Abstract
Objectives
Observational studies have suggested that a higher 25-hydroxyvitamin D concentration may be associated with longer telomere length; however, this has not been investigated in randomised controlled trials. We conducted an ancillary study within a randomised, double-blind, placebo-controlled trial of monthly vitamin D (the D-Health Trial) for the prevention of all-cause mortality, conducted from 2014 to 2020, to assess the effect of vitamin D supplementation on telomere length (measured as the telomere to single copy gene (T/S) ratio).
Design, Setting, Participants, and Intervention
Participants were Australians aged 60–84 years and we randomly selected 1,519 D-Health participants (vitamin D: n=744; placebo: n=775) for this analysis. We used quantitative polymerase chain reaction to measure the relative telomere length (T/S ratio) at 4 or 5 years after randomisation. We compared the mean T/S ratio between the vitamin D and placebo groups to assess the effect of vitamin D supplementation on relative telomere length, using a linear regression model with adjustment for age, sex, and state which were used to stratify the randomisation.
Results
The mean T/S ratio was 0.70 for both groups (standard deviation 0.18 and 0.16 for the vitamin D and placebo groups respectively). The adjusted mean difference (vitamin D minus placebo) was −0.001 (95% CI −0.02 to 0.02). There was no effect modification by age, sex, body mass index, or predicted baseline 25-hydroxyvitamin D concentration.
Conclusion
In conclusion, routinely supplementing older adults, who are largely vitamin D replete, with monthly doses of vitamin D is unlikely to influence telomere length.
Key words: Vitamin D, cellular ageing, relative telomere length, T/S ratio, gerontology, epidemiology
Introduction
Telomeres are nucleoprotein structures comprising repetitive deoxyribonucleic acid (DNA) sequences at the end of chromosomes that protect the chromosomal ends from being recognized as DNA double strand breaks. During each cell division, bases are lost from the telomere termini, eventually resulting in an accumulation of dysfunctional telomeres and the onset of replicative senescence (1). Thus, telomere length is a marker of cellular ageing. The median leukocyte telomere length ranges from 7 to 11.6 kilobases in a healthy newborn and shortens at a rate of 30–35 base pairs (1000 base pairs=1 kilobase) per year (2). Relative telomere length is quantified by telomere to single-copy gene (T/S) ratio. Chronic inflammation, and factors that influence a pro-inflammatory milieu, such as obesity and smoking, are associated with shorter leukocyte telomere length (3, 4). Shortened leukocyte telomere length is associated with increased risk of infections (5), diabetes (6), cardiac diseases (7), and all-cause mortality (8).
Vitamin D may help reduce telomere shortening through anti-inflammatory and anti-proliferative mechanisms (9). The active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), decreases the mediators of systemic inflammation (including interleukin-2 and tumor necrosis factor-a) (10). In addition, inverse associations have been shown between both 25-hydroxyvitamin D (25(OH)D) and 1,25(OH)2D concentrations and C-reactive protein, a marker of inflammation (11, 12).
Observational studies assessing the relationship between 25(OH)D concentration and leukocyte telomere length have generated inconclusive findings. Some cross-sectional studies (13, 14, 15, 16, 17, 18), but not all (19, 20, 21), have suggested that higher 25(OH)D concentration is associated with longer leukocyte telomere length. However, a cohort study from Germany that included 9,940 participants aged 50–74 years, found no association between 25(OH)D concentration and telomere length (22). In a more recent cohort study, in which 775 older adults (≥85 years) were followed up for 36 months, associations between 25(OH)D concentration and telomere length were inconsistent over time (23).
One randomised controlled trial (RCT) found no beneficial effect of vitamin D supplementation on telomere length; however, this study included only 102 postmenopausal women with vitamin D deficiency (<50 nmol/L) and treatment duration was short (50,000 international units/week for 8 weeks) (24). Another RCT, performed in 37 overweight African American adults (aged 19–50 years), found that supplementation with 60,000 international units (IU) of vitamin D per month for 16 weeks resulted in increased telomerase activity in peripheral blood mononuclear cells (PBMCs) (25). Telomerase maintains telomere length by adding telomeric sequences onto the chromosome termini (26); thus, results of this trial suggest that vitamin D may help maintain telomere length.
We used data from a large, population-based trial for the prevention of all-cause mortality (the D-Health Trial) to examine whether supplementing older Australians with monthly doses of 60,000 IU of vitamin D3 for at least 4 years has beneficial effects on relative telomere length.
Method
Trial design and participants
The D-Health Trial was a randomised, placebo-controlled, double-blind trial of vitamin D, the primary outcome of which was all-cause mortality (27). Telomere length was pre-specified as a tertiary outcome in the published analysis plan (28). Detailed trial methods have already been published (29). Briefly, men and women aged 60 to 79 years, randomly selected from the Australian Commonwealth electoral roll (voter registration is compulsory), were invited to participate, and we also included volunteers aged 60 to 84 years. Exclusion criteria included self-reported history of hypercalcaemia, hyperparathyroidism, kidney stones, osteomalacia, or sarcoidosis, or daily intake of more than 500 IU of supplementary vitamin D.
Randomisation and intervention
We used computer-generated permuted block randomisation (stratified by age, sex, and state of residence) to randomly allocate participants in a 1:1 ratio to either 60,000 IU of cholecalciferol (vitamin D3) or identical placebo (containing excipient only), taken orally once a month for a maximum of 5 years (29). The dose was chosen based on a pilot trial that found supplementation with 60,000 IU of vitamin D resulted in a substantial increase in 25(OH)D concentration (33 nmol/L) (30). We elected to use a bolus over a daily dose as pharmacokinetic studies showed an equivalent increase in 25(OH)D (31, 32), and there is evidence that monthly dosing results in better adherence than daily dosing (33). Blister packs containing 12 capsules were posted to participants at enrolment, and annually thereafter. Following recruitment, participants were encouraged to restrict their off-trial vitamin D use to 500 IU/day, but were allowed to remain in the trial provided that the supplementary intake did not exceed 2000 IU/day, which could have resulted in them exceeding the tolerable upper limit of 4,000 IU per day (34). This approach was preferable to withdrawal, as it enabled capture of participant-reported outcomes, and information about compliance and off-study use of vitamin D supplements.
Baseline characteristics
Participants completed a self-administered survey at baseline, providing information about socio-demographic characteristics, lifestyle factors, and pre-existing health conditions. Body mass index (BMI) was calculated by dividing weight (in kilograms) by height (in metres) squared. We predicted deseasonalised serum 25(OH)D concentration at baseline using a model that was developed and internally validated using data collected from a random subset of placebo participants during the trial (35). We did not measure telomere length at baseline.
Monitoring adherence and adverse events
In each annual survey, we asked participants to report the number of trial tablets they had taken in the previous 12 months. For the current analysis, adherence to the intervention was calculated by dividing the number of tablets that participants reported taking in the first four or five years by the number they would have taken if they were fully compliant. For those who did not die during the trial, the expected number for those who had blood collected after 4 years was 48 (1 tablet a month); for those who had blood collected after 5 years the expected number was 60.
Each year, beginning one year after recruitment started, approximately 800 participants received with their annual survey an invitation to provide a blood sample. Sampling of these participants was stratified by randomisation group, age, sex, state, and month of recruitment. Those who consented were instructed to attend their local pathology collection centre, where a blood sample was collected. Liquid chromatography tandem mass spectroscopy was used to measure serum 25(OH)D concentrations (35).
Participants were asked to contact the D-Health Trial staff if they experienced any adverse events. Kidney stones, hypercalcaemia and hyperparathyroidism diagnoses were also captured through the annual surveys.
Measurement of telomere length
Telomere content was analysed in blood samples collected from participants at 4 or 5 years after randomisation. After collection in the field and transport to QIMR Berghofer at room temperature, samples were stored at −80 °C. DNA was extracted at QIMR Berghofer on a Chemagen 360 magnetic bead platform, using a 4ml protocol with kit CMG-1074. We used a Biotek Synergy HTX and Take-3 plate for spectrophotometric analyses, and normalised samples using 10 nM Tris-HCl ph 8.0. We measured telomere length at the Telomere Length Regulation Unit (Children's Medical Research Institute, New South Wales, Australia) using quantitative polymerase chain reaction (qPCR), and calculated the ratio of telomere repeat content to the human beta-globin (HBG) single copy gene (hereafter written as T/S ratio) (36). qPCR was performed on a CFX384 Touch PCR detection system (BioRad) in 384-well plates, using a monochrome multiplex technique described previously (37), with modifications. Each sample was assayed in quadruplicate and analysis was conducted by plotting cycle threshold (Ct) against log[DNA concentration] and fitting unknown patient samples to standard curves for telomere and HBG amplifications.
Eligibility for the analysis
All D-Health Trial participants who were randomly selected to provide blood samples at 4 or 5 years after randomisation were eligible for inclusion. From this group we excluded anyone for whom the T/S ratio could not be determined.
Blinding
Participants, investigators, and analysts were blinded to study group allocation during the intervention. Participants were notified of their group allocation in March 2020 (i.e., after all participants completed the intervention). The investigators and analysts remained blinded until the analyses of mortality, the primary outcome of the D-Health Trial (27), were finalised. For the current analysis, we prepared statistical code using a dataset in which the randomisation allocation and participants' identification numbers were removed, and participants were randomly assigned to two equal-sized groups. Once all code was verified, the analyst was provided with the dataset that contained the true randomisation allocation.
Statistical analysis
All analyses were pre-specified before the final dataset was provided to the analyst. We conducted the analyses in SAS version 9.4 (SAS Institute, Inc., Cary, NC), and R version 4.1.1 (R Foundation for Statistical Computing, Vienna, Austria). All analyses followed the intention-to-treat principle (i.e., participants were analysed according to the study group to which they were randomised). We used mean and standard deviation (SD) to summarise continuous variables. To estimate the effect of vitamin D supplementation on T/S ratio, we fitted linear regression models. Effect estimates were adjusted for age, sex, and state of residence at baseline, and we report 95% confidence intervals (CIs) for the effect estimates and use a statistical significance level of p<005 with no adjustment for multiple testing (38, 39).
Since this analysis uses a subset of the D-Health cohort, we used chi-squared tests to investigate differences in baseline characteristics between participants included and excluded from the analysis, and between the study arms for those included in the analysis. There was slight imbalance in alcohol consumption between the study arms among included participants (p-value=0.12). We therefore performed a pre-specified sensitivity analysis in which alcohol consumption was included in the linear regression models; since this made negligible difference to the results, we do not present these data.
Subgroup analyses
We assessed whether the effect of vitamin D supplementation on T/S ratio was modified by age (<70 years; ≥70 years), sex, BMI (<25 kg/m2; ≥25 kg/m2), and predicted baseline 25(OH)D concentration (<50 nmol/L; ≥50 nmol/L). We present the effect estimate for each level of the characteristics and report the p-value for the interaction, calculated using a likelihood ratio test comparing models with and without the interaction term.
Baseline characteristics associated with telomere length
We calculated the mean and standard deviation of the T/S ratio within subgroups of selected baseline characteristics and used linear regression to estimate the associations between baseline characteristics and T/S ratio. Those with missing data were excluded from the relevant analysis. We present the age-and sex-adjusted estimates.
Results
We invited 421,207 people between January 2014 and May 2015 to participate in the D-Health Trial, and 1,896 volunteers who were not specifically invited also expressed interest; 21,315 eligible people were randomised either to vitamin D or placebo (Figure 1). Overall, 79% of those randomised were still taking trial tablets at the end of the 5-year intervention period; 866 people died before the end of the intervention period. More than 80% of participants in each group took at least 80% of their tablets, and the occurrence of adverse events did not vary between the two randomisation groups (35).
Figure 1.
Flow of the participants included in the analyses of leukocyte telomere length (CONSORT flow diagram)
EOI – Expression of interest; † Those who self-reported a previous or current diagnosis of hypercalcaemia, hyperparathyroidism, kidney stones, osteomalacia or sarcoidosis, or who were taking >500 IU supplemental vitamin D per day were ineligible for randomisation.
We randomly selected 1,927 participants (n=958 in the vitamin D and n=969 in the placebo group) for blood collection at 4 and/or 5 years after randomisation. We obtained at least one blood sample from 1,540 participants (80%; vitamin D: n=752; placebo: n=788); 387 refused to provide a blood sample (vitamin D; n=206; placebo: n=181). For the analysis of T/S ratio, we included 1,519 participants (99% of those with at least one blood sample; vitamin D: n=744; placebo n=775) after excluding those whose blood sample were not suitable for analysis (n=21) (Figure 1). Due to the way we sampled participants for blood collection, the sex distribution was more balanced among participants included in this analysis (men vs. women: 50.8% vs. 49.2%) when compared with those not included (men vs. women: 54.4 vs. 45.6%) (p=0.01) (eTable 1 in the supplement). There were also some differences in the distributions of state of residence (p<0.01), and smoking history (p=0.02) (eTable 1 in the supplement).
Table 1.
Baseline characteristics of the participants according to randomisation groupa
| BASELINE CHARACTERISTICS | Vitamin D (N=744) N (%) | Placebo (N=775) N (%) | p-valueb |
|---|---|---|---|
| Sex | |||
| Men | 385 (51.7) | 387 (49.9) | 0.48 |
| Women | 359 (48.3) | 388 (50.1) | |
| Age (years) | |||
| 60–64 | 182 (24.5) | 187 (24.1) | 0.71 |
| 65–69 | 209 (28.1) | 203 (26.2) | |
| 70–74 | 215 (28.9) | 225 (29.0) | |
| ≥ 75 | 138 (18.5) | 160 (20.6) | |
| Highest qualification obtained | |||
| None | 75 (10.2) | 83 (10.8) | 0.79 |
| School or intermediate certificate | 131 (17.8) | 122 (15.9) | |
| Higher school or leaving certificate | 98 (13.3) | 93 (12.1) | |
| Apprenticeship or certificate | 251 (34.0) | 275 (35.9) | |
| University degree or higher | 183 (24.8) | 193 (25.2) | |
| Missing | 6 | 9 | |
| State of residence | |||
| Queensland | 177 (23.8) | 198 (25.5) | 0.71 |
| New South Wales | 204 (27.4) | 197 (25.4) | |
| Victoria | 111 (14.9) | 99 (12.8) | |
| Tasmania | 74 (9.9) | 84 (10.8) | |
| South Australia | 79 (10.6) | 88 (11.4) | |
| Western Australia | 99 (13.3) | 109 (14.1) | |
| Predicted 25(OH)D concentration (nmol/L) | |||
| < 50 | 178 (23.9) | 174 (22.5) | 0.50 |
| ≥ 50 | 566 (76.1) | 601 (77.5) | |
| Body mass index (kg/m2) | |||
| < 25 | 234 (31.5) | 251 (32.4) | 0.93 |
| 25 to < 30 | 324 (43.7) | 336 (43.4) | |
| ≥ 30 | 184 (24.8) | 188 (24.3) | |
| Missing | 2 | ||
| Smoking history | |||
| Never | 421 (57.0) | 441 (57.5) | 0.84 |
| Ex-smoker | 294 (39.8) | 305 (39.8) | |
| Current | 24 (3.2) | 21 (2.7) | |
| Missing | 5 | 8 | |
| Alcohol consumption (drinks/week) | |||
| < 1 | 167 (23.3) | 213 (28.2) | 0.12 |
| 1 to 7 | 330 (46.1) | 318 (42.2) | |
| > 7 to 14 | 145 (20.3) | 137 (18.2) | |
| > 14 | 74 (10.3) | 86 (11.4) | |
| Missing | 28 | 21 | |
| Living alone | |||
| No | 594 (79.9) | 636 (82.5) | 0.21 |
| Yes | 149 (20.1) | 135 (17.5) | |
| Missing | 1 | 4 | |
| Self-rated overall health | |||
| Excellent | 94 (12.9) | 90 (11.7) | 0.88 |
| Very good | 325 (44.5) | 333 (43.4) | |
| Good | 258 (35.3) | 287 (37.4) | |
| Fair | 50 (6.8) | 54 (7.0) | |
| Poor | 4 (0.5) | 3 (0.4) | |
| Missing | 13 | 8 | |
| Self-rated quality of life | |||
| Excellent | 162 (22.2) | 149 (19.6) | 0.68 |
| Very good | 344 (47.2) | 380 (50.0) | |
| Good | 191 (26.2) | 195 (25.7) | |
| Fair | 28 (3.8) | 33 (4.3) | |
| Poor | 4 (0.5) | 3 (0.4) | |
| Missing | 15 | 15 | |
Note: a. Participants whose blood samples were sent for telomere length measurement and who have data for telomere content (expressed as a ratio between the telomere (T) repeat copy number and single copy gene (S) copy number) are included in the analysis; b. p-value from chi-squared test
The mean ages of included participants at baseline and at blood collection were 69 years and 74 years, respectively; 49% of the included participants were women. The mean serum 25(OH)D concentrations of the included participants in the vitamin D and placebo groups during follow-up were 123 (SD 30) nmol/L and 79 (SD 25) nmol/L respectively. The two groups were well balanced in terms of baseline characteristics (Table 1).
Older age was associated with lower T/S ratio (adjusted mean difference −0.078; 95% CI −0.10 to −0.05; ≥ 75 vs. 60–64 years), and the mean T/S ratio was higher in women than in men (adjusted mean difference 0.033; 95% CI 0.02 to 0.05). Predicted 25(OH)D concentration at baseline was not associated with T/S ratio (adjusted mean difference 0.005; 95% CI −0.02 to 0.03) (eTable 2 in the supplement).
Table 2.
Effect of vitamin D supplementation on leukocyte telomere length
| T/S RATIO | |
|---|---|
| Mean (SD) | |
| Placebo (N=775) | 0.70 (0.16) |
| Vitamin D (N=744) | 0.70 (0.18) |
| Adjusted mean difference (95% CI)a | −0.001 (−0.02 to 0.02) |
Note: Abbreviation: CI – confidence interval; a. The mean difference (vitamin D versus placebo) and 95% CI was estimated using a linear regression model that included randomisation group, age, sex, and state of residence at baseline.
We did not find any difference in T/S ratio between the vitamin D and placebo groups. The mean (SD) T/S ratios were 0.70 (0.18) and 0.70 (0.16) in the vitamin D and placebo groups, respectively (adjusted mean difference −0.001; 95% CI −0.02 to 0.02) (Table 2). There was no effect modification by age, sex, body mass index, or predicted baseline 25(OH)D concentration (Figure 2).
Figure 2.
Effect of vitamin D supplementation on leukocyte telomere length within subgroups of baseline characteristics
Adjusted mean difference (vitamin D versus placebo) and 95% CIs were estimated using linear regression models. Models included randomisation group, age, sex, state of residence at baseline, subgroup variable of interest, and an interaction between randomisation group and subgroup variable. The interaction between randomisation group and subgroup variable was assessed using a likelihood ratio test that compared models with and without an interaction term between randomisation group and the subgroup variable. Abbreviation: CI – confidence interval; T/S - telomere to single copy gene
Discussion
Using data from the population-based D-Health Trial, we found no evidence that monthly supplementation with 60,000 IU of vitamin D3 for at least 4 years affects the telomere length of older Australians overall or in pre-specified subgroup analyses.
Findings from previous observational studies of vitamin D and telomere length have been mixed (13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 25). One cross-sectional analysis, using data from the United States Nurses' Health Study (N=1,337, mean age ∼59 years) reported that a higher plasma 25(OH)D concentration was associated with longer T/S ratio (average change in T/S ratio per one unit increase in 25(OH)D = 0.005; p = 0.05) (13). However, in a longitudinal study from the United Kingdom, in which 775 adults aged ≥85 years were followed up for 36 months, the association between 25(OH)D concentration and telomere length was inconsistent over time (23). Compared to participants with 25(OH)D concentration 25–50 nmol/L, those with 25(OH)D >50 nmol/L had longer telomere length (measured in kilobase pairs) at baseline (mean difference 61.2; p-value=0.02), but this association was reversed at 18 months (mean difference −33.7; p-value=0.01) and 36 months (mean difference −36.8; p-value=0.23) of follow-up (23). We found no evidence that low predicted 25(OH)D concentration at baseline (<50 nmol/L) was associated with shorter relative telomere length, and the results of our intention-to-treat analysis suggest that the associations reported in some of the prior observational studies may not be causal.
The D-Health Trial is the largest RCT to report on the effect of vitamin D supplementation on telomere length to date. Strengths include population-based recruitment including both women and men, a high dose of oral vitamin D3 supplementation resulting in considerable difference in 25(OH)D concentration between the study groups, the long treatment duration with low loss to follow-up, and high adherence to study tablets. Nevertheless, there are some limitations. First, we did not have telomere length measured at baseline, so we could not calculate the difference in lengths between the two groups over the follow-up period or adjust for baseline telomere length. However, the balance on all other measured variables strongly suggests that the mean telomere length would also have been similar between the groups at baseline, so any difference at 4/5 years would be attributable to the intervention. Further, differences in post-intervention values are considered equivalent to differences in changes from baseline when pooling results for meta-analysis (40). Second, we sent invitations for blood collection to a sample of randomly selected participants who had not withdrawn from the trial, and not everyone responded to the invitation. This could have potentially subverted the randomisation, thereby introducing bias. However, baseline characteristics were balanced between the two groups analysed, suggesting any bias was minimal. Third, the length of follow-up duration varied as we included participants who gave blood samples at 4 and/or 5 years of randomisation. The timing of collection was equally distributed across the two groups. Finally, we used predicted rather than measured baseline 25(OH)D concentration. As the positive predictive value for 25(OH)D concentration <50 nmol/L was modest (23%), misclassification of participants' baseline vitamin D status may have attenuated any effect in people predicted to have low vitamin D status at baseline (35). Nevertheless, in the absence of an overall effect, any subgroup effects are highly unlikely.
Compared with the Australian general population, the participants included in this analysis were less likely to report having fair or poor overall health, and were less likely to be current smokers (41). The mean serum 25(OH)D concentration in samples collected from placebo participants during the trial was slightly higher than that in Australians aged ≥65 years (77 nmol/L vs. ∼69 nmol/L) and the percentage of people with 25(OH)D concentration <50 nmol/L was lower (13% vs. 18%) (42). These differences, however, may be due to differences in the latitude distribution of participants (43), the analytic laboratory, and the season of blood collection. While the results from the D-Health Trial are likely generalisable to the Australian setting and other communities with a similar prevalence of vitamin D deficiency, they are less informative about the effect of vitamin D supplementation on telomere length in populations where a higher proportion of people are vitamin D deficient.
In conclusion, supplementing older adults with high-dose vitamin D3, particularly in a setting of low vitamin D deficiency, is unlikely to influence telomere length.
Acknowledgments
We would like to acknowledge: • The D-Health participants who took part in this research. • Susan List-Armitage and the QIMR Berghofer Central Processing Laboratory for processing the blood samples and extracting the DNA. • The D-Health Trial staff and members of the Data and Safety Monitoring Board (Patricia Valery, Ie-Wen Sim, Kerrie Sanders).
Acknowledgements
Author contributions: DW, PW, GH, DE, JV, AV, CB, BDR, PE, DM, RO'C, and RN were involved in designing the trial. BDR, CB, MW, DM, and RN were involved in the recruitment, data collection and curation. HAP and JW were responsible for measuring relative telomere length using the quantitative polymerase chain reaction. SR, HP, MW, and RN were involved in the development of an analysis plan. SR, HP, MW, and RN were involved in formal analysis, and validation of the study. SR wrote the first draft of the report with input from MW and RN. All authors were involved in reviewing and editing the manuscript. MW and RN provided supervision.
Funding: The D-Health Trial is funded by National Health and Medical Research Council (NHMRC) project grants (GNT1046681; GNT1120682). PM Webb, DC Whiteman, and PR Ebeling are supported by fellowships from the NHMRC (GNT1173346; GNT1155413; GNT1197958). DSA McLeod is supported by a Metro North Clinician Research Fellowship and the Queensland Advancing Clinical Research Fellowship. The vitamin D assays were performed at the University of Western Australia, supported by infrastructure funding from the Western Australian State Government in partnership with the Australian Federal Government, through Bioplatforms Australia and the National Collaborative Research Infrastructure Strategy (NCRIS). The funding source had no role in the study design, collection, analysis, or interpretation of data, in writing the report, or in the decision to submit the manuscript for publication.
Ethics Approval and Participants' Consent: The D-Health Trial was approved by the QIMR Berghofer Medical Research Institute Human Research Ethics Committee (HREC) and was monitored by an external Data and Safety Monitoring Board. The trial adhered to the tenets of the Declaration of Helsinki. All participants gave written or online consent to participate. The trial is registered on the Australian New Zealand Clinical Trials Registry: ACTRN12613000743763. https://www.anzctr.org.au/.
Conflict of interest statement: PM Webb has funding from Astra Zeneca for an unrelated study of ovarian cancer. PR Ebeling reports grants and other from Amgen, other from Sanofi, grants and other from Novartis, grants from Eli-Lilly, and grants from Alexion. RE Neale has funding from Viatris for an unrelated study of pancreatic cancer. All other authors declare no competing interests.
Electronic Supplementary Material
Supplementary material is available in the online version of this article at https://doi.org/10.1007/s12603-023-1948-3
Effects of vitamin D supplementation on telomere length: an analysis of data from the randomised controlled D-Health Trial
References
- 1.Kahl VFS, Allen JAM, Nelson CB, Sobinoff AP, Lee M, Kilo T, et al. Telomere length measurement by molecular combing. Front Cell Dev Biol. 2020;8:493. doi: 10.3389/fcell.2020.00493. PubMed PMID: 32612998; PMCID 7308456. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Vaiserman A, Krasnienkov D. Telomere length as a marker of biological age: state-of-the-art, open issues, and future perspectives. Front Genet. 2021;11:630186. doi: 10.3389/fgene.2020.630186. PubMed PMID: 33552142; PMCID 7859450. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Nawrot TS, Staessen JA, Gardner JP, Aviv A. Telomere length and possible link to X chromosome. Lancet. 2004;363:507–510. doi: 10.1016/S0140-6736(04)15535-9. PubMed PMID: 14975611. [DOI] [PubMed] [Google Scholar]
- 4.Valdes AM, Andrew T, Gardner JP, Kimura M, Oelsner E, Cherkas LF, et al. Obesity, cigarette smoking, and telomere length in women. Lancet. 2005;366:662–664. doi: 10.1016/S0140-6736(05)66630-5. PubMed PMID: 16112303. [DOI] [PubMed] [Google Scholar]
- 5.Helby J, Nordestgaard BG, Benfield T, Bojesen SE. Shorter leukocyte telomere length is associated with higher risk of infections: a prospective study of 75,309 individuals from the general population. Haematologica. 2017;102:1457–1465. doi: 10.3324/haematol.2016.161943. PubMed PMID: 28522577; PMCID 5541879. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Zhao J, Miao K, Wang H, Ding H, Wang DW. Association between telomere length and type 2 diabetes mellitus: a meta-analysis. PLoS One. 2013;8:e79993. doi: 10.1371/journal.pone.0079993. PubMed PMID: 24278229; PMCID 3836967. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Haycock PC, Heydon EE, Kaptoge S, Butterworth AS, Thompson A, Willeit P. Leucocyte telomere length and risk of cardiovascular disease: systematic review and meta-analysis. BMJ. 2014;349:g4227. doi: 10.1136/bmj.g4227. PubMed PMID: 25006006; PMCID 4086028. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Cawthon RM, Smith KR, O'Brien E, Sivatchenko A, Kerber RA. Association between telomere length in blood and mortality in people aged 60 years or older. Lancet. 2003;361:393–395. doi: 10.1016/S0140-6736(03)12384-7. PubMed PMID: 12573379. [DOI] [PubMed] [Google Scholar]
- 9.Nagpal S, Na S, Rathnachalam R. Noncalcemic actions of vitamin D receptor ligands. Endocr Rev. 2005;26:662–687. doi: 10.1210/er.2004-0002. PubMed PMID: 15798098. [DOI] [PubMed] [Google Scholar]
- 10.Cyprian F, Lefkou E, Varoudi K, Girardi G. Immunomodulatory effects of vitamin D in pregnancy and beyond. Front Immunol. 2019;10:2739. doi: 10.3389/fimmu.2019.02739. PubMed PMID: 31824513; PMCID 6883724. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Puts MT, Visser M, Twisk JW, Deeg DJ, Lips P. Endocrine and inflammatory markers as predictors of frailty. Clin Endocrinol. 2005;63:403–411. doi: 10.1111/j.1365-2265.2005.02355.x. [DOI] [PubMed] [Google Scholar]
- 12.Oelzner P, Müller A, Deschner F, Hüller M, Abendroth K, Hein G, et al. Relationship between disease activity and serum levels of vitamin D metabolites and PTH in rheumatoid arthritis. Calcif Tissue Int. 1998;62:193–198. doi: 10.1007/s002239900416. PubMed PMID: 9501950. [DOI] [PubMed] [Google Scholar]
- 13.Liu JJ, Prescott J, Giovannucci E, Hankinson SE, Rosner B, Han J, et al. Plasma vitamin D biomarkers and leukocyte telomere length. Am J Epidemiol. 2013;177:1411–1417. doi: 10.1093/aje/kws435. PubMed PMID: 23660800; PMCID 3676154. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Richards JB, Valdes AM, Gardner JP, Paximadas D, Kimura M, Nessa A, et al. Higher serum vitamin D concentrations are associated with longer leukocyte telomere length in women. Am J Clin Nutr. 2007;86:1420–1425. doi: 10.1093/ajcn/86.5.1420. PubMed PMID: 17991655. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Mazidi M, Michos ED, Banach M. The association of telomere length and serum 25-hydroxyvitamin D levels in US adults: the National Health and Nutrition Examination Survey. Arch Med Sci. 2017;13:61–65. doi: 10.5114/aoms.2017.64714. PubMed PMID: 28144256. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Borras M, Panizo S, Sarró F, Valdivielso JM, Fernandez E. Assessment of the potential role of active vitamin D treatment in telomere length: a case-control study in hemodialysis patients. Clin Ther. 2012;34:849–856. doi: 10.1016/j.clinthera.2012.02.016. PubMed PMID: 22417715. [DOI] [PubMed] [Google Scholar]
- 17.Hoffecker BM, Raffield LM, Kamen DL, Nowling TK. Systemic lupus erythematosus and vitamin D deficiency are associated with shorter telomere length among African Americans: a case-control study. PLoS One. 2013;8:e63725. doi: 10.1371/journal.pone.0063725. PubMed PMID: 23700431; PMCID 3658981. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Lau H, Mat Ludin AF, Rajab NF, Shahar S. Identification of neuroprotective factors aAssociated with successful ageing and risk of cognitive impairment among Malaysia older adults. Curr Gerontol Geriatr Res. 2017;2017:4218756. doi: 10.1155/2017/4218756. PubMed PMID: 29109736; PMCID 5646302. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Liu JJ, Cahoon EK, Linet MS, Little MP, Dagnall CL, Higson H, et al. Relationship between plasma 25-hydroxyvitamin D and leucocyte telomere length by sex and race in a US study. Br J Nutr. 2016;116:953–960. doi: 10.1017/S0007114516002075. PubMed PMID: 27515444. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Williams DM, Palaniswamy S, Sebert S, Buxton JL, Blakemore AI, Hyppönen E, et al. 25-hydroxyvitamin D concentration and leukocyte telomere length in young adults: findings from the Northern Finland birth cohort 1966. Am J Epidemiol. 2016;183:191–198. doi: 10.1093/aje/kwv203. PubMed PMID: 26797572; PMCID 4724094. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Julin B, Shui IM, Prescott J, Giovannucci EL, De Vivo I. Plasma vitamin D biomarkers and leukocyte telomere length in men. Eur J Nutr. 2017;56:501–508. doi: 10.1007/s00394-015-1095-7. PubMed PMID: 26558766. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Schöttker B, Hagen L, Zhang Y, Gào X, Holleczek B, Gao X, et al. Serum 25-Hydroxyvitamin D levels as an aging marker: strong associations with age and all-cause mortality independent from telomere length, epigenetic age acceleration, and 8-isoprostane levels. J Gerontol A Biol Sci Med Sci. 2019;74:121–128. doi: 10.1093/gerona/gly253. PubMed PMID: 30371905. [DOI] [PubMed] [Google Scholar]
- 23.Hakeem S, Mendonça N, Aspray T, Kingston A, Martin-Ruiz C, Robinson L, et al. The association between 25-Hydroxyvitamin D concentration and telomere length in the very-old: the Newcastle 85+ study. Nutrients. 2021;13:4341. doi: 10.3390/nu13124341. PubMed PMID: 34959893; PMCID 8707859. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Agirbasli D, Kalyoncu M, Muftuoglu M, Aksungar FB, Agirbasli M. Leukocyte telomere length as a compensatory mechanism in vitamin D metabolism. PLoS One. 2022;17:e0264337. doi: 10.1371/journal.pone.0264337. PubMed PMID: 35202418; PMCID 8870535. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Zhu H, Guo D, Li K, Pedersen-White J, Stallmann-Jorgensen IS, Huang Y, et al. Increased telomerase activity and vitamin D supplementation in overweight African Americans. Int J Obes. 2012;36:805–809. doi: 10.1038/ijo.2011.197. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Gomez DE, Armando RG, Farina HG, Menna PL, Cerrudo CS, Ghiringhelli PD, et al. Telomere structure and telomerase in health and disease. Int J Oncol. 2012;41:1561–1569. doi: 10.3892/ijo.2012.1611. PubMed PMID: 22941386; PMCID 3583695. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Neale RE, Baxter C, Duarte Romero B, McLeod DSA, English DR, Armstrong BK, et al. The D-Health Trial: a randomised controlled trial of the effect of vitamin D on mortality. Lancet Diabetes Endocrinol. 2022;10:120–128. doi: 10.1016/S2213-8587(21)00345-4. PubMed PMID: 35026158. [DOI] [PubMed] [Google Scholar]
- 28.Waterhouse M, English DR, Armstrong BK, Baxter C, Duarte Romero B, Ebeling PR, et al. A randomized placebo-controlled trial of vitamin D supplementation for reduction of mortality and cancer: statistical analysis plan for the D-Health Trial. Contemp Clin Trials Commun. 2019;14:100333. doi: 10.1016/j.conctc.2019.100333. PubMed PMID: 30886934; PMCID 6402378. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Neale RE, Armstrong BK, Baxter C, Duarte Romero B, Ebeling P, English DR, et al. The D-Health Trial: a randomized trial of vitamin D for prevention of mortality and cancer. Contemp Clin Trials. 2016;48:83–90. doi: 10.1016/j.cct.2016.04.005. PubMed PMID: 27086041. [DOI] [PubMed] [Google Scholar]
- 30.Tran B, Armstrong BK, Carlin JB, Ebeling PR, English DR, Kimlin MG, et al. Recruitment and results of a pilot trial of vitamin D supplementation in the general population of Australia. J Clin Endocrinol Metab. 2012;97:4473–4480. doi: 10.1210/jc.2012-2682. PubMed PMID: 23066119. [DOI] [PubMed] [Google Scholar]
- 31.Ish-Shalom S, Segal E, Salganik T, Raz B, Bromberg IL, Vieth R. Comparison of daily, weekly, and monthly vitamin D3 in ethanol dosing protocols for two months in elderly hip fracture patients. J Clin Endocrinol Metab. 2008;93:3430–3435. doi: 10.1210/jc.2008-0241. PubMed PMID: 18544622. [DOI] [PubMed] [Google Scholar]
- 32.Takács I, Tóth BE, Szekeres L, Szabó B, Bakos B, Lakatos P. Randomized clinical trial to comparing efficacy of daily, weekly and monthly administration of vitamin D3. Endocrine. 2017;55:60–65. doi: 10.1007/s12020-016-1137-9. PubMed PMID: 27718150. [DOI] [PubMed] [Google Scholar]
- 33.Hassan AB, Hozayen RF, Alotaibi RA, Tayem YI. Therapeutic and maintenance regimens of vitamin D3 supplementation in healthy adults: a systematic review. Cell Mol Biol. 2018;64:8–14. PubMed PMID: 30511630. [PubMed] [Google Scholar]
- 34.Ross AC, Manson JE, Abrams SA, Aloia JF, Brannon PM, Clinton SK, et al. The 2011 report on dietary reference intakes for calcium and vitamin D from the Institute of Medicine: what clinicians need to know. J Clin Endocrinol Metab. 2011;96:53–58. doi: 10.1210/jc.2010-2704. PubMed PMID: 21118827. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 35.Waterhouse M, Baxter C, Duarte Romero B, McLeod DSA, English DR, Armstrong BK, et al. Predicting deseasonalised serum 25 hydroxy vitamin D concentrations in the D-Health Trial: an analysis using boosted regression trees. Contemp Clin Trials. 2021;104:106347. doi: 10.1016/j.cct.2021.106347. PubMed PMID: 33684596. [DOI] [PubMed] [Google Scholar]
- 36.Cawthon RM. Telomere measurement by quantitative PCR. Nucleic Acids Res. 2002;30:e47. doi: 10.1093/nar/30.10.e47. PubMed PMID: 12000852; PMCID 115301. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 37.Cawthon RM. Telomere length measurement by a novel monochrome multiplex quantitative PCR method. Nucleic Acids Res. 2009;37:e21. doi: 10.1093/nar/gkn1027. PubMed PMID: 19129229; PMCID 2647324. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 38.Rothman KJ. No adjustments are needed for multiple comparisons. Epidemiology. 1990;1:43–46. PubMed PMID: 2081237. [PubMed] [Google Scholar]
- 39.Li G, Taljaard M, Van den Heuvel ER, Levine MA, Cook DJ, Wells GA, et al. An introduction to multiplicity issues in clinical trials: the what, why, when and how. Int J Epidemiol. 2017;46:746–755. doi: 10.1093/ije/dyw320. PubMed PMID: 28025257. [DOI] [PubMed] [Google Scholar]
- 40.Deeks JJ, Higgins JPT, DG A. Chapter 10: Analysing data and undertaking meta-analyses. In: Higgins JPT, Thomas J, Chandler J, Cumpston M, Li T, Page MJ, et al., eds. Cochrane Handbook for Systematic Reviews of Interventions. 2022.
- 41.AIHW . Older Australians. Australian Institute of Health Welfare; Canberra: 2021. [Google Scholar]
- 42.Malacova E, Cheang PR, Dunlop E, Sherriff JL, Lucas RM, Daly RM, et al. Prevalence and predictors of vitamin D deficiency in a nationally representative sample of adults participating in the 2011–2013 Australian Health Survey. Br J Nutr. 2019;121:894–904. doi: 10.1017/S0007114519000151. PubMed PMID: 30674358. [DOI] [PubMed] [Google Scholar]
- 43.Kimlin MG, Olds WJ, Moore MR. Location and vitamin D synthesis: is the hypothesis validated by geophysical data? J Photochem Photobiol B. 2007;86:234–239. doi: 10.1016/j.jphotobiol.2006.10.004. PubMed PMID: 17142054. [DOI] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Effects of vitamin D supplementation on telomere length: an analysis of data from the randomised controlled D-Health Trial