Introduction

The coronavirus disease (COVID-19) pandemic was caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Methods to reduce the risk of COVID-19 for patients undergoing hemodialysis have been rapidly implemented worldwide [1]. In a study involving approximately 1,300 dialysis facilities across the United States, the analysis of plasma levels of total antibodies against the spike protein receptor-binding domain in 28,503 adult patients on dialysis revealed that the seroprevalence of SARS-CoV-2 antibodies was 8.3% [2]. SARS-CoV-2 has four major structural proteins: spike (S), membrane, envelope, and nucleocapsid proteins. The S protein comprises an S1 subunit that mediates cell surface binding via the receptor-binding domain, and an S2 subunit that induces viral host cell membrane fusion. Antibodies to the highly immunogenic receptor-binding domain account for approximately 90% of neutralizing SARS-CoV-2specific antibodies, whereas few studies have reported neutralizing antibodies to viral structural proteins, such as the nucleocapsid protein [3]. Speer et al. reported a reduced antibody response to the first and second doses of the mRNA vaccine BNT162b2 (BioNTech) in patients on hemodialysis treatment. Most patients undergoing hemodialysis HD (82%) developed neutralizing antibodies after the second dose, but healthy controls had lower levels [4]. Japanese patients undergoing hemodialysis have exhibited evidence of immunity to SARS-CoV-2 after BNT162b2 mRNA vaccination; the majority of these patients develop spike antibodies after the second dose. The mean SARS-CoV-2 spike antibody levels are significantly higher after the third BNT162b2 vaccine dose than after the second dose [5].

Zinc is an essential trace element that plays various important roles in the body to maintain immune function and block the invasion and proliferation of pathogenic viruses. Symptoms of hypozincemia include general fatigue, dysosmia, dysgeusia, loss of appetite, hair loss, anemia, and headache [6]. A link between SARS-CoV-2 infection and zinc has been suggested [7-9]. Razzaque reported that zinc exerts antiviral effects by suppressing viral replication and enhancing immune responses [10].Rheingold et al. reported that zinc supplementation is associated with decreased mortality in those with COVID-19. This is promising as zinc is widely available and may be valuable as a cost-effective way to prevent poor outcomes for those with COVID-19 [11]. However, the serum zinc levels of patients on hemodialysis treatment with SARS-CoV-2 infection have not been investigated. Hence, we investigated the serum zinc levels of Japanese patients with COVID-19 undergoing hemodialysis.

Materials and Methods

Study design, population, and data sources This was an observational cohort study.

Study 1

We quantitatively measured zinc levels in the serum of patients who underwent hemodialysis in the dialysis facilities of the Aobadai Jin Clinic, Takada Jin Clinic, and Yokohama Dai-ichi Hospital between August 1 and December 1, 2023.

We quantitatively measured SARS-CoV-2 spike antibodies in the serum of patients with COVID-19 who underwent HD in the dialysis facilities of the Aobadai Jin Clinic, Takada Jin Clinic, and Yokohama Dai-ichi Hospital between August 1 and December 1,

2023 (Table 1)

Study 2

We measured zinc levels in the serum of nine adults with normal renal function (four males and five females) in the department of Clinical Inspection, Prime Health Partners, Zenjinkai Group between July 1 and August 31, 2023.

Blood sampling and measurement of serum zinc

Serum zinc levels were measured several times during blood sampling at the start of hemodialysis. Serum zinc concentrations were analyzed using the Accuras Auto Zn kit (Shino-Test Corporation, Tokyo, Japan), which was developed to measure serum zinc levels, with an automatic analyzer at the clinical laboratory in our hospital. Serum zinc levels were classified per the Japanese Society of Clinical Nutrition, as follows: 80-130 μg/ dL, normal; 60-80 μg/dL, subclinical zinc deficiency; and <60 μg/dL, clinical zinc deficiency [12].In Harrison’s Principles of Internal Medicine, an international standard indicator, clinical zinc deficiency is defined as serum zinc levels of <70 μg/dL [13]. Serum zinc levels tend to be affected by sex, age, and the time at which blood is sampled. We defined serum zinc concentrations of <80 and <60 μg/dL as mild and severe hypozincemia, respectively.

Antibody detection

We quantified the SARS-CoV-2 spike antibodies in human plasma. SARS-CoV-2 spike antibodies levels were evaluated with a fully automated Cobas e801 analyzer (Roche Diagnostics, Santa Clara, California, USA), using an Elecsys^® anti-SARS-CoV-2 electrochemiluminescence immunoassay (Roche Diagnostics), which has 100% sensitivity and 99.81% specificity (Roche Diagnostics) to qualitatively detect SARS-CoV-2 spike antibodies in human plasma [14]. A semiquantitative index of <0.8 U/mL was classified as negative, and one of ≧0.8 U/mL was classified as positive.

Statistical analysis

Data are expressed as mean ± standard deviation or median (interquartile range) for continuous variables and as frequency (percentage) for categorical variables. The Wilcoxon signed–rank test was used for proportional assessments. Statistical significance was set at p<0.05, and all statistical analyses were performed using SAS Statistics software (version 9.4; SAS Institute, Cary, North Carolina, USA).

Ethics statement

The study was approved by the ethics committee of the Zenjinkai Group (approval number: 0001008422) and was conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from all patients before the study.

Results

Data from 91 patients undergoing hemodialysis and 9 adults with normal renal function were analyzed in this study.

Study 1

The background characteristics of patients undergoing hemodialysis with and without COVID-19 are shown in Table1. Overall, 24 (26.4%) patients undergoing hemodialysis  tested positive for

SARS-CoV-2 on polymerase chain reaction testing, whereas 67 (73.6%) patients tested negative. Serum zinc concentrations in the patients on hemodialysis treatment are shown in Table 2. All 24 patients with COVID-19 had hypozincemia, of whom 9 (37.5%) had mild hypozincemia, and 15 (62.5%) had severe hypozincemia. The mean titer of SARS-CoV-2 spike antibodies was 10,032±4,695 U/mL. The mean serum zinc level in patients with COVID-19 undergoing hemodialysis was significantly lower than that in patients without COVID-19 (58.9±7.5 vs. 63.3±11.4 μg/dL; p=0.03436).

Study 2

The daily variations in serum zinc concentration in adults with normal renal function are shown in Table 3. In Japanese adults (mean age: 32.2±3.4 years; 4 females, 5 males) with normal renal function, the mean serum zinc concentrations were 78.0±8.3, 74.7±13.6, and 75.6±11.2 μg/dL at 10 am, 2 pm, and 4 pm, respectively, indicating that the daily variations in serum zinc levels were minor. The mean serum zinc concentration in patients with COVID-19 undergoing hemodialysis was significantly lower than that in adults with normal renal function (58.9±7.5 vs. 78.0±8.3 μg/dL; p=0.00042). The mean serum zinc concentration in patients without COVID-19 undergoing hemodialysis  was significantly lower than that in adults with normal renal function (63.3±11.4 μg/ dL vs. 78.0±8.3 μg/dL; p=0.00049).

Table 3: Daily variations in the serum zinc concentration of adults with normal renal function.

Discussion

Zinc is an essential mineral that plays various important roles in the body, such as maintaining immune function and blocking the invasion and proliferation of pathogenic viruses [6]. Brewer et al. reported that zinc may be effective against SARS-CoV-2 infection [7]. Serum zinc levels are low in severe cases, suggesting a link between COVID-19 infection and zinc levels [8, 9].Tsuchiya et al. reported that serum zinc levels are significantly lower in SARSCoV-2-infected patients with pneumonia than in those without pneumonia [15]. Vogal-González et al. reported that serum zinc levels of <50 μg/dL at admission are correlated with a worse clinical presentation, longer time to reach stability, and higher mortality [16]. In vitro results have indicated that low zinc levels favor viral expansion in SARS-CoV-2-infected cells [16].Yasui et al. reported that prolonged hypozincemia is a risk factor for severe COVID-19 [17].Matsuda et al. reported that the most frequent symptom in patients with long COVID with hypozincemia is general fatigue. Serum zinc levels should be measured in patients with long COVID with general fatigue [18].

Japanese patients with COVID-19 undergoing hemodialysis had low serum zinc levels. Hypozincemia was detected in most patients undergoing hemodialysis and may be a risk factor for COVID-19. Weiss et al. reported that patients undergoing hemodialysis have a higher risk of SARS-CoV-2 infection than the general population [19].Prasad reported that zinc deficiency is related to smell dysfunction, taste abnormalities, delayed wound healing, skin changes, hair loss, and mental lethargy [20].Therefore, patients with COVID-19 undergoing hemodialysis with hypozincemia may have more complaints, including dysgeusia, general fatigue, and hair loss, which are common complains among patients with COVID-19 undergoing hemodialysis. Fromonot et al. reported that hypozincemia is more frequently observed in patients with COVID-19 than in those without [21]. In the present study, hypozincemia was detected in most patients undergoing hemodialysis. Highly predictive clinical factors must be examined to forecast the outcome immediately after COVID-19. Yasui et al. reported that almost all severe cases show subclinical or clinical zinc deficiency and that prolonged hypozincemia is a risk factor for critical illness in COVID-19 [17].Razzaque reported that zinc can exert antiviral effects by suppressing viral replication and enhancing immune responses [10].Rheingold et al. reported that zinc supplementation is associated with decreased mortality in those with COVID-19 but does not change symptomatology. This is promising because zinc is widely available and may be valuable as a cost-effective way to prevent poor outcomes for those with COVID-19 [11].Ota reported that acquired immunity against primary SARS-CoV-2 confers protection from re-infection [22]. Sethuraman et al. found that a SARS-CoV-2 infection can also be detected indirectly by measuring the host’s immune response. Serological diagnosis is particularly important for patients with mild to moderate illness who may present late in the course of their infection, particularly more than 2 weeks after the onset of illness [23]. To et al. reported that enzyme-linked immunosorbent assay-based immunoglobin M and immunoglobin IgG antibody tests had a >95% specificity for the diagnosis of SARS-CoV-2. The receptor-binding domain of S is a host-attachment protein, and antibodies to RBD-S are more specific and are expected to be neutralizing [24].

We aimed to examine the relationship between serum zinc levels and COVID-19 in patients undergoing hemodialysis to evaluate whether hypozincemia could be a predictive factor for critical illness in patients with COVID-19 undergoing hemodialysis. Urinary epidermal growth factor levels have been associated with acute kidney injury [25]. Menez et al. reported that lower urinary epidermal growth factor levels are associated with poor prognosis in patients hospitalized with COVID-19. Urinary biomarkers are associated with adverse kidney outcomes in patients hospitalized with COVID-19 and provide valuable information to monitor kidney disease progression and recovery [26]. Further studies are needed to validate the association between urinary epidermal growth factor levels and serum zinc levels of patients undergoing hemodialysis with COVID-19.

Our observational cohort study was limited by its design, small number of patients on hemodialysis treatment, and timing of serological testing. Our preliminary data requires confirmation through further testing.

Conclusions

We revealed that Japanese patients with COVID-19 undergoing hemodialysis had low serum zinc levels. Most patients undergoing hemodialysis had hypozincemia. Among patients undergoing hemodialysis, the mean serum zinc level in those with COVID-19 was significantly lower than that in patients without COVID-19. Hypozincemia therefore may be a risk factor for COVID-19 in patients undergoing hemodialysis.

Author Contributions: Data analysis: TT, MH, AS, GO, RO, MK, and YH; supervision/mentorship: KA, TC, and AY. Each author contributed important intellectual content during manuscript drafting or revision and accepted accountability for the overall work by ensuring that questions pertaining to the accuracy or integrity of any portion of the work were appropriately investigated and resolved.

Acknowledgments: The authors thank the staff of the renal dialysis facilities of the Zenjinkai Group for their support and the patients for taking the time to complete the research protocol. The authors are also grateful for the support of Ms. Kaori Soneda, Hiroshi Yamamoto, Hiroshi Shinada, and Yuya Okazaki of the Division of Total Management System in the Yokohama Daiichi Hospital. We thank Editage (www. editage.com) for the English language editing.

Funding: None.

Institutional Review Board Statement: The study was approved by the ethics committee of the Zenjinkai Group (approval number: 0001008422) and was conducted in accordance with the Declaration of Helsinki.

Informed Consent Statement: Written informed consent was obtained from all patients before the study.

Data Availability Statement: The data that support the findings of this study are available from the corresponding author upon reasonable request.

Conflicts of Interest: None.

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