Journal of Human Nutrition

Hypovitaminosis D is Associated with the Components of Metabolic Syndrome in Brazilian Women

Abstract

Low vitamin D status and oxidative/nitrosative stress have been recognized as risk factor for chronic diseases such as metabolic syndrome (MetS). The objective of this study was to evaluate the metabolic and oxidative/nitrosative markers for association with hypovitaminoses D in Brazilian women with MetS. The following included 88 women with MetS and evaluated metabolic biomarkers (total cholesterol, LDL-cholesterol, HDL-cholesterol and triglycerides, uric acid, plasma glucose, insulin), and oxidative/nitrosative stress biomarkers (hidroperoxides-CL-LOOH, carbonyl protein, nitric oxide metabolites-NOx, sulfhydryl groups-SH of proteins, total radical-trapping antioxidant parameter-TRAP, total serum activity of PON1). The homeostasis model assessment (HOMA-IR) was calculated. Vitamin D was determined as 25(OH)D and categorized as hipovitaminosis (MetSHD) and suficient vitamin D (MetSD). Levels of vitamin D were lower in the MetSHD (p < 0.0001). MetSHD group showed increase in fasting glucose (p = 0.03), TG (p = 0.04), HOMA-IR (p = 0.03), CT/HDL (p = 0.04), LDL/HDL (p = 0.03), non-HDL (p = 0.04). There were increased levels of AOPP (p = 0.03) and NOx (p = 0.04) levels in MetSHD group. In MetSHD, the SH group were negatively correlated with TC (r = -0.36, p < 0.05) and LDL-C (r = -0.347, p < 0.05) whereas AOPP was negatively correlated with HDL (r = -0.37, p < 0.05), and positively correlated with fasting glucose (r = 0.32, p < 0.05), TG (r = 0.66, p < 0.0001) insulin levels (r = 0.50, p < 0.001) and HOMA-IR (r = 0.54, p < 0.001). NOx was positively correlated with HOMA-IR (r = 0.34, p < 0.05) and TRAP/uric acid was negatively correlated with BMI, WC, and insulin levels (r = -0.33, -0.34, -0.366; p < 0.05), respectively. In conclusion, the present study showed that hypovitaminosis D in women with MetS is associated metabolic and oxidative/nitrosativo biomarkers, which are important factors for cardiovascular risk.

Keywords

Hypovitaminosis D, Oxidative stress, Metabolic syndrome, Nitrosativo stress, Metabolic markers

Introduction

Metabolic syndrome (MetS) is a complex disease characterized by a distinct process and cardiovascular risks factors that could culminate in cardiovascular disease and type 2 diabetes (D2M) [1].

Vitamin D has a pleiotropic action and can affect multiples organs and metabolic processes, including the cardiovascular, renal and immune systems [2]. Some studies showed that hypovitaminosis D is associated with several risk components of MetS, individually. The abdominal obesity, characteristic of individuals with MetS, is associated with lower levels of vitamin D [1,3,4]. MetS affects approximately 50% of the female and is associated with a threefold increase in morbidity and mortality due to cardiovascular disease [5-8]. A transversal study showed that hypovitaminosis D is associated with cardiovascular disease independently of the degree obesity [1]. Another study showed that individuals with low levels of vitamin D presented higher risks of cardiovascular diseases after 10-15 years of segment [9].

An imbalance between reactive oxygen/nitrogen species (ROS/RNS) production and antioxidant substances is main characteristic of oxidative stress [10-12]. Inflammation and oxidative stress occurs when the energy supply begins to exceed the storage capacity of the adipocytes and, as result, hypertrophy occurs. This hypertrophy leads to a higher release of adipokines as proinflammatory cytokines, resulting in low-grade inflammation, which begins in the adipose tissue and eventually reaches the circulation system and other organs [10-12].

Additionally, paraoxonase (PON) is a family of Ca++ dependent enzymes, namely PON1, PON2 and PON3 [13,14]. Paraoxonase1 (PON1) is exclusively associated with HDLc and is a genetically polymorphic enzyme. It plays a vital role in the prevention of microvascular complications due to oxidative stress and against various toxic chemicals. HDL oxidation is stopped by PON1-mediated hydrolysis of lipid peroxides [15].

Oxidative stress plays an important role in the pathogenesis of insulin resistance by disrupting the release of adipokines by adipose tissue that can trigger inflammation. Thus, it seems that MetS is a factor associated with inflammation and oxidative stress. The possible importance of vitamin D status as a novel risk factor for various chronic diseases has gained more interest. In addition, one area of recent study has been the investigation of the association between vitamin D status and MetS. However, despite evidence of the association of the serum vitamin D and MetS in women, some studies have demonstrated contradictory results. More clinical studies are needed to confirm the association between Vitamin D and women with MetS. Thus, in this context, the aim of the present study was to evaluate the oxidative and nitrosative markers, and the association with hypovitaminosis D in Brazilian women with MetS.

Materials and Methods

Participants

The study was conducted according to the guidelines laid down in the Declaration of Helsinki and all procedures involving human patients were approved by the Ethical Committee of the University of Londrina, Paraná, Brazil (CAAE 41718014.9.0000.5231). Written informed consent was obtained from all patients. This study included 88 women with MetS from the ambulatory centre of the University Hospital of Londrina, Parana, Brazil. Patient motivation was related to the intake of a nonpharmacologic therapy that was practically without side effects. The exclusion criteria were cardiovascular disease (CVDs) (except hypertension); thyroid, renal, hepatic, gastrointestinal, and oncologic diseases; or acute infection and utilization of lipid lowering drugs, estrogens replacement therapy, drugs for hyperglycaemia; and antioxidant supplements. Patients who were taking antihypertensive drugs were not excluded and were allowed to continue taking their prescribed dosage. None of the participants followed a specific diet before the start of the study. The patients were instructed not to change their usual diets, alcohol intake, level of physical activity, or other lifestyle factors throughout the study.

Anthropometric Measurements

MetS was defined following the Adult Treatment Panel III criteria [16]. When three of the following five of the characteristics were verified, a diagnosis of MetS was given: 1. Abdominal obesity: Waist circumference > 88 cm in women; 2. Hypertriglyceridemia ≥ 150 mg/dL (1.695 mmol/L); 3. Low levels of HDL-C: ≤ 50 mg/dL (1.295 mmol/L) in women; 4. High blood pressure: ≥ 130/85 mm Hg; and 5. High fasting glucose: ≥ 100 mg/dL (5.5 mmol/L). Height and weight were measured in the morning with participants wearing light clothing, but no shoes. After 20 min of rest, each participant had her blood pressure measured on the left arm while in a sitting position. We considered the current use of antihypertensive medication as an indication of high blood pressure. Body mass index (BMI) was calculated as weight (kg) divided by height (m) squared. Waist circumference was measured at the umbilical level with the participants standing after normal expiration and the hip girth was measured at the widest part of the hip and, the waist-to-hip ratio was calculated.

Blood sample

Peripheral blood samples were collected from each participant with EDTA as anticoagulant and without anticoagulant. All samples were immediately centrifuged at 3,000 rpm for 15 minutes and plasma, serum, and buffy coat aliquots were stored at freezer -80℃ until use. The samples were identified consecutively by number to guarantee the confidentiality.

Metabolic markers

The 25 hidroxi (OH) were determined using a quimioluminescence assay (CMIA) (Architech™, Abbott Laboratory, Abbott Park, IL, USA) and the results were expressed as ng/mL. The MetS patients were categorized as Vitamin D levels < 30 ng/mL (MetSHD) and ≥ 30 ng/mL (MetSD).The metabolic biomarkers were evaluated by serum levels of lipids (total cholesterol, LDL-cholesterol, HDL-cholesterol and triglycerides), uric acid and by plasma glucose using Dade Behring™ reagents in a biochemical autoanalyser (DimensionTM Dade AR Dade Behring, Deerfield, IL, USA). Plasma insulin levels were determined by chemiluminescence microparticle immunoassay (Architech™, Abbott Laboratory, Abbott Park, IL, USA). The homeostasis model assessment (HOMA-IR) was used as a surrogate measurement of insulin sensitivity [17]. The homeostasis model of assessment in insulin resistance (HOMA-IR) was used as a surrogate measure of insulin sensitivity using HOMA-IR = insulin (mU/mL) × glucose (nmol/L/22.5) (HAFFNER, 2003).

Oxidative Stress Biomarkers

Tert-butyl hydroperoxide-initiated chemiluminescence (CL-LOOH) was evaluated as described previously by Gonzalez-Flecha [18] and the results were expressed in counts per minute (cpm). Carbonyl protein content was measured as an estimate of protein oxidative injury as described by Reznick and Packer [19]. Nitric oxide metabolites (NOx) were assessed by nitrite (NO₂^-) and nitrate (NO₃^-) concentration according to the Griess reaction supplemented by the reduction of nitrate to nitrite with cadmium [20] and the results were expressed in µM. The sulfhydryl group of proteins was evaluated in the plasma by a spectrophotometric assay based on 5,5-dithiobisnitrobenzoic acid (DTNB) as reported previously [21] and the results were expressed in µM. Total radical-trapping antioxidant parameter (TRAP) was determined as reported previously [22]. This method detects hydrosoluble and/or liposoluble plasma antioxidants by measuring the chemiluminescence inhibition time induced by 2,2-azobis (2-amidinopropane). The system was calibrated with the vitamin E analogue Trolox and TRAP values were expressed in the equivalent of µM Trolox and the results were expressed as TRAP/AU. Total serum activity of PON1 was determined by the method described by Richter, Jarvik, and Furlong [23]. The rate of hydrolysis of phenyl acetate was determined in a microplate reader EnSpire, Perkin Elmer® (Walthman, MA, USA) at 270 nm and the temperature was maintained at 25 ℃. Measures were recorded for 4 min each 15 s. The activity was expressed in U/mL on the phenyl acetate molar extinction coefficient of 1.31 mMol/L cm^-1.

Statistical analysis

The data were evaluated by the statistical analysis program GraphpadStatemate 2.0 (GrahpPad Software, San Diego, CA). Continuous variables were evaluated using the Mann-Whitney test and data were expressed as the median and interquartile range (25%-75%). Correlations were evaluated by Spearman's rank correlation. All the results were considered significant when p < 0.05.

Results

Clinical, anthropometric and metabolic parameters in women categorizes as MetSHD and MetSD are shown in Table 1. The mean age o MetSHD group was 50.61 ± 7.65 years and MetSD group was 53.7 ± 4.35 years. According to BMI, the MetSHD and MetSD group presented 33.5 (25.3-61.0) Kg/m² and 30.6 (24.0-39.0) Kg/m², respectively. MetSHD group presented WC and AC as 95.0 (81.0-140.0) cm and 103.5 (89.0-170.0) cm, respectively, while MetSD group presented 95.0 (67.0-115.0) cm and 96.2 (90-127.0) cm, respectively. No differences about age, BMI, WC, and AC were found between the groups. MetSHD group showed a statistically significant increases in fasting glucose (p = 0.03), TG (p = 0.04), HOMA-IR (p = 0.03), CT/HDL (p = 0.04), LDL/HDL (p = 0.03), non-HDL (p = 0.05) when compared with MetSD. As expected, levels of vitamin D were lower in the group women with MetSHD compared with those with MetSD (p < 0.0001).

Oxidative stress markers in MetSHD and MetSD groups were described in Table 2. There was a significant increase in AOPP (p = 0.03) and NOx (p = 0.04) levels in MetSHD group when compared to MetSD. No difference was found in the SH group, hydroperoxides (CL-LOOH), TRAP/uric acid, and AOPP/TRAP (p < 0.05). Additionally, there was a tendency to higher levels of PON in the MetSHD group (p = 0.06).

A correlation rank between clinical, metabolic, and oxidative stress parameters in women with MetS and D hypovitaminosis was performed and the results were described in Table 3. The SH group was negatively correlated with TC (r = -0.36, p < 0.05) and LDL-C (r = -0.35, p < 0.05) whereas AOPP was negatively correlated with HDL (r = -0.37, p < 0.05), and positively correlated with fasting glucose (r = 0.32, p < 0.05), TG (r = 0.66, p < 0.0001), insulin levels (r = 0.50, p < 0.001) and HOMA-IR (r = 0.54, p < 0.001). NOx was positively correlated with HOMA-IR (r = 0.34, p < 0.05) and TRAP/uric acid was negatively correlated with BMI, WC, and insulin levels (r = -0.33, -0.34, -0.37, p < 0.05), respectively [24-26].

Discussion

The main finding of this study was that women with MetSHD presented higher levels of fasting glucose, HOMA-IR, triglycerides, and biomarkers of oxidative stress than did women without hypovitaminosis D. In addition, it seems that hypovitaminosis D is a determinant factor for cardiovascular risk and oxidative stress, once all women presenting MetS were assessed.

Our results agree with previous research's involving hypovitaminosis D and disturbance in metabolic and oxidative stress markers, showing that serum vitamin D levels seems to be inversely correlated with measures of obesity, BMI, fat mass and WC [4,27-34].

There is an inverse correlation between serum 25(OH)D levels and cardiovascular disease, MetS and their complications, such as glucose intolerance, obesity, hypertension, insulin resistance, ischaemic heart disease, and stroke [35,36]. Schimitt, et al. [37] showed that women postmenopausal with vitamin D deficiency had a higher risk of developing MetS, hypertriglyceridemia and low HDL levels than did women with adequate levels of vitamin D (p < 0.05).

In this study, women with MetSHD presented higher levels of fasting glucose and HOMA-IR, and these results agree with previous studies [37-40]. The most plausible explanation is that vitamin D influences insulin secretion and sensitivity, which plays a major role in MetS. The vitamin D receptor is expressed in insulin-secreting pancreatic beta cells and in peripheral target tissues such as skeletal muscles and adipose tissue. Vitamin D can compromise the capacity of beta cells to convert pro-insulin in insulin [41,42]. Vitamin D indirectly affects insulin sensitivity in skeletal muscles and adipose tissue by regulating the levels of extracellular calcium, which is essential for insulin-mediated intracellular processes [41].

A study showed that vitamin D stimulated the expression of insulin receptor and low vitamin D levels were associated with beta-cell dysfunction. Vitamin D deficiency was independently associated with insulin resistance and beta-cell function in patients at risk for diabetes [43-46]. Individuals with lower levels of vitamin D presented a higher incidence of type 2 diabetes due to insulin secretion and sensibility. In vitro studies showed that 1,25(OH)-vitamin could stimulate insulin secretion directly by enhancing calcio levels due to adequate vitamin D levels [44,47].

Vitamin D also has an immunomodulation effect than can protect against type 1 diabetes [48]. In addition, studies support a direct correlation between vitamin D and insulin resistance [44]. The relationship between hypovitaminosis D and hypertension is clear, and the mechanism of this association is due to the interaction of Vitamin D with the renin-angiotensin-aldosterone system, the immune inflammation in MetS and the effects of vitamin D in the vascular endothelium and smooth muscle [49].

Diabetes is not only about the body's inability to handle glucose properly but is also an inflammatory disease. Because vitamin D has anti-inflammatory effects, it is not surprising that it has beneficial effects on improving islet cell functions, insulin release, ad decreasing insulin resistance [38,50-55].

Our results showed that women with MetSHD presented higher triglycerides levels. The relationship between vitamin D deficiency and dyslipidaemia may be due, in part, to vitamin D's effects on hepatic lipid metabolism. Vitamin D promotes intestinal calcium absorption, and calcium may bind to fatty acids to form insoluble complexes that inhibit lipid absorption. Thus, vitamin D deficiency may lead to abnormal processing of lipids due to calcium available [56]. Linear regression analysis demonstrated a significant inverse association between vitamin D and LDL-cholesterol, and TG. In addition, vitamin D was independently associated with greater odds of hyperlipidaemia [43]. Schimitt, et al. [37] found that women with vitamin D deficiency had a higher risk of hypertriglyceridemia and low HDL. Other review study also showed that subjects with high serum levels of vitamin D had a more favorable lipid profile than those with vitamin D deficiency [57]. A longitudinal study demonstrated that an increased in serum 25(OH)D levels was associated with a significant reduction of triglycerides levels and that the mechanism underlying the inverse association is a reduction in the intestinal absorption and synthesis of lipid, as well as decreased in lipolysis with increasing vitamin D concentrations [34,58].

The inflammatory process seems to be responsible for oxidative stress generation and may induce gene expression related with inflammatory cytokines [59]. On the other hand, vitamin D has a potent immunoregulatory action, such as inhibiting the production of interleukin-6, interleukin-8, interferon-γ by peripheral blood mononuclear cells in autoimmune disease [60]. Reported results support the concept that increased oxidative stress may play an important role in MetS [61,62].

In our study, women with MetS and hypovitaminoses D presented higher levels of AOPP. In addition, a significantly and positive correlation between AOPP and triglycerides, fasting glucose, insulin and HOMA-IR values were found. We also showed a negative correlation between AOPP and HDL-cholesterol. Korkmaz, et al. [27], showed that AOPP values are higher in MetS patients group when compared with control group and found a positive correlation between AOPP levels and glucose, triglycerides, insulin levels, and HOMA-IR values (p < 0.0001). Cakatay [63] reported that patients with type 2 diabetes exhibit elevated protein oxidation, indicated by elevated plasma protein carbonyl and AOPP levels, this can be correlated glycemia and with glycemic control. Another study permed a multiple regression analysis and revealed that AOPP levels are the most important independent determinants of the MetS. This finding confirms that high AOPP levels are indicative of an increase in oxidative stress and can cause direct oxidative damage in proteins in MetS patients [63-65]. Our group of research demonstrated a positive correlation between AOPPs and waist circumference (p < 0.01), fasting glucose (p < 0.05), homeostasis model assessment insulin resistance (p < 0.001), triacylglycerol (p < 0.0001), and uric acid (p < 0.01), whereas there was an inverse correlation with high-density lipoprotein cholesterol (p < 0.001) [66].

Women with MetSHD presented higher levels NOx compared with those with MetSD. In addition, NOx metabolites were positively associated with HOMA-IR in women with MetS and lower levels of vitamin D. The role of insulin resistance at the level of the endothelial cell in vascular pathophysiology is unclear. Several studies in humans and gene-modified mice have demonstrated a close association between insulin resistance and nitric oxide bioactivity [67]. Endothelial dysfunction and vascular insulin resistance usually coexist, and chronic inflammation engenders both. Growing evidence from both clinical and animal studies has suggested that endothelial dysfunction and vascular insulin resistance coexist in obesity and diabetes and they may play a causative role in the development of metabolic insulin resistance [68-71].

Choi, et al. [72], concluded that uric acid induced endothelial dysfunction by contributing to vascular insulin resistance in terms of insulin-induced NO production, potentially leading to the development of hypertension. Montagnani, et al. [73] dissected the pathway by which insulin stimulate the NO release from endothelial tissue in vitro. In addition, Kuboki, et al. [74] demonstrated that insulin regulates the eNOS transcription in the endothelium.

MetSHD group showed a negative correlation between SH groups and TC, and HDL. Several oxidative modifications of proteins may be introduced by ROS-RNS. The thiol (-SH) group of cysteine residues is a particularly sensitive target of oxidant species, forming sulfenic acid, mixed disulfide, S-glutathiolated derivatives, as well as sulfinic and sulfonic acid [75]. Cysteine-bound thiol may also be nitrosylated through the addition of a NO group. Such S-nitrosylation is a reversible modification playing essential roles in modulating the function of a great number of cellular proteins [76,77]. Tyrosine residues may be affected by peroxynitrite-mediated nitration, that is, the addition of an NO2 group to the phenolic ring of tyrosine [78].

Oxidized proteins may be subject to accelerated degradation and loss of function, with potentially significant cytotoxic consequences [79]. A study showed suggest AOPP as the most appropriate parameter for determination of oxidative stress by the action of the chloraminated oxidants, mainly by the action of chloramines, produced by myeloperoxidase un activated neutrophils. They also determined that group with more MetS components (with 5 components) presented higher AOPP an lower TRAP when compared with another group of MetS with less components (4 components) [80]. Taken together, MetSHD group presented higher AOPP levels when compared with MetSD group, which are in agreement with previous results.

Studies have found decreases in individual antioxidants, such carotenoids, vitamin C, and vitamin E, as well as TRAP in MetS subjects [81,82]. A study showed lower TRAP concentration compared with control group [82]. Althought no difference was found between TRAP concentrations in MetSHD and MetSD groups, MetSHD group showed a negative correlation between TRAP/uric acid and BMI, AC, and insulin.

Our research group showed that hypertriacyglycerolemia, hyperglycemia, hypertension, and lower HDL-cholesterol values are important factors in increasing oxidative stress, which leads to increase production of superoxide anion via the nicotinamide adenosine phosphate oxidase pathway. This anion reacts rapidly with NO to form peroxynitrite, thus inactivating NO and leading to endothelial disfunction. Thus, the individuals with MetSHD of our study showed a similar metabolic profile of the previous cited study, and also increased levels oxidative markers such AOPP, NOx, and a tendency to increased antioxidant defense evaluated by PON activity [82].

Conclusions

In conclusion, hypovitaminoses D in women with MetS is associated with elevated levels of fasting glucose, HOMA-IR, triglycerides, and biomarkers of oxidative stress than women with MetSD. These are important factors for cardiovascular risk.

Conflict of Interest

The authors declare none conflict of interest.

Authorship

The authors' responsibilities were as follows: M.C.S., A.M.O. collected the data, designed the study, interpreted the results, wrote the manuscript, D.V. performed the statistical analysis. R.D.B.M, F.K.G, A.C.F.M., A.K.M, B.S.A. performed the oxidative stress parameters. D.V and D.S.B developed the hypothesis tested in the study, designed the study, interpreted the results, and wrote the manuscript.

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