Development and Validation of Accurate HPLC-MS-MS Based Methods to Investigate the Vitamin D Status Involved in COVID-19

Abstract Background: Vitamin D status has been suggested to play a possible role in human skeletal and non-skeletal health problems. Recent literature has focused on the role of vitamin D3 in the immune system, asserting its involvement in regulation, modulation of innate and adaptive responses, and the production of antibacterial and antiviral proteins. Recent studies have also shown an inverse correlation between Vitamin D status and increased susceptibility to respiratory infections, such as acute respiratory distress syndrome (ARDS), one of the most severe complications of coronavirus disease 2019 (COVID-19). In March 2020, COVID-19 spread as a pandemic emergence, with uncertain outcomes in the absence of definite effective therapies and vaccines. Other than specific treatments, public health measures were required to characterize risk factors and prevent the infection or the progression of the disease to reach its most aggressive form. Among multiple factors that could eventually contribute to COVID-19, the vitamin D status was proposed as a credible candidate. This research project focused on the role of vitamin D3 in COVID-19. Commonly, to define Vitamin D status, most routine analytical methods quantify 25-hydroxyvitamin D3 (25OHD3), the most abundant circulating metabolite. However, vitamin D3 is characterized by complex metabolic and catabolic pathways, and simultaneous measurement of vitamin D3 in its biologically active and inactive forms may provide a more accurate interpretation of vitamin D status in the pathophysiological context of COVID-19. Methods: This study firstly aimed to investigate the blood levels of Vitamin D metabolites and catabolites in COVID-19 positive patients and in healthy subjects to evaluate any differences in the vitamin D3 status and obtain some information about vitamin D3 metabolism and catabolism during SARS-CoV2 infection. In this retrospective study, we analyzed 30 plasma samples from healthy subjects, used as controls, and 69 samples from COVID-19 patients hospitalized at the University Hospital in Pisa between March and May 2020. Secondly, the study focused on the relationship between vitamin D status, considered as 25-OHD3, and a biochemical panel of inflammatory markers in a cohort of patients (n=93) with COVID-19-related pneumonia. For the first aim of the study, a selective analytical method based on liquid chromatography coupled to tandem mass spectrometry (LC-MS-MS) to quantify vitamin D3 and three of its metabolites, such as 25-OHD3, 24,25-(OH)2D3, 1,25-(OH)2D3, in plasma samples, was developed and validated. All analyses were performed with an Agilent Infinity UHPLC system, coupled to Sciex QTrap 6500+ mass spectrometer, equipped with an IonDrive Turbo-V electrospray (ESI) ion source. Chromatographic separation was achieved by a phenyl column, under gradient conditions. The MS method to assay 25-OHD3, 24,25-(OH)2D3, vitamin D3, and their ISs was based on positive ion mode multiple reaction monitoring (MRM), while the MS the quantification of both 1,25-(OH)2D3 and its IS was based on positive ion mode MS3 (MS/MS/MS). Regarding the relationship between 25-OHD3 levels and inflammatory markers, the analysis of cytokines (IL-6, and IL-10; tumor necrosis factor-a, TNF-α) was performed by a fully automated ELISA processing system, using commercial ELISA assays. Hemogasanalysis was performed by GEM Premier 4000 Blood Gas Analyzer, and gas exchange impairment was evaluated using arterial partial pressure of oxygen (PaO2) to fraction of inspired oxygen (FiO2) (P/F). Results: The LC-MS-MS method provided satisfactory results in terms of selectivity, reproducibility, sensitivity as well as in recovery, accuracy and stability. The high linearity allows an accurate quantification of the analytes. The method was used for the simultaneous quantification of vitamin D3 and three of its metabolites in plasma samples. Our data indicated a statistically significance decrease in the concentration levels of the four analytes of interest in COVID-19 positive patients compared to the controls. As regards the relationship between 25OHD3 and inflammatory markers, sixty-five percent of patients presented hypovitaminosis D3 (25OHD3 ≤ 20 ng/ml) and showed significantly higher levels of IL-6, CRP, TNF-a, D-dimer, and IL-10. A considerable inverse correlation was found between 25OHD and all these markers, even adjusted for age and sex. Hypovitaminosis D3 was prevalent in patients with severe ARDS, compared with the other groups, and 25OHD3 levels were lower in non-survivor patients. Conclusions: The LC-MS-MS method developed in our laboratory revealed to be very useful instrument to understand the role of vitamin D metabolites in COVID-19 disease. Nevertheless, this powerful analytical tool could be exploited to examine in depth on routine or research basis the complex interaction between vitamin D3, bone homeostasis and immune system. However, this thesis work is a preliminary study, carried out on a small sample population; the extension of the study to a larger number of samples could allow the identification of a reliable biomarker, among the metabolites of vitamin D3, that may be related to the degree of severity of COVID-19 disease. The relationship between 25OHD3 levels and inflammatory markers suggests that vitamin D status needs to be considered in the management of these patients. If vitamin D is a marker of poor prognosis or a possible risk factor with beneficial effects from supplementation, this still needs to be elucidated.