential role in regulating the immune system through the interaction between 1 and 22-dihydroxyvitamin D and the vitamin D 1 receptor (VDR) (21).
In "A Review on the Role of Vitamin D in Asthma", Adite et al. (2009) found that it reduces respiratory infections, prevents asthma attacks, steroid resistance, reduces osteoporosis, and controls asthma. It becomes chronic and also plays an immune role in increasing the antimicrobial peptide and the production of interleukin-10 (22)Studies on the association of serum levels of vitamin D, calcium, and zinc with the severity of COVID-19 have yielded con icting results. The results of some studies show that serum levels of vitamin D affect the COVID-19 severity, although studies in some areas do not con rm this relationship. This study intends to investigate the relationship between disease severity and serum levels of zinc, vitamin D, and calcium in COVID-19 patients.

Subjects and data collection
This cross-sectional study was conducted in 2020 to evaluate the serum levels of vitamin D, calcium, and zinc on the severity of the disease in patients with COVID-19, after obtaining the necessary permits from Shoushtar University of Medical Sciences. After receiving the required permission to collect data, the researcher went to the hospital and performed sampling by explaining the research objectives, obtaining patient satisfaction, and obtaining informed consent from the subjects.
This study was conducted from May 13 to May 30, 2020, on 93 COVID-19 patients admitted to Khatam al-Anbia Hospital in Shoushtar in southwestern Iran. For SARS-CoV-2 nucleic acid detection, the real-time reverse-transcriptase-polymerase-reaction (RT-PCR) was performed on nasopharyngeal and oropharyngeal swabs.
The subjects were selected via a purposive sampling method among COVID-19 patients admitted to Shoushtar Khatam Al-Anbia Hospital. Blood samples were taken from the patients by a hospital nurse (a project colleague) and sent to a private laboratory for more investigation.
Inclusion criteria were as follows: 1. having an Iranian ethnicity and being a resident of Shoushtar city; 2.
being infected with COVID-19 based on PCR test; 3. having the willingness to participate in the study; 4. being over 15 years old, and 5. being fasting during sampling.
Exclusion criteria were as follows: 1-living outside of Shoushtar city 2-having dissatisfaction to participate in the study; 3-taking Vitamin D, calcium, and zinc supplements during the last six months before the study; 4-taking corticosteroid drugs, cholesterol-lowering drugs such as cholesterol and barbiturates and phenytoin (which they can reduce the serum levels of vitamin D, calcium and zinc in the body); 5. having parathyroid disease, bone disease, chronic liver disease, kidney disease, and cancer; and 6. having been treated with vitamin D, calcium and zinc.

Laboratory and clinical results
In this study, we measured serum levels of vitamin D, calcium, and zinc. Serum vitamin D levels were measured by ELISA (Enzyme-Linked Immunosorbent Assay). Serum levels of vitamin D were divided into 30ng/ml-100 ng/ml as su cient, 10 ng/ml -30 ng/ml as insu cient, and < 10 ng/ml as de cient. Calcium Arsenazo measured serum levels of calcium with a reference range of 8.6-10.3 mg/dl. Serum levels of zinc were measured by Atomic Absorption Spectrophotometry (AAS) with a reference range of 70-127 µg/dl.
In this study, information such as age, gender, place of residence, calcium, zinc, and vitamin D supplementation, history of underlying diseases (diabetes, hypertension, heart disease, lung disease, hyperlipidemia); symptoms and possible complications of the disease (cough, shortness of breath).

Statistical analysis
According to the research objectives, previous studies (55), and the parameter of respiratory problems in COVID-19 patients in the two normal serum calcium and low serum calcium groups, the subjects' proportions with respiratory problems in the two groups were P1 = 0.06 and P = 0.016. Also, given β = 0.9, α = 0.05, d = 0.08 and using the sample volume ratio formula, the case group was estimated to consist of 93 subjects.
Blood test results, demographics, and clinical information were entered into IBM SPSS Statistics 18.0 software. Quantitative variables are reported as mean (average), standard deviation (mid-quarter range), minimum and maximum, and qualitative variables are reported as number (percentage). The Kolmogorov-Smirnov test was employed to con rm the normality of quantitative variables, the Spearman rank-order correlation coe cient to examine the relationship between quantitative variables, the independent t-test to compare means, the Kruskal-Wallis test to compare the levels of variables, the Chisquared test to compare qualitative variables in terms of number, and sequential regression to predict some factors affecting the severity of COVID-19. The signi cance level of the above tests is considered less than 0.05.
Serum vitamin D levels were not statistically signi cant between the male and female subjects (P = 0.182); however, serum levels of vitamin D were higher in male than female subjects. Serum levels of calcium were not signi cantly different between the male and female subjects (P = 0.059), but in the female subjects, serum calcium levels were lower than the male subjects. Serum zinc levels were not signi cantly different between the male and female subjects (P = 0.591). The disease's severity was higher in the male than female subjects (P = 0.009) (See Table 2). The severity of the disease in coronary arteries was not signi cantly different according to the serum level of vitamin D (P = 0.102). The COVID-19 severity in patients was signi cantly different according to serum calcium levels (P = 0.005). The lower the serum levels of calcium, the more severe the disease. The severity of the COVID-19 was not signi cantly different in the subjects in terms of their serum levels of zinc (P = 0.216). There was a statistically signi cant and direct relationship between the severity of the disease with the age of patients (P < 0.001) and length of hospital stay (P = 0.026). With increasing age, the disease severity increases. Also, with increasing the disease severity, hospitalization prolongs. There was a statistically signi cant and direct relationship between BS and disease severity (P = 0.007). The higher the blood sugar level is, the more severe the disease is. People with more severe diseases had higher blood ESR levels (P = 0.048) (See Tables 3&4).    a. This parameter is set to zero because it is redundant.

Discussion
This study aimed to investigate the relationship between disease severity and serum levels of zinc, calcium, and vitamin D in COVID-19 patients. The ndings show that serum levels of vitamin D and zinc did not affect the COVID-19 severity, given that serum levels are both lower than normal. However, serum calcium levels have a signi cant and direct effect on disease severity and may help predict the risk of more severe disease in COVID-19 patients.
Existing studies suggest that the nutrients zinc, calcium, and vitamin D may play a role in the course and severity of COVID-19 disease. However, based on SARS and other viral infections, the present research observations and results suggest nutritional supplements increase host resistance to RNA viral infections, including severe COVID-19. In this study, the severity of the disease in 37 patients (40%) was associated with their pulmonary involvement. In Cao X et al. (2020), most COVID-19 patients showed mild to moderate symptoms, but approximately 15% suffered from severe pneumonia (23).
Moreover, Huang (2020) indicated that the disease's severity resulted in mortality (24).The present study showed that serum levels of zinc in all three groups were lower than normal. Still, the severity of the disease in the COVID-19 patients did not differ signi cantly according to their zinc serum levels. Yao et al.
(2021) did not observe hospital survival time and disease severity (25).In an RCT study, Bansal et al. in their study that VITD levels did not differ signi cantly between disease severity groups (29). Hollams et al. (2011) showed that vitamin D de ciency is associated with asthma severity in children with the disease (19). In Brehm Childcare Asthma Management (CAMP) (2010) conducted on children with asthma in North America, low levels of vitamin D were directly associated with an increased risk of hospitalization (30). Barassi (2021) showed serum levels of vitamin D in patients with severe COVID-19, and more need for arti cial ventilation was low (31) In a randomized clinical trial study of COVID-19 hospitalized patients, Murai (2021) concluded that a high dose of vitamin D3, compared with placebo, did not signi cantly reduce hospital stays, and the use of high doses of vitamin D3 For the treatment of COVID-19 does not con rm moderate to severe (32).
Among hospitalized patients with COVID-19, a single high dose of vitamin D3, compared with placebo, did not signi cantly reduce hospital length of stay. The ndings do not support the use of vitamin D3 for the treatment of moderate to severe COVID-19.
The results also showed that the COVID-19 severity in patients was signi cantly different according to serum calcium levels (P = 0.037). The lower the calcium level, the more severe the disease. Recently, a high prevalence of hypocalcemia in COVID-19 patients was reported in several other articles. Sun (2020) stated that serum levels of calcium were associated with disease severity and prognosis in COVID-19 patients (33). Our study results showed that people whose disease was more severe had higher blood ESR levels. Deng SQ (2020) Most of the subjects had high ESR (2).
There was a statistically signi cant and direct relationship between BS and disease severity (P = 0.007).

Conclusion
Our ndings show that serum levels of vitamin D and zinc are lower than normal in all patients and have no effect on the COVID-19 severity. However, serum levels of calcium have a signi cant and direct effect on disease severity and may help predict the risk of more severe disease in COVID-19 patients. Because no study con rms the supplementation to help prevent COVID-19 and no research on the appropriate dose of the supplements in COVID-19 have not been published so far, the consumption of calcium-rich food sources, such as dairy, are economically viable, is recommended.

Suggestions
Because COVID-19 is a pandemic and there is currently no de nitive cure for it, we must look for ways to prevent it. It is suggested that other studies with larger sample sizes in different parts of the world evaluate whether de ciency of these supplements is associated with an increase in the COVID-19 severity.
Also, more immediate clinical trials are recommended to reach a de nitive conclusion about taking vitamin D, Calcium, and zinc supplements to reduce the risk of getting infected by COVID-19, and its severity.