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Abstract

Introduction: Micronutrient deficiencies are quite common worlwide and have a major impact on public health. Vitamin D deficiency is among the risk factors for many diseases. Vitamin B12 deficiency has been associated with a wide variety of neurological, hematological, and gastrointestinal abnormalities. Iron deficiency can affect many organ functions of the human body and is the most common cause of anemia [1]. Recent studies suggest that these micronutrient deficiencies may be related to disorders in the structure and functions of the gut microbiota [2-4]. The imbalance in the intestinal microbiota, can cause intestinal barrier disorder and bacterial translocation and trigger a permanent systemic inflammation state, affect the immune system and metabolism [5]. Gut microbiota metabolites are considered potential disease biomarkers. In adition, these metabolites are of great importance in identfying therapeutic drug targets. Trimethylamine-N-oxide (TMAO) is known as a metabolite derived from choline, L-carnitine, and betaine found in foods of animal origin. These dietary molecules are metabolized to trimethylamine (TMA) in the colon by the gut microbiota. TMA, which then comes to the liver by portal circulation, is oxidized to TMAO by flavin monooxygenase 3 (FMO3) [6]. Many factors such as age, gender, dietary patterns, intestinal microflora composition, renal function and liver FMO activity have been reported that affect the circulating level of TMAO. Studies to date have shown that circulating TMAO concentrations are closely associated with various cardiometabolic diseases [7]. It is thought that endothelial dysfunction, inflammation, platelet activation and lipid metabolism alterations triggered by high TMAO levels may impact cardiovascular function. TMAO activates multiple intracellular signaling pathways that promote cardiovascular pathological changes. Additionally, TMAO induces the release of inflammatory cytokines [8]. TMAO is also related to a variety of central nervous system diseases, cancer and progresion of kidney diseases. However, the mechanisms related to the role of TMAO in the etiopathogenesis of diseases have not been fully revealed [9]. Therefore, more data are needed to fully understand the role of TMAO concentrations in various pathophysiological conditions. Lipopolysaccharide binding protein (LBP) is the first protein involved in the recognition of lipopolysaccharide (LPS) in the outer wall of gram negative bacteria. Increased intestinal permeability leads to leakage of LPS into the bloodstream and low grade inflammation. LBP is recommended as a biomarker for intestinal permeability [10]. The intestinal barrier system allows nutrients and fluids to be absorbed, preventing harmful molecules such as endo-toxins from crossing the intestinal epithelium and reaching the organs [11,12].Emerging evidence links vitamin D with microbiota status. It has been reported that vitamin D may have a regulatory effect on the intestinal microbiota composition, and that administration of vitamin D therapy may be effective in improving intestinal permeability [4]. Studies have reported that vitamin B12 deficiency may be related to changes in the intestinal microbiota [2,13]. Because vitamin B12 is synthesized by bacteria in the human gutmicrobiome, altered gut microbiota may also be considered an critical factor in vitamin B12 deficiency. In addition, it is thought that there may be changes in gut microbiome composition and function due to vitamin B12 deficiency [13]. Recent research suggests that there is a biface interaction between the gut microbiota and iron. The bioavailability and absorption of iron can be affected by microorganism activity in the gut, and microbiota imbalance may have a rol in iron deficiency, while iron deficiency or iron overload can also affect the gut microbiota [3]. Based on these data in the literature, we aim to investigate the role of TMAO and LBP in cases with vitamin D, vitamin B12, and iron deficiency. Methods: This case-control study included the patients in 33 with iron deficiency, 30 with vitamin B12 deficiency, 33 with vitamin D deficiency, 32 with combined deficiency (subjects with concurrent deficiencies of vitamin D, vitamin B12 and iron), 24 taking vitamin D supplements, and 32 age- and body mass index (BMI)-matched healthy controls were in current. The patient and control groups consisted of volunteers who applied to the outpatient clinic for routine control. The cases with newly diagnosed iron deficiency (serum ferritin concentration <30 ng/ml, transferrin saturation <20%), vitamin D deficiency (vitamin D concentrations of <20 ng/ml) and vitamin B12 deficiency (vitamin B12concentrations of <200 pg/ml) were included in the patient group. Vitamin D deficient patients were treated with oral vitamin D at 50000 IU/week for 2 months. After vitamin D treatment, 24 patients with a vitamin D concencentrations of >30 ng/ml were included. Exclusion criteria were obesity, diabetes, cardiovascular and gastrointestinal disease, renal failure, liver or kidney disfunction, having a pregnancy, using additional medicine including antibiotics, corticosteroids, immunosupresif drugs. All volunteers provided informed consent before participating in the study. The study was approved by Ethics Committee (2021/3376). Fasting venous blood specimens were taken between 9:00 a.m. and 11:00 a.m. The blood samples were centrifuged and serum was extracted and portioned into tubes. Serum biochemical variables were analyzed immediately by photometric methods on the Roche Cobas c501 device (Roche Diagnostics, Mannheim Germany). Vitamin D, B12 and ferritin levels were measured by electrochemiluminescence immunoassay (ECLIA) on the Roche Cobas e601 biochemistry analyzer. The measurement of serum TMAO and LBP levels were performed using by Enzyme-Linked ImmunoSorbent Assay (ELISA) technique (BT Lab Bioassay Technology Laboratory Human Elisa Kits, Shanghai Korain Biotech, China). TMAO catalog number: E4733Hu, standard curve range: 0.2-60 ng/mL, intra-assay: CV<8%, inter-assay: CV<10%. LBP catalog number: E0360Hu, standard curve range: 0.2-60 ng/mL, sensitivity: 0.12 ng/mL, intra-assay: CV<8%, inter-assay: CV<10%. Absorbance measurements were performed at 450 nm. Statistically analyses were carried out using SPSS v. 22.0 (SPSS Inc., IL, USA). The chi-squared test was used for categorical variables such as gender. Normality distribution was checked with the Kolmogorov-Smirnov test. Continuous variables were compared with the Student’s t and Mann-Whitney U test. Furthermore, correlations were calculated by Spearman’s correlation test. A value of p<0.05 was recognized as significant. Results: Demographic and biochemical characteristics of the patients groups and control cases are presented in Table 1. Iron and ferritin values of the iron deficiency patients and combined deficiency patients were lower than those of controls (p<0.001). Unsaturated iron binding capacity values of the iron deficiency patients and combined deficiency patients were higher than those of controls (p<0.001). Vitamin B12 values of the vitamin B12 deficiency patients and combined deficiency patients were lower than those of controls (p<0.001). Vitamin D values of the vitamin D deficiency patients and combined deficiency patients were lower than those of controls (p<0.001). Serum TMAO values were significantly lower in the iron deficiency group and combined deficiency group than in the control group (p<0.01). No statistically differences were found in TMAO levels in those with vitamin B12 deficiency and those with vitamin D deficiency compared to the control group. We found lower serum TMAO levels after vitamin D supplementation in those with vitamin D deficiency. There was no significant difference in serum LBP concentrations between the patient groups and the control group except for the group receiving vitamin D therapy. Serum LBP concentrations were significantly lower in the vitamin D suplemented group compared to the vitamin D deficiency group and the control group (Table 2). Spearman’s Rho correlation analysis was performed. There was a positive correlation between TMAO and creatinine (p<0.05), ferritin (p<0.05) and iron (p<0.001) levels. TMAO levels were negatively correlated with glucose levels (p<0.05). There was a significant negative correlation between serum LBP concentrations and ferritin levels(Table 3) Discussion: Recently, the functions of vitamin D other than to control of bone metabolism have attracted attention. It has been reported that vitamin D may have a regulatory effect on the gut microbiota, whose changes are associated with many dissease. Recent researchs show that vitamin D can directly affect the gut microbiome and alleviate dysbiosis [4]. In our study, we wanted to analyze the TMAO levels, one of the intestinal microbiota metabolites, before and after vitamin D suplementation in patients with vitamin D deficiency. In a research investigating the effect of cholecalciferol suplementation on biomarkers associated with carrdiovascular disease in obese people with vitamin D deficiency, a significant reduction in TMAO levels was observed after supplementation [14]. In another study, high TMAO levels were thought to be associated with vitamin D deficiency and non-alcoholic fatty liver disease (NAFLD). TMAO concentrations were highest in patients with both vitamin D deficiency and NAFLD. Vitamin D values showed important opposite associations with circulating TMAO values [15]. Obeid at al showed that TMAO plasma levels were reduced after vitamins B plus vitamin D supplementation [16]. A study in mice reported that with vitamin D supplementation, the intestinal microbiota was regulated, and TMA and TMAO levels were significantly reduced [17]. On the other hand, in a study conducted in HIV-1 infected individuals, it was found that dietary supplementation containing vitamin D had no effect on TMAO level and intestinal microbial composition. In this study, there was no important difference in TMAO concentrations in those with vitamin D deficiency compared to the healthy group [18]. Although not statisticaly significant, in our investigation, we observed a decrease in serum TMAO levels after vitamin D supplementation in cases with vitamin D deficiency. There are few studies investigating the relationship between vitamin B12 and intestinal microbial metabolites TMAO. Obeid at al. observed that plasma levels of TMAO decreased after B vitamins plus vitamin D supplementation [15]. Another study in vegetarians found no effect of intervention with vitamin B12 on plasma TMAO levels [19]. According to the results of our research, there was no considerable difference between the TMAO levels of the vitamin B12 deficient group and the control group. There is a complex relationship between iron and gut microbiota. Iron is of great importance for microbial growth in gut. It has been reported that gut microbial metabolites regulate the host’s iron homeostasis by inhibiting iron transport and storage [20]. The presence of iron in the intestinal lumen can prevent or promote intestinal dysbiosis [21]. In a study in mice, it was reported that iron overload led to intestinal dysbiosis and increased TMAO levels [22]. In another study, an inverse relationship was observed between transferrin and TMAO [23]. It has been reported that paraferritin containing FMO functions as a ferrireductase and iron plays a role in cellular uptake and, abnormalities of FMO enzymes may be associated with sideroblastic anemia [24]. In our study, we found TMAO levels to be significantly lower than the control in cases with iron deficiency and combined deficiency. In addition, there was a positive correlation between TMAO values and ferritin and iron values. Serum LBP concentrations can be measured to assess changes in the gut microbiome [11,12]. Circulating LBP has been found to be an innate immune related component, associated with pathophysiological conditions such as obesity, metabolic syndrome [25]. It has been reported that it may be an important factor in the emergence of the metabolic syndrome through low-grade endotoxemia, systemic chronic inflammation and insulin resistance [26]. It has also been reported that LBP is associated with fatty liver [27]. In a study of patients with liver cirrhosis, they found an inverse interaction between vitamin D concentrations and LBP [28]. We did not observe a statistically important difference in LBP values between our study groups, except for the group that received vitamin D supplementation. Serum LBP levels were significantly lower in the vitamin D suplemented group compared to the vitamin D deficiency group and the control group. In addition, we found a negative correlation between LBP values and ferritin values. Based on the limited information about the roles of LBP in micronutrient deficiencies, we are unable to explaine the present findings at this time. The limitation of our study is that we did not evaluate the participant’s dietary intake and fecal microbiome composition in this study and the sample size was limited. In conclusion we measured the levels of TMAO, a compound produced by the gut microbiome and associated with various chronic diseases, and LBP, a biomarker for intestinal permeability, in vitamin D, vitamin B12 and iron deficiency. Our findings support that although TMAO and LBP are not strong predictors of vitamin D, Vitamin B12 and iron deficiency, they are associated with the pathogenesis of these micronutrient deficiencies.