The Effect of Calcium and Vitamin D Supplements as an Adjuvant Therapy to Metformin on Lipid Profile

ABSTRACT:

Objective:

This study aims to investigate the impact of combining calcium and vitamin D supplements with metformin on lipid profile in vitamin D deficient/insufficient polycystic ovary syndrome (PCOS) women. Study design: A randomized, placebo-controlled clinical trial. Setting: Damascus University of Obstetrics and Gynecology Hospital and Orient Hospital, in Damascus, Syria. Materials and methods: Forty PCOS women with 25-hydroxyvitamin D < 30 ng/mL were randomly assigned to take either metformin (1500 mg/daily) plus placebo or metformin (1500 mg/daily) plus calcium (1000 mg/daily) and vitamin D3 (6000 IU/daily) orally for 8 weeks. Fasting serum levels of total cholesterol (TC), high-density lipoprotein cholesterol (HDL), low-density lipoprotein cholesterol (LDL), triglyceride (TG) and non-HDL cholesterol (non-HDL) were detected at the baseline and after 8 weeks of intervention. Results: Of all participants, thirty-four completed the study. After 8 weeks of intervention, calcium and vitamin D levels increased significantly in the supplementation group, but no significant changes in the lipid profile were noticed in both groups. Conclusions: Adding calcium and vitamin D to metformin therapy had no superior effect on improving lipid profile in vitamin D deficient/insufficient subjects with PCOS.

KEYWORDS:

Polycystic Ovary Syndrome; Lipid Profile; Calcium; Vitamin D; Metformin.

INTRODUCTION:

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder among females of reproductive age, with a worldwide prevalence of 5-20% depending on the criteria is used [1,2]. The main manifestations of this syndrome are ovulatory dysfunction, hyperandrogenism, and polycystic ovarian morphology [2]. Noticeably, PCOS is associated with several metabolic disturbances such as insulin resistance, compensatory hyperinsulinemia, dyslipidemia and central obesity, which increase the risk for long-term complications like type 2 diabetes mellitus, metabolic syndrome, and cardiovascular diseases [3]. However, the exact aetiology of PCOS remains unclear and current treatments are only moderately effective at controlling PCOS symptoms and preventing its complications [4]. Thus, seeking for alternative or adjuvant therapies, numerous studies investigated the impact of dietary supplements such as zinc [5], omega-3 fatty acids [6], and vitamins [7] in management this syndrome. Some of those studies focused on vitamin D. Vitamin D is no longer considered a vitamin solely responsible for bone metabolism and calcium homeostasis as several studies demonstrated its influence on cell proliferation, differentiation, apoptosis, immune regulation, genome stability, and neurogenesis [8]. Growing evidence suggests a role of vitamin D in female reproductive diseases, as the expression of Vitamin D Receptors (VDR) was identified in many organs throughout the female reproductive tract, such as ovary (particularly granulosa cells), uterus, and placenta [9]. On the top of that, vitamin D regulates over 300 genes, including genes that are important for glucose and lipid metabolism. Thus, vitamin D deficiency may be the missing link between insulin resistance and PCOS [3]. Moreover, vitamin D deficiency is a common condition among women with PCOS [10,11], and several studies indicated an association between low levels of serum 25-hydroxyvitamin D (25-OH-Vitamin D) and manifestations of PCOS including insulin resistance [10“14], hyperandrogenism [12], and infertility [15,16]. However, Although pre-clinical data have suggested a possibility for calcium and vitamin D to modulate lipid profile [17“19], clinical studies about the impact of calcium and vitamin D supplements on lipid profile in PCOS subjects are scarce and inconsistent [20“23]. Considering the aforementioned data, we conducted this placebo-controlled clinical trial to assess the effect of calcium and vitamin D supplements as an adjuvant therapy to metformin on lipid profile in vitamin D deficient/insufficient PCOS women.

MATERIAL AND METHODS:

Study design: This randomized, single-blinded, placebo-controlled clinical trial was conducted on women with PCOS who referred to the outpatient clinic at Damascus University of Obstetrics and Gynecology Hospital, and Orient Hospital, in Damascus, Syria, from December 2016 to December 2017. The Ethical Committee of Damascus University approved the study protocol, and a written informed consent was obtained from all participants. Participants: In this study, we included PCOS women diagnosed according to the Rotterdam criteria [24], which require the presence of at least two of the following three criteria: 1) Oligo or anovulation, 2) Clinical and/or biochemical signs of hyperandrogenism, 3) Polycystic ovarian morphology on ultrasound examination (defined as the presence of 12 or more follicles in each ovary measuring 2“9 mm in diameter and/or an ovarian volume >10 mL). Patients who were diagnosed with androgen-secreting tumours, Cushing's syndrome, congenital adrenal hyperplasia, hyperprolactinemia, hypercalcemia, malabsorption disorders, diabetes mellitus, thyroid disorders, liver disease, renal disease, history of kidney stones, epilepsy, or cardiovascular disease were excluded. Pregnant, postpartum or breastfeeding women were excluded as well. All women at the baseline were vitamin D deficient or insufficient according to the Endocrine Society Clinical Practice Guideline [25]. All study participants reported no use of any hormonal therapy, corticosteroids (other than topical corticosteroids forms), insulin sensitizers, hypolipidemic agents, anti-obesity medications, vitamin D or calcium supplements, anti-epileptic drugs, or any other drugs known to affect endocrine parameters, carbohydrate metabolism, or calciotropic hormone concentrations during the last 3 months. Every patient who met the inclusion criteria and approved to participate in this trial had a face-to-face interview at the baseline to answer a comprehensive questionnaire, which embraced; age, smoking and alcohol drinking habits, dress code, skin colour, outdoor exposure to the sunlight and the use of sunscreens. All women were advised to maintain their usual dietary habits, and not to modify other lifestyle factors such as sunlight exposure or physical activity during the study. Intervention: Patients were randomly assigned into two groups when they came back in the early follicular phase (2-5 days of menses) using a randomization table. Patients in group A received metformin and placebo; patients in group B received metformin, calcium carbonate and vitamin D3 (Cholecalciferol), orally for 8 weeks. The metformin dose was increased stepwise, starting with 500 mg once daily for the 1st week, 500 mg twice daily in the 2nd week, followed by 500 mg 3 times daily from the 3rd week onward. The dose of calcium carbonate (1000 mg/daily) and vitamin D3 (6000 IU/daily) remained constant throughout the study period.

Clinical Assessment:

Standard anthropometric data were obtained from each subject (height, weight, waist circumference, and Hip circumference), in an overnight fasting status without shoes with light clothes, at the baseline and after 8 weeks of intervention. Body mass index (BMI) was calculated as weight in kilograms divided by height in meters squared (kg/m2). Menstrual regularity was assessed as the presence of a menstrual cycle with 21-35 days. Hirsutism was evaluated using the modified Ferriman-Gallwey score (m-FGS) [26], with a threshold (m-FGS) ?‰?6. Assessment of biochemical variables: All assays were conducted at the laboratories of Damascus University of Obstetrics and Gynecology Hospital. All blood samples were taken after an overnight fast. We took 10 millilitres of venous blood from each participant at the baseline and after 8 weeks of intervention. Samples were centrifuged at 4000 rpm for 10 min to separate serum. Then, the serum was stored at -60??c until assayed. Serum concentrations of calcium and phosphorus were assayed by colourimetric method using kits from AMS (AMS S.p.A., Italy). Serum concentrations of LDL and HDL were assayed by direct method using kits from AMS (AMS S.p.A., Italy). Serum concentrations of TC and TG were assayed by enzymatic colourimetric method using kits from AMS (AMS S.p.A., Italy). Serum concentrations of 25-OH-Vitamin D were assayed using Immunofluorescence kits from I-CHROMA (Boditech Med Inc., Korea). Statistical analysis: All statistical analyses were performed using a Statistical Package for Social Science software (SPSS) version 24.0 (IBM Corp., Armonk, N.Y., USA).

Continuous variables were expressed as mean ?± standard deviation and categorical variables as counts with percentages. The Kolmogorov“Smirnov test was used to evaluate the normality of data distribution. Between groups comparisons were performed using the independent t-test for normally distributed variables, the Mann“Whitney U test for non-normally distributed variables, and Chi-square or Fisher's exact test as appropriate for categorical variables. Within-group comparisons were performed using the paired t-test for normally distributed variables and the Wilcoxon paired rank test for non-normally distributed variables. For testing all hypotheses, tests were two-tailed, and p values less than 0.05 were considered statistically significant.RESULTS: Of 82 patients with PCOS, 45 women met the inclusion criteria and participated in the study, of which 5 patients were lost before randomization while waiting for their menses. Forty Patients were randomly assigned into the two groups, 20 patients in each group. However, only 34 patients (85%) completed the study (group A: metformin and placebo, n = 16; group B: metformin, calcium carbonate and vitamin D3, n = 18). The details about the study design and subjects lost to follow-up are illustrated in Figure 1. At the baseline, the two groups did not differ significantly in age, BMI, or other baseline characteristics as shown in Table 1. The mean age was 23.38 ?± 3.54 and 23.06 ?± 3.32 years in group A and B, respectively. The mean BMI was 28.01 ?± 4.41 and 25.48 ?± 4.97 kg/m2 in group A and B, respectively. After 8 weeks of intervention, vitamin D3 and calcium supplementation, compared to placebo, led to a significant increase in 25-OH-vitamin D levels (+19.38 ?± 7.78 vs +0.11 ?± 4.79 ng/mL, respectively), and calcium levels (+0.83 ?± 0.82 vs +0.01 ?± 0.86 mg/dL, respectively), but no significant change was detected in phosphorus levels (+0.38 ?± 0.85 vs +0.26 ?± 1.78 mg/dL, respectively) (Table 3). Notably, 25-OH-vitamin D levels normalized after supplementation in all patients of group B. Weight, BMI and waist to hip ratio decreased significantly in both groups with a higher reduction in the group received metformin plus calcium and vitamin D (table 2), but the means of changes from baseline didn't differ significantly between them (table 3). However, no significant changes were noticed in any studied lipid profile parameters in both groups (table 2 and 3).DISCUSSION: The results of our study indicated that adding calcium and vitamin D to metformin therapy had no superior effect on improving lipid profile in vitamin D deficient/insufficient subjects with PCOS. Previous studies have suggested several mechanisms by which vitamin D may affect lipid profile like increasing the expression of very low-density lipoprotein cholesterol receptors (VLDL-R), reducing PTH concentration and improving insulin sensitivity [17].

Besides, vitamin D enhances intestinal calcium absorption which reduces lipids solubility and absorption from the gut [18,19]. On the other hand, a recent study disclosed that vitamin D might inhibit the expression of apolipoprotein AI gene, the major apoprotein of HDL [27], and animal studies showed an increase in TC levels in both male and female vitamin D knock-out mice, but HDL levels increased only in the male vitamin D knock-out mice [28]. So, it is still unknown whether combining these elements will lead to a beneficial, detrimental, or even null effect on lipid profile. Concerning clinical studies on PCOS subjects, previously the single arm study of Wehr et al.[20] showed that supplementation with vitamin D3 (20000 IU/weekly) caused a significant increase in LDL and TC and a significant decrease in TG levels. On the other hand, Irani et al study [22] demonstrated that 8 weeks of vitamin D3 supplementation (50000 IU/weekly) in vitamin d deficient PCOS subjects led to a significant decrease in TG levels, but no effects on other lipid profile parameters were detected compared to placebo. On the contrary, our results did not indicate any beneficial (nor detrimental) effects of supplementation with calcium and vitamin D supplements on serum lipid profile in vitamin D deficient/insufficient PCOS subjects, which is consistent with the study of Raja-Khan et al.[21] as they could not detect notable effects on lipid profile when PCOS patients were treated with a high dose of vitamin D3 (12000 IU/daily) for 12 weeks compared to placebo. Moreover, although Asemi et al. study [23] demonstrated that calcium carbonate (1000 mg/day) and vitamin D (50000 IU/week) co-supplementation for 8 weeks in overweight-obese vitamin D deficient PCOS patients caused a significant decrease in serum TG (P value=0.02) and VLDL levels (P value=0.02), these effects disappeared after adjustment for baseline values (P value=0.12 for both TG and VLDL). Nevertheless, none of those studies concerned about the impact of combining calcium and vitamin D supplements with metformin on lipid profile. A recent study showed that vitamin D3 analogs can modulate glucose parameters and lipid metabolism in a diabetic rat model with additional protective effects when combined with metformin [29]. However, our study does not support the effects on lipid profile in vitamin D deficient/insufficient PCOS subjects.

CONCLUSIONS:

Adding calcium and vitamin D to metformin therapy had no superior effect on lipid profile in vitamin D deficient/insufficient subjects with PCOS.CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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