Leukocyte telomere length as a compensatory mechanism in vitamin D metabolism.

Vitamin D deficiency is common among postmenopausal women. Telomere length can be a potential protective mechanism for age-related diseases. The objective of our study is to examine the association of vitamin D supplementation on leukocyte telomere length (LTL) in healthy postmenopausal women with vitamin D deficiency. The study was designed as a placebo-controlled study to investigate the short-term effects of vitamin D supplementation and seasonal changes on vitamin D related parameters, including 25(OH)D, 1,25(OH)2D parathormone (PTH), Vitamin D binding protein (VDBP), vitamin D receptor (VDR), and telomere length in a cohort of postmenopausal women (n = 102). The group was divided as supplementation (n = 52) and placebo groups (n = 50). All parameters were measured before and after treatment. Serum VDBP levels were measured by ELISA method and VDR, GC (VDBP) gene expressions and relative telomere lengths were measured in peripheral blood mononuclear cells (PBMC) using a quantitative real-time PCR method. The results demonstrate that baseline levels were similar between the groups. After vitamin D supplementation 25(OH)D, 1,25(OH)2D, PTH and VDBP levels were changed significantly compared to the placebo group. At the end of the study period, LTL levels were significantly increased in both groups and this change was more prominent in placebo group. The change in GC expression was significant between treatment and placebo groups but VDR expression remained unchanged. Even though the study was designed to solely assess the effects of vitamin D supplementation, LTL was significantly increased in the whole study group in summer months suggesting that LTL levels are affected by sun exposure and seasonal changes rather than supplementation. The study displayed the short-term effect of Vitamin D supplementation on vitamin D, PTH levels, LTL and vitamin D associated gene expressions. The relation between Vitamin D and LTL is not linear and could be confounded by several factors such as the population differences, regional and seasonal changes in sun exposure.

Introduction

Vitamin D is a steroid hormone synthesized in the skin via sunlight. It plays an important role in biological systems including mineralization of bones, stabilization of blood calcium levels, modulating the innate and adaptive immune responses, and nerve conduction. Vitamin D deficiency is defined as 25-hydroxy-vitamin D (25(OH)D) concentrations lower than 20 ng/ml (50 nmol/l) whereas insufficiency is defined as levels ranging from 20 to 29.9 ng/ml [1]. Population studies indicate that 25(OH)D deficiency is a risk factor for common chronic complex diseases such as cardiovascular disease, diabetes mellitus, neuropsychiatric disorders, and autoimmune diseases. Obesity and vitamin D deficiency in women are important public health problems [2, 3]. Vitamin D deficiency can be seen at any age and is highly prevalent in postmenopausal women due to the decrease in estrogen levels [4]. Aging, insufficient exposure to sunlight, obesity, diet, and hyperlipidemia are also risk factors for vitamin D deficiency. For instance, among the elderly population, 61% in the United States, 90% in Turkey, 96% in India, 72% in Pakistan, and 67% in Iran are vitamin D deficient [5].

Although there is still controversy about the amount and duration of vitamin D supplementation, vitamin D deficient adults require 6000 IU/day of vitamin D3 for 8 weeks or 50,000 IU of vitamin D3 once weekly for 8 weeks [1]. Seasonal variations affect vitamin D levels but as a carrier protein for vitamin D metabolites; vitamin D binding protein (VDBP) levels remain mostly stable [6].

Telomere length is an important parameter in chromosome stability and chronic diseases. Telomeres are repetitive DNA sequences at the ends of the chromosomes and protect the chromosomal integrity. Telomeres maintain the genomic and cellular stability. Telomere length decreases with aging, inflammation and oxidative stress. Telomere length shortening is associated with many chronic diseases [7, 8].

Vitamin D is known to be protective for aging and age-related chronic diseases. It also plays a role in the cell’s vital activities such as differentiation, proliferation and apoptosis [9]. Vitamin D levels are related to aging and telomere length as it reduces inflammation and is related to genomic stability [10, 11]. 1,25- Dihydroxyvitamin D (1,25(OH)2D) receptors are present in leukocytes and this may support the effect of vitamin D in leukocyte telomere length (LTL) [12]. There are numerous studies investigating the LTL in postmenopausal women. Although ethnicity does not play a role in LTL, age and gender are among the determinants of LTL [13, 14]. Decreased estrogen levels after menopause, a pivotal factor in the biology of aging, were positively associated with LTL [13]. Studies also indicate from the premenopausal period through the perimenopausal period to the postmenopausal period there is gradual attrition in LTL which then turned out to be more stable after the postmenopausal period [15].

Previous studies demonstrated the association between telomere length and vitamin D levels in cross sectional studies [16]; however, the short-term effects of vitamin D supplementation on telomere length remain to be elucidated. The effects of Vitamin D supplementation or seasonal changes on telomere length remain largely unknown. Therefore, this study was designed as a placebo-controlled study to investigate the short-term effects of vitamin D supplementation on vitamin D related parameters, including 25(OH)D, 1,25(OH)2D and PTH, VDBP, VDR, and telomere length in a cohort of postmenopausal women.

Materials and methods

The study population

Healthy postmenopausal women with serum vitamin D levels < 20 ng/ml (<50 nmol/l) (n = 102) were included in the study. According to the definition of the World Health Organization (WHO); health is a state of complete physical, mental, and social well-being. The study population was defined as healthy, indicating that they are independent of any known diseases without any prior discerning medical history in accordance with the definition of WHO. The participants were chosen from the volunteers who have not been exposed to vitamin D supplements for at least one year prior to the study. Menopause was defined as the cessation of menstrual periods for twelve consecutive months [17].

The participants were all chosen from the primary health care center in Manyas, Balikesir; a city located in the south Marmara region of Turkey. We selected all participants from the same region to avoid differences in sunlight exposure. Subjects were given case numbers and identities were kept confidential. Information on height, weight, dietary habits, physical activity, alcohol intake, and smoking habits was collected by self-administered questionnaires. All patients underwent physical examination. Body mass index (BMI) was calculated as weight (kg)/height (m2). Cigarette smoking status was defined as the consumption of 100 cigarettes per lifetime for a current cigarette smoker. Never-smoker was defined as consumption of less than 100 cigarettes per lifetime. Subjects were divided into three groups by self-reports for the level of physical activity as non-exercisers, mild exercisers (<4 hours/week), moderate to high exercisers (> 4 hours/week). Physical activity was defined as the performance of any structured activity on a regular basis such as walking, lifting weights, doing aerobics, resistance training, or riding a stationary bike [18]. Subjects with chronic diseases (renal insufficiency, megaloblastic anemia, cardiovascular disease, history of cancer, cardiovascular disease, thyroid or parathyroid disease, liver dysfunction, kidney disease, extensive skin disease, extreme stress / depression, malabsorption / malnutrition, chronic inflammatory disease, hyperlipidemia requiring statin therapy) and subjects using medications that alter serum vitamin D levels within one year, carriers of mutations that reduces vitamin D levels and causing hereditary diseases, women who follow a strict vegan diet were excluded from the study. The study was conducted according to the principles expressed in the Declaration of Helsinki. Acıbadem University Institutional Review Board (ATADEK) approved the study protocol with the decision number 2016-1/17. Written informed consent was obtained from all participants. When the type 1 error is set as 5% with study power 80%, with a hypothetical difference of 10% between the telomere lengths of the study groups, the minimum sample size is calculated as 38 for each group.

Vitamin D supplementation

Serum vitamin D (25(OH)D) levels of all subjects were measured at the beginning of the study. The subjects who were eligible to participate in the study (vitamin D levels < 20 ng/ml), (n = 102) were randomly selected. Randomization was performed by toss of a coin method. The treatment group (n = 52) received oral vitamin D3 supplementation (cholecalciferol) at a dose of 50,000 IU/week for 8 weeks (Devit-3® Deva oral solution, Turkey) and the placebo group (n = 50) received sunflower oil as a placebo of which participants consumed 15 ml once a week for 8 weeks.

Sunflower oil was chosen as placebo, since prior studies stated that it did not have a significant effect on vitamin D levels [19-21]. Subjects and the laboratory staff were blinded to the study arm. Vitamin D supplementation bottles were packaged to look identical to ensure that the participants were truly blinded. After the study, the placebo group received vitamin D supplements free of charge at the same amount given to the treatment group. Since 25(OH)D had a long half-life and was the predominant form in the circulation, it was used as the primary marker of vitamin D status [22].

To detect the effect of seasonal variations on vitamin D supplementation, we analyzed LTL according to the season of treatment. The summer group was the participants taking vitamin D or placebo starting in May-June; the winter group was the participants taking vitamin D or placebo starting in October-January. After 2 months of treatment and 1 month waiting period, peripheral blood was taken and the parameters were re-measured.

Laboratory assessment

Blood samples and peripheral blood mononuclear cell (PBMC) isolation

Peripheral blood samples were drawn at 9 a.m. Blood samples were taken into BD Vacutainer® SST ™ Tubes (8 cc) for vitamin D measurements and ELISA analysis; and purple-cap EDTA tubes (10 cc) for genetic measurements. Serum was obtained by centrifugation for 6 minutes at 3000 rpm, +4°C within 1 hour after the blood collection. Serum was aliquoted into 1.5 ml Eppendorf tubes and stored at -80°C for ELISA and vitamin D measurements.

PBMCs were isolated from peripheral blood using the Ficoll-Paque gradient centrifugation method within 6 hours after blood collection [23]. PBMCs were used for DNA and RNA isolation.

Biochemical analysis

All biochemical measurements were carried out in ISO-15189 accredited Acibadem Labmed Clinical Laboratories, Istanbul, Turkey. Biochemical measurements of 25(OH)D levels were carried out by CLIA method on ADVIA Centaur® XPT Immunoassay System, Siemens, Germany. Briefly, serum samples in eppendorf tubes were centrifuged at 1000 g for 10 minutes before being placed in the instrument, transferred to cuvettes and incubated for 15 seconds. After incubation with 200 μl reactive auxiliary pack reagent at 37°C for 4.5 minutes, with 50 μl lite reagent at 37°C for 5.5 minutes, with 100 μl solid phase reagent and with 50 μl auxiliary well reagent at 37°C for 3 minutes, aspiration of the reagent by separating the solid phase from the mixture was performed by the device. The chemiluminescence reaction was initiated and the results were reported. Intra-assay and inter-assay CV of the measurements were 2.29 and 5.16 respectively.

PTH serum levels were measured by the ADVIA Centaur PTH test which is a direct method using fixed amounts of 2 anti-human PTH antibodies. It is a sandwich immunoassay using chemiluminometric technology. The first (N-terminal) antibody in Lite Reagent is a monoclonal mouse anti-human PTH labeled with acridinium ester. The second antibody is a biotinylated monoclonal mouse anti-human PTH (C-terminal) antibody bound to Streptavidin coated paramagnetic latex particles in Solid Phase. The reference values for PTH levels were 18.5–88 pg/ml. Serum 1,25(OH)2D levels were measured by IDS-iSYS 1,25-Dihydroxy Vitamin D assay involving immunopurification followed by quantitative determination of serum 1,25(OH)2D on IDS-iSYS system (IDs-iSYS Multi-Discipline Automated System, Immunodiagnostic Systems Limited, Tyne & Wear, UK). Intra-assay and inter-assay CV of the measurements were 3.76 and 7.59 respectively. The reference values for 1,25(OH)2D were 26.1–95.0 pg/ml.

Measurement of leukocyte telomere length

DNA was isolated from mononuclear cells using Quick-DNATM Miniprep Plus Kit (Zymo Research®, Irvine CA, USA). The purity and concentration of DNA samples were measured by NanoDrop ™ 2000 /2000c spectrophotometer (Thermo Scientific Inc). DNA samples with A260/280 ratio between 1.8–2 nm and A260/230 ratio between 2–2.2 nm were selected and stored at -20°C for the measurement of telomere length. The quantitative PCR (qPCR) method was performed for the determination of LTL [24]. The primer sequences for the amplification of telomeres (T), single copy gene 36B4 (S), and β-globin were described by Cawthon, 2002. QPCR was performed on the BIORAD CFX96 Touch ™ Real Time PCR detection system using the SensiFAST ™ SYBR® No-ROX Kit (Bioline Meridian, USA). The amount of DNA for qPCR was calculated from the standard curve obtained from the dilution series as described [24]. Each sample was studied in duplicate. Telomere length, expressed as telomere to single-copy gene ratio (T/S), was assessed by qPCR. The ΔCt values were calculated for each subject. (ΔCt = averageCt36B4 –average Cttelomere).

cDNA synthesis and vitamin D receptor (VDR), vitamin D-binding protein gene (GC/VDBP) expressions

RNA was isolated from mononuclear cells using Quick RNA Miniprep Plus Kit (Zymo Research®, Irvine CA, USA). The purity and concentration of DNA samples were measured by NanoDrop ™ 2000/2000c spectrophotometer (Thermo Scientific Inc). RNA samples with A260/280 ratio between 1.9–2.2 nm were selected and stored at -80°C for VDR and GC mRNA expression studies. In order to determine GC and VDR expression, the same amount of RNA was taken from each sample and cDNA synthesis was performed. cDNA synthesis was performed with SensiFAST cDNA synthesis kit (Zymo Research®, Irvine CA, USA). Quantitative real-time PCR (qRT-PCR) was performed using DNA specific primers (at least one of the primer pairs was designed with exon-intron junction) (Table 1). Primers were designed by Primary BLAST software available at https://www.ncbi.nlm.nih.gov/tools/primer-blast/. B-Actin (ACTB) was used as an internal control. B-Actin primers were prepared as described [25]. Fluorescence detection of samples in qRT-PCR was performed on the BIORAD CFX96 Touch ™ Real-Time PCR detection system using Sensi FASTTM SYBR® No-ROX Kit (Bioline Meridian, USA). The amount of expression of the genes was determined by ΔΔCT method.

Table 1

Designed primers for VDR and GC genes.

ACTB gene was used for internal control.

Gene	Primer Sequence
VDR	F: 5′-CCAGGATTCAGAGACCTCACC-3′
	R: 5′-AATCAGCTCCAGGCTGTGTC-3′
GC (VDBP)	F: 5′-CAAGGCTCAGCAATCTCAT-3′
	R: 5′-CTCTTTGGCCATGCAATC-3′
ACTB(B-ACTIN)	F: 5′-GCACAGAGCCTCGCCTT-3′
	R: 5′-GTTGTCGACGACGAGCG-3′

Measurement of VDBP levels by ELISA

VDBP serum levels were measured by a commercially available sandwich-type ELISA (ELISA VDBP, SEB810Hu, Cloud Clone, USA), according to the manufacturer’s protocol. Briefly, standards and sample sera, respectively, were added to the wells on the plate coated with antibodies specific for VDBP. After incubation at room temperature, unbound material was washed off, secondary antibodies specific to the target protein, conjugated with horse radish peroxidase (HRP) was added. After incubation and addition of stop solution after washing, the plate was read at an appropriate wavelength (450 nm) on a spectrophotometer (Biotek, Vermont, USA). A standard curve was generated with standard absorbance values and the amount of target proteins in the subject samples was determined.

In the ELISA test, how close the absorbance values were to the standard values was calculated with the correlation coefficient predisposed to 0.95. Values above R2 = 0.9967 showed that the positive standards of all samples were working properly.

Statistics

All statistical analyzes were performed using SPSS program (version 20.0 for Windows, SPSS Inc. Chicago, IL). The Kolmogorov-Smirnov or Shapiro-Wilk test was used to analyze the normality of the data. Continuous variables with normal distribution were expressed as mean ± standard deviation, and variables with nonparametric distribution were expressed as median and interquartile range (IQR) and categorical variables were expressed as percentages when appropriate. Student’s t-test or Mann Whitney U test was used to compare unpaired samples as needed. The relationships among parameters were assessed using Pearson’s or Spearman’s correlation coefficient according to the normality of the data. A p-value of <0.05 was considered statistically significant.

Results

Demographic and biochemical results of the study group

This study included healthy 102 postmenopausal women (25(OH)D<20 ng/ml (<50 nmol/l)) who had never used vitamin D supplements or hadn’t used vitamin supplements for at least a year. Subjects were divided into two groups as Vitamin D group taking vitamin D supplements (n = 52) and the placebo group (n = 50). Subjects were randomly selected for allocating vitamin D supplement/placebo by toss of a coin method.

Enrollment of subjects started in October 2017, ended in June 2018. Demographic variables were displayed in Table 2. Baseline characteristics were similar between the groups. The distribution between Vitamin D and placebo groups was similar for smoking and physical activity status (Table 2). Baseline biochemical parameters were distributed homogenously in both groups and were shown in Table 3.

Table 2

Demographic parameters in subjects.

Variables	Study group (n = 102)	Vitamin D group (n = 52)	Placebo group (n = 50)	p-value*
Age (years)	58.4 ± 8.0	59.2 ± 8.6	57.7 ± 7.4	0.34
Weight (kg)	69.6 ± 9.1	68.8 ± 9.5	70.4 ± 8.6	0.58
Height (cm)	161.2 ± 5.5	161.4 ± 5.8	161.1 ± 5.2	0.76
BMI (kg/m 2 )	26.7 ± 3	26.4 ± 3.2	27 ± 2.7	0.26
Menopause age	47.5 ± 4	47 ± 4.9	48.1 ± 4.3	0.21
Smoking status	n (%)	n (%)	n (%)	0.20
Smoker	26 (24.5)	13 (23)	13 (26)
Nonsmoker	77 (75.5)	40 (77)	37 (74)
Exercise status	n (%)	n (%)	n (%)	0.94
Moderate-exercisers	9 (8.8)	5 (9.6)	4 (8)
Mild-exercisers	19 (18.6)	10 (19.2)	9 (18)
Non-exercisers	74 (72.5)	37 (71.2)	37 (74)

Mean values ± standard deviations were shown when parameters were distributed normally. Median and (IQR) were shown when the parameters were in abnormal distribution.

*Significance between groups is shown as p-value <0.05.

Table 3

Baseline biochemical characteristics of subjects.

Laboratory	Study group	Vitamin D group	Placebo group	p-value*
Total cholesterol (mg/dl)	208.8±41.2	199.6±31.8	213.6±45.5	0.42
LDL-cholesterol (mg/dl)	115.1±35.9	107.3±30.5	119.3±38.8	0.45
HDL-cholesterol (mg/dl)	62.5±12.5	59.3±11.8	64.4±12.8	0.34
Triglyceride (mg/dl)	173.8±137.4	126 (180)	117.5 (104.8)	0.37
Fasting glucose (mg/dl)	102.2±22.2	94 (36.5)	96.5 (26.9)	0.84
Creatinine (mg/dl)	0.8±0.1	0.85 ± 0.2	0.9± 0.1	0.53
Alanine transaminase (ALT) (U/l)	15.8±6.2	15.0 (11)	13.0 (5.0)	0.29
Aspartate transaminase (AST) (U/l)	19,1±6.7	17.3 (3.3)	17.6 (4.8)	0.79

Mean values ± standard deviations were shown when parameters were distributed normally. Median and (IQR) were shown when the parameters were in abnormal distribution.

*Significance between groups is shown as p-value <0.05 and labeled as bold.

Vitamin D supplementation and placebo groups have similar 25(OH)D levels before vitamin D treatment (Table 4). The change in vitamin D levels was statistically significant both in the placebo (11.8 ± 4.2 vs 15.2 ± 5.9 before and after treatment, respectively, p <0.001) and in the vitamin D supplementation (11.3 ± 3.6 vs 28.6 ± 10.3 before and after treatment, respectively, p <0.0001) groups. Table 4 demonstrates that vitamin D related parameters were similar at baseline between the study groups.

Table 4

Baseline levels of variables in vitamin D supplementation and placebo groups.

Variables	Vitamin D group (n = 52)	Placebo group (n = 50)	p-value*
25(OH)D (ng/ml)	11.2±3.6	11,8±4,2	0.534
1,25 (OH) 2 D (pg/ml)	70.2±22.7	74.5±34.3	0.454
PTH (pg/ml)	47.5(29.6)	54.4(33.9)	0.084
LTL	6.0±1.4	6.1±1.3	0.751
VDBP levels (mg/l)	538.0±224.1	465.6±297.1	0.180

Mean values ± standard deviations were shown when parameters were distributed normally. Median and (IQR) were shown when the parameters were in abnormal distribution.

*Significance between groups is shown as p-value <0.05 and labeled as bold.

Table 5 displays same parameters after vitamin D (or placebo) supplementation. At the end of the study period 25(OH)D and 1,25(OH)2D levels increase and, PTH, VDBP levels decrease after vitamin D supplementation, yet, although LTL levels increase in both groups, the increase is more prominent in the placebo group (Table 5).

Table 5

Change in variables after treatment in vitamin D supplementation and placebo groups.

Variables	Vitamin D group (n = 52)	Placebo group (n = 50)	p-value*
25(OH)D (ng/ml)	28.6±10.3	15.2±5.9	<0.0001
1,25 (OH) 2 D (pg/ml)	93.0±30.8	81.3±27.4	0.046
PTH (pg/ml)	29.9(26.8)	41.1 (24.9)	0.019
LTL	7.3±0.9	7.7±0.9	0.01
VDBP levels (mg/l)	433.7±198.1	352.9±170.1	0.034
GC expression change (2 -ΔΔCt )	0.58(0.42)	0.9(0.9)	0.012
VDR expression change (2 -ΔΔCt )	0.17(0.9)	0.4 (1.5)	0.18

Mean values ± standard deviations were shown when parameters were distributed normally. Median and (IQR) were shown when the parameters were in abnormal distribution.

*Significance between groups is shown as p-value <0.05 and labeled as bold.

Relative leukocyte telomere length

LTL was similar between supplementation and placebo groups before treatment. After the treatment, there was a statistically significant difference between the treatment and placebo groups (Table 6). Within the groups, there were significant seasonal changes in LTL (Table 6). LTL increased significantly in summer in both groups, yet no significant difference was noted in winter (Table 6).

Table 6

Comparison of relative telomere length (ΔCt), in treatment and placebo groups according to the season of treatment.

Variables	Vitamin D group (n = 52)	Placebo group (n = 50)	p-value*
LTL
before treatment (summer)	5.29±1.06 (n = 25)	5.67±1.16 (n = 39)	0.20
after treatment (summer)	7.46±0.63 (n = 25)	7.72±0.73 (n = 39)	0.14
p-value	<0.0001	<0.0001
before treatment (winter)	6.69±1.24 (n = 27)	7.67± 0.47 (n = 11)	0.001
after treatment (winter)	7.08±1.06 (n = 27)	7.72±1.28 (n = 11)	0.16
p-value	0.22	0.90

Mean values ± standard deviations were shown as parameters were distributed normally.

*Significance between groups is shown as p-value <0.05 and labeled as bold.

VDR and GC mRNA expressions and VDBP levels

The concentrations of VDBP protein in human plasma are normally maintained within a relatively narrow range (350–550 mg/l [6.25 to9.8 pmol] [26].

VDBP levels were similar at baseline (Table 4). After treatment, VDBP levels were higher in the treatment group compared to the placebo (Table 5).

When treatment vs placebo groups were compared, the decrease in VDBP levels and change in GC (VDBP) mRNA expression were statistically significant (Table 5).

The fold change (2-ΔΔCt) in groups was compared as the change in VDR expression. The difference in mRNA expressions of VDR was not statistically significant in treatment vs placebo groups. When we compared VDR expression with smoking in the whole group, VDR expression did not change in smokers (p = 0.786), but significantly decreased in non-smokers (p = 0.007). GC (VDBP) expression did not change in smoking (p = 0.498) vs non-smoking (0.373) groups. Physical activity had no effect on VDR expression.

Discussion

Vitamin D deficiency is a common risk factor for aging and age-related diseases [11, 27]. Telomere length is one of the potential protective mechanisms especially for age-related diseases including cancer, diabetes and cardiovascular diseases [28]. The short-term effects of Vitamin D supplementation on telomere length are largely unknown. We evaluated the short-term effects of vitamin D supplementation on LTL and on the expressions of vitamin D associated genes in postmenopausal women. We have chosen post-menopausal women to exclude the estrogen effects on vitamin D [29, 30]. In order to establish homogeneity of the groups, we selected a village in western Anatolia with ample seasonal sun exposure. The homogeneity of the group enables us to observe the effects of treatment better, minimizing the confounding effects.

At the end of the study period 25(OH)D and 1,25(OH)2D levels increase and, PTH, VDBP levels decrease after vitamin D supplementation, yet, although LTL levels increase in both groups, the increase is more prominent in the placebo group. Given the study design we cannot make conclusions about the effects of seasonal changes on LTL. However, LTL significantly increases in both groups in summer months, suggesting that the relation between LTL and vitamin D levels is complex and can be affected by sun exposure and seasonal changes. The study region experienced particularly hot summers in 2018–2019, which could have affected the study results [31].

The characteristics of the study location and population i.e. obesity, diet and socioeconomic factors can be other confounding factors [32, 33]. Our findings support prior studies. The seasonal changes may cause variability in vitamin D levels among subjects with high BMI. 1,25(OH)2D levels are lower in obese and elderly persons [32].

The study results suggest that telomere length can be a potential compensatory mechanism in persistent vitamin D deficient states. The active form of vitamin D is 1,25(OH)2D. 1,25(OH)2D levels are increased after vitamin D supplementation compared to the placebo with borderline statistical significance. Prior study reports that 1,25(OH)2D levels remain constant throughout the year. Age and BMI can affect 1,25(OH)2D levels [33]. Our study subjects were overweight and postmenopausal which can affect the 1,25(OH)2D levels.

PTH levels decreased with vitamin D supplementation. PTH plays a role in the regulation of vitamin D metabolism and stimulates the formation of 1,25(OH)2D in the kidneys. There is an inverse association between PTH levels and vitamin D deficiency and up to a 20% decrease is expected with vitamin D supplementation in normal weight individuals [34, 35]. However, the dose of vitamin D supplementation to suppress PTH levels may differ in overweight and obese adults [36].

Vitamin D is reported to have an effect on telomere length due to its role in cell proliferation, aging and apoptosis [16, 37, 38]. The molecular mechanisms of interaction between Vitamin D and telomere length are largely unknown. Telomere shortening is directly proportional to age and oxidative stress. Vitamin D replacement reduces nuclear factor–kB activity, increases anti-inflammatory IL10 levels, decreases pro-inflammatory interleukins, therefore acts on cell survival [39]. However, although 25(OH)D is associated with telomere levels, it is not known whether vitamin D replacement has any effect on LTL. There are conflicting reports about vitamin D levels and LTL. A positive correlation between 25(OH)D levels with LTL is reported [37, 38]. Yet, these studies are cross sectional and did not examine the seasonal changes and/or vitamin D supplementation on LTL levels.

Our observations suggest that the relation between Vitamin D and LTL is not linear and can be affected by several confounding factors such as the populational, regional and seasonal changes. Prospective studies are needed to monitor the LTL in relation to vitamin D levels. Our observations can add to the literature as it is a placebo-controlled study among postmenopausal women showing the short-term effect of Vitamin D supplementation on LTL together with vitamin D associated gene expressions.

A prior study that demonstrates the effects of vitamin D supplementation in Afro-American subjects associated the effects of vitamin D supplementation with telomerase activity and telomerase activity increases with vitamin D supplementation. However, telomere length can also be preserved independent of the activity of the telomerase enzyme [40]. Julin et al. reported that no correlation exists between 25(OH)D, 1,25(OH)2D and LTL in men [41]. Yang et al. demonstrate that vitamin D supplementation for 12 months increases the telomere length in elderly subjects with mild cognitive impairment (MCI) who received oral vitamin D3 daily for 12-month period [16].

VDBP is the primary vitamin D carrier; binding almost 90% of circulating vitamin D and the unbound (or bound to albumin) is the bioavailable part. Menopause represents an important transition in vitamin D requirement [42]. VDBP and 25(OH)D levels are significantly higher in premenopausal women than postmenopausal women and estradiol levels are correlated with VDBP and 25(OH)D [43]. Individual and ethnic differences can affect VDBP level and attachment capacity. In our study, we observed a that VDBP levels decrease and 25(OH)D levels increase with treatment in both placebo and supplementation groups. The change in mRNA expression of GC(VDBP) is significant when treatment and placebo groups were compared. The placebo group showed a more prominent increase in expression compared to the treatment group. The gene expression and protein levels may be discordant according to differences in regulation at the protein or transcriptional level. Protein degradation may be slowed or accelerated according to changing transcript levels [44]. VDR mediates 1,25 (OH)2D and its functions. VDR expression remained stable independent of treatment.

In our study, we observe the short-term effects of vitamin D supplementation. The homogeneity of the group enables us to observe the effects of treatment better, minimizing the confounding effects. The limitations of our study include the small size of the study. In Turkey almost 50% of postmenopausal women have obesity and metabolic syndrome [45]. The study included subjects with high BMI which can limit the generalizability of results to the lean population [46]. We applied the same dose of vitamin D to all participants but metabolism of vitamin D varies between individuals, therefore dose adjustments can yield different results in overweight individuals. We did not have detailed dietary and physical activity scores from the participants. Socioeconomic factors, household income, and ecological and environmental factors can all confound the study findings Even though we mention the seasonal effects on telomere length, the design of the study precludes us to make any conclusive statements on seasonal changes. An appropriate design would have been to include the season of recruitment in the stratification matrix at the time of randomization to exclude the confounding factors. Unfortunately, due to limited funding for a precise period of time, the speed of enrollment, it remained to be underpowered to investigate the effects of seasons on telomere length. We cannot claim that the findings apply to global populations, i.e. unlike western European populations. Due to the climate, the location of the study receives ample amount of sunlight even in the winter months. The results can be different in a different location with limited sun exposure in the winter months.

Conclusions and future directives

LTL displays dynamic changes in postmenopausal women with vitamin D deficiency. Yet the interaction appears more complex than a linear relationship possibly attributable to persistent vitamin D deficiency, and other confounding factors. This study provides a short term evaluation of the effects of vitamin D supplementation on LTL and vitamin D related biomarkers in postmenopausal women with vitamin D deficiency. The results indicate that vitamin D supplementation and seasonal changes can insert bidirectional effects on vitamin D related parameters including LTL and VDBP. Large prospective studies in diverse populations are needed to understand the effect of vitamin D levels and seasonal changes on LTL and vitamin D related parameters.

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1 Dec 2021

PONE-D-21-17303

Leukocyte telomere length as a compensatory mechanism in vitamin D metabolism

PLOS ONE

Dear Dr. Agirbasli,

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ACADEMIC EDITOR: The study and manuscript have merit, but need a better description of sample size determination and other improvements (Introduction etc.). Please adhere closely and address the concerns of the reviewers.

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Additional Editor Comments:

The pilot study seems interesting and of good quality.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: No

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The topic of this paper is of scientific interest. The paper by Agirbasli et al. deals with leucocyte telomere length (LTL) and vitamin D, especially the effect of vitamin D supplementation on LTL in postmenopausal women having vitamin D deficiency. This short-term placebo-controlled study investigates vitamin D supplementation and seasonal changes of vitamin D related parameters. The following parameters were measured before and after treatment: 25(OH)D, 1,25(OH)2D, PTH, VDBP, VDR, and LTL.

It has been shown that serum vitamin D levels increase by its supplementation while LTL is influenced by sun exposure and seasonal periods. In the summer time LTL is higher than in winter season. VDBP decreases with vitamin D supplementation. The findings demonstrate that vitamin D levels are related to aging and to genomic stability. As vitamin D reduces inflammation it is related to telomere length and in that way to genomic integrity. The findings underline that vitamin D plays an important role in LTL changes by vitamin D supplementation but also by variation in sun exposure, summer vs. winter time.

This short-term study describes effects of vitamin D supplementation on LTL and vitamin D related biomarkers in postmenopausal women with vitamin D deficiency. The findings indicate that vitamin D supplementation and seasonal changes have bidirectional effects on vitamin D related parameters including LTL and VDBP. The paper is well written. The tables are informative and demonstrate the findings very well.

Reviewer #2: PONE-D-21-17303

Leukocyte telomere length as a compensatory mechanism in vitamin D metabolism

The role of vitamin D in preventing genome damage has been associated with a multitude of disease conditions. Telomeres play a major role is capping chromosome ends to prevent DNA shortening, thus playing a vital role in preserving genome integrity. There have been some evidence to suggest that vitamin D may prevent genome damage and this effect may also be apparent for maintaining telomere length (TL). The authors have conducted a clinical trial to determine if vitamin D supplementation was associated with TL. They also attempted to examine the seasonal effect of this supplementation. The authors have recognised the role of latitude on vitamin D levels and thus selected participants from the one region to minimise this effect. The topic is topical however I have some concerns regarding the study design and interpretation of results.

Major:

1. The authors have not included a sample size calculation. Their primary aim was to determine if there was an association between D-supplementation and TL. A secondary aim was to additionally examine a seasonal effect which they would have been under powered to examine with a sample size of 102. An appropriate design would have been to include season of recruitment in the stratification matrix at time of randomisation. Instead, their results have been stratified by season at time of analysis which is not appropriate, with no analysis on whole group analysis. As a consequence the placebo group only contained a fifth of the recruited participants (n=11) and would have been way under powered to generate anything meaningful. They should only be publishing data on whole group analysis.

2. Additionally the authors should not be comparing inter group analysis as this is not a phase 3 trial. They should be reporting results from intra group analysis.

3. The authors have not described how the blinding and randomisation was carried out. They claim that the participants and laboratory staff were blinded to the study arm, where the intervention was Devit-3 Deva oral solution and placebo was sunflower oil. Were these ampules/bottles packaged to look identical to ensure that the participants were truly blinded. If so, they need to describe this process.

4. The authors have not described the vitamin D metabolite used for intervention i.e. is it cholecalciferol or ergocalciferol?

Minor:

1. Authors need to include I their Introduction what the evidence is on TL status in postmenopausal women, else what is the purpose of studying this relationship in this group.

2. Line 86: they have described using ‘healthy” postmenopausal women. Can they please describe definition for ‘healthy”.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: No

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6 Jan 2022

Dear Editor,

We greatly appreciate the time, effort, and substantive comments of the reviewers. We have incorporated changes in accordance with reviewer suggestions, or provided rationale for not incorporating the respective suggestion. We would like to thank the reviewers for helping us to improve the clarity and substance of our paper. The reviewers’ valuable comments helped us refine the manuscript in preparation for publication. The reviewers’ comments have been included below for reference with a subsequent response.

Reviewer #1:

The topic of this paper is of scientific interest. The paper by Agirbasli et al. deals with leucocyte telomere length (LTL) and vitamin D, especially the effect of vitamin D supplementation on LTL in postmenopausal women having vitamin D deficiency. This short-term placebo-controlled study investigates vitamin D supplementation and seasonal changes of vitamin D related parameters. The following parameters were measured before and after treatment: 25(OH)D, 1,25(OH)2D, PTH, VDBP, VDR, and LTL.

It has been shown that serum vitamin D levels increase by its supplementation while LTL is influenced by sun exposure and seasonal periods. In the summer time LTL is higher than in winter season. VDBP decreases with vitamin D supplementation. The findings demonstrate that vitamin D levels are related to aging and to genomic stability. As vitamin D reduces inflammation it is related to telomere length and in that way to genomic integrity. The findings underline that vitamin D plays an important role in LTL changes by vitamin D supplementation but also by variation in sun exposure, summer vs. winter time.

This short-term study describes effects of vitamin D supplementation on LTL and vitamin D related biomarkers in postmenopausal women with vitamin D deficiency. The findings indicate that vitamin D supplementation and seasonal changes have bidirectional effects on vitamin D related parameters including LTL and VDBP. The paper is well written. The tables are informative and demonstrate the findings very well.

We appreciate the kind comments of the reviewer.

Reviewer #2:

PONE-D-21-17303

Leukocyte telomere length as a compensatory mechanism in vitamin D metabolism

The role of vitamin D in preventing genome damage has been associated with a multitude of disease conditions. Telomeres play a major role is capping chromosome ends to prevent DNA shortening, thus playing a vital role in preserving genome integrity. There have been some evidence to suggest that vitamin D may prevent genome damage and this effect may also be apparent for maintaining telomere length (TL). The authors have conducted a clinical trial to determine if vitamin D supplementation was associated with TL. They also attempted to examine the seasonal effect of this supplementation. The authors have recognised the role of latitude on vitamin D levels and thus selected participants from the one region to minimise this effect. The topic is topical however I have some concerns regarding the study design and interpretation of results.

Major:

1. The authors have not included a sample size calculation.

We agree with the reviewer. We added the power analysis to the methods section.

‘When the type 1 error is set as 5% with study power 80%, with a hypothetical difference of 10% between the telomere length of the study groups, the minimum sample size is calculated as 38 for each group.’ (page 6, lines 125-127).

Their primary aim was to determine if there was an association between D-supplementation and TL. A secondary aim was to additionally examine a seasonal effect which they would have been under powered to examine with a sample size of 102. An appropriate design would have been to include season of recruitment in the stratification matrix at time of randomisation. Instead, their results have been stratified by season at time of analysis which is not appropriate, with no analysis on whole group analysis. As a consequence the placebo group only contained a fifth of the recruited participants (n=11) and would have been way under powered to generate anything meaningful. They should only be publishing data on whole group analysis.

We agree with insightful comments of the reviewer. To provide the whole group analysis comparing the groups vitamin D supplementation with placebo, we revised the table 4 and added table 5 as suggested by the reviewer and deleted results associated to seasonal changes. We rewrote the discussion after excluding the statistics on seasonal effects and added the limitations to the discussion. We gave the results of the telomere length both in the placebo and treatment groups before and after 2 months of treatment and 1 month waiting period. This remains to be the main finding of the study. Despite an increase in vitamin D levels and decrease in PTH levels in the treatment group compared to the placebo arm, the telomere length was longer in the placebo group compared to the active treatment arm at the end of study period. We agree with the reviewer that an appropriate design would definitely have been to include the season of recruitment in the stratification matrix at time of randomization to exclude the confounding factors. Unfortunately, due to limited funding for a precise period of time, and the speed of enrollment, study could not be designed as suggested by the reviewer, and therefore remained to be underpowered to investigate the effects of seasons on telomere length. We acknowledge the limitations as indicated by the reviewer. The subgroup analysis remains in telomere length with intra group analysis in table 6 as suggested by the reviewer. We omitted previous table 6 showing the seasonal effects of vitamin D related parameters as suggested by the reviewer. We will be willing to shorten the results and the tables further per reviewer suggestions.

2. Additionally the authors should not be comparing inter group analysis as this is not a phase 3 trial. They should be reporting results from intra group analysis.

We agree with the reviewer. We revised tables 4 and 5 and omitted table 6 as suggested by the reviewer. We added the limitations to the discussion.

3. The authors have not described how the blinding and randomisation was carried out. They claim that the participants and laboratory staff were blinded to the study arm, where the intervention was Devit-3 Deva oral solution and placebo was sunflower oil. Were these ampules/bottles packaged to look identical to ensure that the participants were truly blinded. If so, they need to describe this process.

“Vitamin D supplementation bottles were packaged to look identical to ensure that the participants were truly blinded.” The explanation is added to methods section (page 6, lines 139-140).

4. The authors have not described the vitamin D metabolite used for intervention i.e. is it cholecalciferol or ergocalciferol?

The vitamin D metabolite used for intervention is cholecalciferol. The information is added to the methods section (page 6, line 134).

Minor:

1. Authors need to include I their Introduction what the evidence is on TL status in postmenopausal women, else what is the purpose of studying this relationship in this group.

We thank the reviewer for the careful consideration of the manuscript. According to the comments of the reviewer, a paragraph is added to Introduction section explaining the LTL status in postmenopausal women and the purpose of studying this relationship in this group with associated references (page 4, lines 78-84).

“There are numerous studies investigating the LTL in postmenopausal women. Although ethnicity does not play a role in LTL, age and gender are among the determinants of LTL [13,14]. Decreased estrogen levels after menopause, a pivotal factor in the biology of aging, were positively associated with LTL. [13]. Studies also indicate from the premenopausal period through the perimenopausal period to the postmenopausal period there is gradual attrition in LTL which than turned out to be more stable after the postmenopausal period [15].

Shin YA, Lee KY. Low estrogen levels and obesity are associated with shorter telomere lengths in pre- and postmenopausal women. J Exerc Rehabil. 2016 Jun 30;12(3):238-46.

Jones HJ, Janson SL, Lee KA. Leukocyte Telomere Length in Postmenopausal Women. J Obstet Gynecol Neonatal Nurs. 2017;46(4):567-575.

Dalgård C, Benetos A, Verhulst S, Labat C, Kark JD, Christensen K, Kimura M, Kyvik KO, Aviv A. Leukocyte telomere length dynamics in women and men: menopause vs age effects. Int J Epidemiol. 2015 Oct;44(5):1688-95.

2. Line 86: they have described using ‘healthy” postmenopausal women. Can they please describe definition for ‘healthy”.

We added the explanation under the title “Study Population” in Methods section (page 5, lines 96-100). “According to the definition of World Health Organization (WHO); health is a state of complete physical, mental, and social well-being. The study population was defined as healthy, indicating that they are independent any known diseases without any prior discerning medical history in accordance with the definition of WHO.”

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

9 Feb 2022

Leukocyte telomere length as a compensatory mechanism in vitamin D metabolism

PONE-D-21-17303R1

Dear Dr. Agirbasli,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Gabriele Saretzki, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

All remaining issues have been addressed.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: No

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: PONE-D-21-17303

Leukocyte telomere length as a compensatory mechanism in vitamin D metabolism

The role of vitamin D in preventing genome damage has been associated with a multitude of disease conditions. Telomeres play a major role is capping chromosome ends to prevent DNA shortening, thus playing a vital role in preserving genome integrity. There have been some evidence to suggest that vitamin D may prevent genome damage and this effect may also be apparent for maintaining telomere length (TL). The authors have conducted a clinical trial to determine if vitamin D supplementation was associated with TL. They also attempted to examine the seasonal effect of this supplementation. The authors have recognised the role of latitude on vitamin D levels and thus selected participants from the one region to minimise this effect. These findings will add to our growing knowledge on the relationship between vitaD and genome damage in cancer risk.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

14 Feb 2022

PONE-D-21-17303R1

Leukocyte telomere length as a compensatory mechanism in vitamin D metabolism

Dear Dr. Agirbasli:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

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on behalf of

Dr. Gabriele Saretzki

Academic Editor

PLOS ONE