Data from in vitro experiments suggest that vitamin D reduces the rate of skin aging, whereas population studies suggest the opposite, most likely due to confounding by UV exposure. We investigated whether there are causal associations between 25-hydroxyvitamin D concentrations and features of skin aging in a bidirectional Mendelian randomization study. In the Rotterdam Study (N = 3,831; 58.2% women, median age 66.5 years) and Leiden Longevity Study (N = 661; 50.5% women, median age 63.1 years), facial skin aging features (perceived age, wrinkling, pigmented spots) were assessed either manually or digitally. Associations between 25-hydroxyvitamin D and skin aging features were tested by multivariable linear regression. Mendelian randomization analyses were performed using single nucleotide polymorphisms identified from previous genome-wide association studies. After meta-analysis of the two cohorts, we observed that higher serum 25-hydroxyvitamin D was associated with a higher perceived age (P-value = 3.6 × 10 –7 ), more skin wrinkling (P-value = 2.6 × 10 –16 ), but not with more pigmented spots (P-value = 0.30). In contrast, a genetically determined 25-hydroxyvitamin D concentration was not associated with any skin aging feature (P-values > 0.05). Furthermore, a genetically determined higher degree of pigmented spots was not associated with higher 25-hydroxyvitamin D (P-values > 0.05). Our study did not indicate that associations between 25-hydroxyvitamin D and features of skin aging are causal.

Causality can be inferred between a certain exposure and outcome using Mendelian randomization studies (). With such analyses, genetic polymorphisms that are strongly related to the exposure are investigated in relation to the outcome, in the absence of confounding. Therefore, we aimed to investigate whether associations between serum 25-hydroxyvitamin D and features of skin aging are causal using a bidirectional Mendelian randomization study.

Different in vitro studies have shown that physiological concentrations of 1,25-hydroxyvitamin D, the active vitamin D metabolite, protect the skin against factors that promote skin aging, including cellular damage induced by UVB irradiation. Vitamin D has been demonstrated to influence keratinocyte proliferation () and differentiation () with the response dependent on vitamin D concentrations and culture conditions (). Although the bioavailable levels of vitamin D in human skin are unknown, a higher serum concentration of 25-hydroxyvitamin D was associated with a higher number of facial pigmented spots in the Leiden Longevity Study (). However, the nature of these studies is observational, and causality cannot be ascertained due to influences of, for example, residual confounding by sunlight.

Although sun exposure contributes to premature skin aging (), it is essential for vitamin D synthesis in the skin () and vitamin D is essential for musculoskeletal health. Moreover, in clinical practice, low serum concentrations of 25-hydroxyvitamin D or vitamin D deficiency is a broadly accepted marker for general health status, and has been associated with multiple extraskeletal age-related diseases (e.g., type 2 diabetes mellitus and cardiovascular disease), and mortality ().

Vitamin D and risk of cause specific death: systematic review and meta-analysis of observational cohort and randomised intervention studies.

A higher perceived age—estimated age based on facial appearance—is associated with an increased risk of morbidity and mortality (), making it a useful marker in aging research. In addition to well-described extrinsic factors, such as smoking and UV exposure (), a higher perceived age also has an intrinsic component (). It has previously been shown that high serum concentrations of glucose and cortisol were associated with a higher perceived age (), whereas a high concentration of insulin-like growth factor-1 was associated with a lower perceived age mainly through skin wrinkling (). Besides skin wrinkling, facial pigmented spots are also an important component of skin aging.

We found no evidence after meta-analyzing the results of the Rotterdam Study and Leiden Longevity Study that any of the genotypes for pigmented spots or perceived age (MC1R gene only) or the genetic risk score for pigmented spots was associated with a higher 25-hydroxyvitamin D concentration ( Table 4 ; e.g., β = 0.146 ln[25-hydroxyvitamin D in nmol/l] per one unit increases in pigmented spots GRS; SE = 0.089; P-value = 0.10).

Effect estimates presented as the increase in the standardized outcomes per one unit increase in the genetic risk score. Analyses adjusted for age and sex. Effect estimates of the meta-analysis obtained using fixed-effect models..

After meta-analyzing the observed estimates of the Rotterdam Study and the Leiden Longevity Study ( Table 3 ), a higher genetically determined 25-hydroxyvitamin D concentration was not associated with (i) a higher perceived age (β = 0.030 SD per one genetically determined ln[25-hydroxyvitamin D]; SE = 0.023; P-value = 0.18); (ii) a higher perceived age additionally adjusted for skin wrinkling (β = 0.017 SD per one genetically determined ln[25-hydroxyvitamin D]; SE = 0.016; P-value = 0.28); (iii) a higher degree of skin wrinkling (β = 0.000 SD per one genetically determined ln[25-hydroxyvitamin D]; SE = 0.028; P-value = 1.00); (iv) a higher degree of pigmented spots (β = 0.055 SD per one genetically determined ln[25-hydroxyvitamin D]; SE = 0.030; P-value = 0.07).

Effect estimates presented as the increase in the standardized outcomes per one unit increase in the genetic risk score.

Analysis based on 2,843 individuals from the Rotterdam Study. Analyses adjusted for age and sex. Effect estimates of the meta-analysis obtained using fixed-effect models.

2 Analysis based on 2,843 individuals from the Rotterdam Study. Analyses adjusted for age and sex. Effect estimates of the meta-analysis obtained using fixed-effect models.

After meta-analysis, all three selected 25-hydroxyvitamin D genotypes were associated with a 25-hydroxyvitamin D concentration ( Supplementary Table S3 online). In line with this, the calculated weighted genetic score for a higher 25-hydroxyvitamin D concentration was associated with a higher 25-hydroxyvitamin D concentration in our study populations and meta-analysis (β = 0.24 units ln[25-hydroxyvitamin D] increase per one unit increase in genetic score; SE = 0.01; P-value = 2.23 × 10).

We calculated, per participant, a weighted genetic score for a 25-hydroxyvitamin D concentration based on the single nucleotide polymorphisms that were identified in a genome-wide association study on a 25-hydroxyvitamin D concentration (notably, rs2282679 [GC], rs3829251 [NADSYN1], and rs2060793 [CYP2R1];). On the basis of the observational effect estimates, we had an 82% and 84% power to detect significant (α = 0.05) associations between the 25-hydroxyvitamin D genetic risk score (GRS) and perceived age and degree of skin wrinkling, respectively.

After meta-analyzing the results of the Rotterdam Study and the Leiden Longevity Study ( Table 2 ), a higher 25-hydroxyvitamin D concentration was associated with a higher perceived age (β = 0.149 standard deviation [SD] per one ln[25-hydroxyvitamin D]; standard error [SE] = 0.029; P-value = 3.58 × 10). However, this association disappeared after additional adjustment for the degree of skin wrinkling (β = 0.020 SD per one ln[25-hydroxyvitamin D]; SE = 0.022; P-value = 0.36). In line with this, a higher 25-hydroxyvitamin D concentration was associated with a higher degree of skin wrinkling (β = 0.250 SD per one ln[25-hydroxyvitamin D]; SE = 0.030; P-value = 2.61 × 10). In contrast, a higher 25-hydroxyvitamin D was only associated with a higher degree of pigmented spots in the Leiden Longevity Study, and not in the Rotterdam Study. After meta-analysis, a higher 25-hydroxyvitamin D was not associated with a higher degree of pigmented spots (β = –0.033 SD per one ln[25-hydroxyvitamin D]; SE = 0.031; P-value = 0.30). These results were similar when we additionally adjusted for UV exposure, physical activity, and dietary vitamin D and any vitamin D supplementation in the Rotterdam Study ( Supplementary Table S1 online), despite that we observed strong associations between these factors and the 25-hydroxyvitamin D level ( Supplementary Table S2 online).

Effect estimates presented as the increase in standardized outcome per one ln-transformed unit increase in 25-hydroxyvitamin D serum concentration.

Analysis based on 2,843 individuals from the Rotterdam Study. Analyses adjusted for chronological age, sex, season, current smoking status, and body mass index. The results of the digitally measured wrinkles and pigmented spots in the Rotterdam Study were additionally adjusted for technical variables.

3 Analysis based on 2,843 individuals from the Rotterdam Study. Analyses adjusted for chronological age, sex, season, current smoking status, and body mass index. The results of the digitally measured wrinkles and pigmented spots in the Rotterdam Study were additionally adjusted for technical variables.

Analyses additionally adjusted for the degree of facial skin wrinkling. Effect estimates of the meta-analysis obtained using fixed-effect models.

1 Analyses additionally adjusted for the degree of facial skin wrinkling. Effect estimates of the meta-analysis obtained using fixed-effect models.

A maximum of 3,831 participants from the Rotterdam Study (median [interquartile range] age: 66.5 [61.0, 71.5] years) and 661 participants from the Leiden Longevity Study (median [interquartile range] age: 63.1 [58.9, 67.5] years) were included in the present study ( Table 1 ). Compared with participants from the Leiden Longevity Study, participants from the Rotterdam Study were more frequently women (58.2% vs. 50.4%), smokers (18.5% vs. 13.8%), and had a lower 25-hydroxyvitamin D concentration (median: 61.0 nmol/l vs. 68.3 nmol/l). Also, in line with the higher mean chronological age, participants from the Rotterdam Study had a higher mean perceived age (mean: 65.9 years vs. 59.4 years).

For the Rotterdam Study, measured digitally as area (wrinkles or pigmented spots) as a percentage of the total facial area. For the Leiden Longevity Study, wrinkle score and pigmented spots were measured manually by two expert dermatologists using a photonumeric scale ranging from 1 to 9.

2 For the Rotterdam Study, measured digitally as area (wrinkles or pigmented spots) as a percentage of the total facial area. For the Leiden Longevity Study, wrinkle score and pigmented spots were measured manually by two expert dermatologists using a photonumeric scale ranging from 1 to 9.

For the Rotterdam Study, measured digitally as area (wrinkles or pigmented spots) as a percentage of the total facial area. For the Leiden Longevity Study, wrinkle score and pigmented spots were measured manually by two expert dermatologists using a photonumeric scale ranging from 1 to 9.

2 For the Rotterdam Study, measured digitally as area (wrinkles or pigmented spots) as a percentage of the total facial area. For the Leiden Longevity Study, wrinkle score and pigmented spots were measured manually by two expert dermatologists using a photonumeric scale ranging from 1 to 9.

Discussion

We found evidence that a higher serum 25-hydroxyvitamin D concentration was associated with a higher perceived age and a higher degree of skin wrinkling. However, we found no evidence that a higher genetically determined 25-hydroxyvitamin D was associated with any of the studied skin aging features, nor was there evidence that a higher genetically determined degree of pigmented spots was associated with a higher 25-hydroxyvitamin D concentration. These results suggest that the association between 25-hydroxyvitamin D and skin aging features is not likely causal.

Bollag et al., 1995 Bollag W.B.

Ducote J.

Harmon C.S. Biphasic effect of 1,25-dihydroxyvitamin D3 on primary mouse epidermal keratinocyte proliferation. Gniadecki, 1996 Gniadecki R. Stimulation versus inhibition of keratinocyte growth by 1,25-Dihydroxyvitamin D3: dependence on cell culture conditions. Manggau et al., 2001 Manggau M.

Kim D.S.

Ruwisch L.

Vogler R.

Korting H.C.

Schafer-Korting M.

et al. 1Alpha,25-dihydroxyvitamin D3 protects human keratinocytes from apoptosis by the formation of sphingosine-1-phosphate. Bollag et al., 1995 Bollag W.B.

Ducote J.

Harmon C.S. Biphasic effect of 1,25-dihydroxyvitamin D3 on primary mouse epidermal keratinocyte proliferation. Gniadecki, 1996 Gniadecki R. Stimulation versus inhibition of keratinocyte growth by 1,25-Dihydroxyvitamin D3: dependence on cell culture conditions. Manggau et al., 2001 Manggau M.

Kim D.S.

Ruwisch L.

Vogler R.

Korting H.C.

Schafer-Korting M.

et al. 1Alpha,25-dihydroxyvitamin D3 protects human keratinocytes from apoptosis by the formation of sphingosine-1-phosphate. We did not find evidence of an association between higher genetically determined 25-hydroxyvitamin D levels and features of facial skin aging. Our findings suggest that the observations in the previously published in vitro experiments () might not have in vivo relevance. This could be because most in vitro studies demonstrate beneficial effects of the most potent vitamin D metabolite (1,25-hydroxyvitamin D) at very high physiological levels (≥100 nmol/l) compared with no vitamin D (). In contrast, most participants in the present study had a 25-hydroxyvitamin D concentration between 40 and 140 nmol/l; hence, the biological effects in this range will likely be lower. However, bioavailable levels of 25-hydroxyvitamin D and 1,25 hydroxyvitamin D in skin need to be ascertained to determine the relevance of the in vitro studies to in vivo conditions.

Saternus et al., 2015 Saternus R.

Pilz S.

Graber S.

Kleber M.

Marz W.

Vogt T.

et al. A closer look at evolution: variants (SNPs) of genes involved in skin pigmentation, including EXOC2, TYR, TYRP1, and DCT, are associated with 25(OH)D serum concentration. There was no significant association between the genetic score for pigmented spots and vitamin D levels. However, there was a borderline significant association between a single nucleotide polymorphism in the IRF4 gene and 25-hydroxyvitamin D concentration, replicating a similar finding in a different cohort (). This finding warrants follow-up particularly because many of the pigmented spot genes are also linked to melanin levels in skin, which protects skin from UVB radiation effects, the key determinant of vitamin D production in skin.

The observational associations between 25-hydroxyvitamin D concentration and a higher perceived age and a higher degree of skin wrinkling could be the result of residual confounding or reverse causality. However, single nucleotide polymorphisms in the MC1R gene associate with a higher perceived age, but were unrelated to 25-hydroxyvitamin D concentration in our study population suggesting that reverse causality is not at play here. We believe that the most likely explanation for the association between 25-hydroxyvitamin D concentration and features of skin aging is residual confounding, probably due to UVB radiation exposure.

Hamer et al., 2015 Hamer M.A.

Jacobs L.C.

Lall J.S.

Wollstein A.

Hollestein L.M.

Rae A.R.

et al. Validation of image analysis techniques to measure skin aging features from facial photographs. The present study has a number of limitations. First, the assessment of the degree of skin wrinkling and pigmented spots was different in the Rotterdam Study and the Leiden Longevity Study, which might have caused increased disparity in the data. The differences in perceived age between the two cohorts (despite having a similar chronological age) might originate from slight methodological differences as well as differences in lifestyle factors and medical history. However, we used the data on comparable scales (Z-scores) and there is large agreement between digital and manual assessment of skin wrinkling and pigmented spots (). The present study populations only comprised individuals from European ancestry, and our study findings might therefore not necessarily be generalizable to populations of different ancestry backgrounds. In addition, regarding the observational associations found between 25-hydroxyvitamin D, the available UV variables used might not have captured cumulative sun exposure accurately. However, this would not affect the Mendelian randomization analyses. Furthermore, although we have validated the GRS for 25-hydrxoyvitamin D against 25-hydroxyvitamin D levels in our study populations, we cannot completely rule out that the lack of evidence for an association between the GRS and features of skin aging is the result of a lack of power for the GRS to detect 25-hydroxyvitamin D effects in skin. Lastly, the facial photographs of the Rotterdam Study and the Leiden Longevity Study were taken at a later moment in time than the blood drawing for 25-hydroxyvitamin D assessment, which could have weakened any observational links.

In summary, we did not find evidence that the previously described beneficial in vitro effects of vitamin D on cellular processes are detectable at a population level. The observational associations in our study between 25-hydroxyvitamin D and features of skin aging are, most likely, predominately due to residual confounding.