*Article* **Vitamin D Synthesis Following a Single Bout of Sun Exposure in Older and Younger Men and Women**

**Jenna R. Chalcraft 1,**†**, Linda M. Cardinal 2,**†**, Perry J. Wechsler 3, Bruce W. Hollis 4, Kenneth G. Gerow 5, Brenda M. Alexander 6, Jill F. Keith <sup>1</sup> and D. Enette Larson-Meyer 1,7,\***


Received: 30 May 2020; Accepted: 16 July 2020; Published: 27 July 2020

**Abstract:** Older adults are frequently cited as an at-risk population for vitamin D deficiency that may in part be due to decreased cutaneous synthesis, a potentially important source of cholecalciferol (vitamin D3). Previous studies found that cutaneous D3 production declines with age; however, most studies have been conducted ex vivo or in the photobiology lab. The purpose of this study was to characterize the response of vitamin D metabolites following a 30-min bout of sun exposure (15-min each to the dorsal and ventral sides) at close to solar noon in younger and older adults. Methods: 30 healthy individuals with skin type II/III were recruited; a younger cohort, aged 20–37 (*n* = 18) and an older cohort (*n* = 12), age 51–69 years. Exposure was at outer limits of sensible sun exposure designed to enhance vitamin D synthesis without increasing risk of photo ageing and non-melanoma skin cancer. Serum D3 concentration was measured at baseline, 24, 48 and 72 h post-exposure. Serum 25(OH)D was measured at baseline and 72 h post-exposure plus 168 h post-exposure in the older cohort. Results: D3 increased in response to sun exposure (time effect; *p* = 0.002) with a trend for a difference in D3 between cohorts (time\*group; *p* = 0.09). By regression modeling of continuous data, age accounted for 20% of the variation in D3 production. D3 production decreased by 13% per decade. Despite changes in D3, however, serum 25(OH)D did not change from baseline to 72 or 168 h post exposure (*p* > 0.10). Conclusions: Serum D3 concentration increased significantly in response to outdoor sun exposure in younger and older adults. While ageing may dampen cutaneous synthesis, sunlight exposure is still a significant source of vitamin D3.

**Keywords:** ageing; older persons; sensible sun exposure; cutaneous synthesis; vitamin D3; natural sunlight; serum 25(OH)D

#### **1. Introduction**

Accumulating evidence has documented that vitamin D deficiency is associated with the onset and progression of a variety of chronic diseases including cardiovascular disease, type 2 diabetes, immune system diseases, neuropsychiatric disorders and certain cancers [1]. Older adults are frequently cited as an at-risk population for vitamin D deficiency [2–4]. Population-based studies suggest a high prevalence of vitamin D deficiency (serum 25(OH)D <20 ng/mL) among older adults (>60 to 65 years) that varies by location and ethnicity, and ranges from 12.1% of individuals with mixed ancestry in Greater Toronto (43◦ N) [5] to 37.3% in Mexico (<32◦ N) [6] and 45% in the Netherlands (52◦ N) [7]. Recent analysis of data from the National Health and Nutrition Examination Survey (NHANES 2011–2014), however, found that while vitamin D status differed by age, the prevalence of at risk of deficiency or inadequacy (defined as serum 25(OH)D <20 and 50 nmol/L, respectively) was highest in adults aged 20–39 years (7.6 and 23.8%) compared to adults ages 40–59 years (5.7 and 18.6) and those 60 years or older (2.9 and 12.3) [8], with no differences between men and women. Indeed, all adults and children may be at risk for vitamin D deficiency, especially when living in high latitude regions (>35◦) [9] or receiving limited sun exposure. Nonetheless, deficiency in older adults is most often a combination of age-related changes in vitamin D metabolism and lifestyle factors. Age-related alterations in vitamin D metabolism include decreased epidermal 7-dehydrocholesterol (DHC) concentration, reduced thickness of the epidermis [10,11], reduced dermal [12] (and epidermal) vitamin D production, increased adiposity [13] (fat sequestration), decreased renal 1,25(OH)2D synthesis [14–16] and increased 1,25(OH)2D catabolism [16]. The reduced dermal capacity to synthesize vitamin D at age 65 for example has been estimated to be ~25% of that of a 20–30-year-old exposed to the same amount of radiation [12,17]. Lifestyle factors associated with deficiency include poor appetite, low vitamin D intake, limited sun exposure/sun avoidance, increased clothing coverage/sunscreen use, reduced physical activity and financial constrains [3,9,14,18–20].

A number of studies in older adults, on the other hand, have demonstrated that exposure to ultraviolet B (UVB, 290–315 nm) radiation is an effective strategy for increasing serum 25(OH)D concentration [21–24]; this may suggest that sun avoidance rather than reduced synthesis per se is an important risk factor for suboptimal status in older individuals. Most of the aforementioned studies, except for that of Reid et al. [24], however, have been conducted in the photobiology lab following delivery of a measured dose of UVB. Further research using ambient sun exposure is necessary to continue to characterize the pattern of cutaneous vitamin D production in older versus younger adults in a natural environment (i.e., the sun is freely available) and determine the effectiveness of sun exposure therapy on vitamin D status.

Despite its potential benefit, however, sunlight exposure is considered a controversial way to maintain vitamin D status [25]. Many health authorities including the World Health Organization [26], the American Cancer Society [27] and the surgeon general [28] emphasize sun abstention, especially during mid-day when the sun's ultra-violet (UV) rays are the most potent. UV damage from too much sun exposure plays a role in the development of skin cancer and is considered a public health risk [25]. General public health efforts target sunscreen use, skin coverage with clothing and/or a sun hat and sun avoidance to reduce exposure to UVA and UVB radiation [3] and prevent cancer and photo ageing. While these public health efforts are understandable from the viewpoint of prevention of nonmelanoma skin cancer, they neglect the potential physiological benefits of mindful sunlight exposure that includes elevation of vitamin D status [25] and the potential influence of sun exposure and vitamin D on health in ageing individuals [29]. "Sensible sun exposure" is the practice of obtaining the minimum sun exposure required for adequate vitamin D synthesis followed by application of sunscreen or clothing coverage; and is often characterized as sun exposure of 5–30 min, two–three times per week to the arms, legs and torso during 10:00 h to 15:00 h [2,30]. The specific duration required within this time frame is dependent on skin type, previous tanning, latitude, season and environmental conditions. The minimal erythemal dose (MED), or the minimum quantity of UVB that induces a slight erythema 16–24 h post exposure, is a common dosage employed for sensible sun exposure guidelines [31]. The standard erythemal dose (SED) is a standardized method for quantifying erythemal UV radiation (UVR) dose and is becoming more commonly used because the MED varies from person to person even within the same skin type. The SED is defined as 100 J/m2 [32]. For adults with a fair complexion, one MED is equal to about 10–12 min of full body exposure during peak summer sun for all skin types [33]. One MED may be considered equivalent to ~2.5 to 3.5 SEDs depending on skin type [34].

As "sensible sun exposure" may be a simple, cost effective method to obtain optimal 25(OH)D concentration and/or prevent deficiency in older populations, the purpose of this study was to characterize the response of vitamin D3 and 25(OH)D to a single bout of outdoor sun exposure of 15 min to both the dorsal and ventral sides of the body (30 min total) in older and younger adults. We hypothesized, based on previous literature, that a single bout of sun exposure would be effective at increasing serum vitamin D3 concentration but that older individuals would demonstrate a reduced response to natural sunlight exposure compared to younger individuals.

#### **2. Materials and Methods**

This study consisted of exposing younger (19–39 years) and older (51–69 years) adults to a 30-min bout of natural outdoor sunlight (15 min each on the dorsal and frontal sides of the body) at moderate-altitude (2194 m, 41.3◦ N) in late spring/early summer (close to the summer solstice). Both research procedures were reviewed and approved by the Institutional Review Board at the University of Wyoming (Protocols # 20150317EL 00717 and #20180219EL01882) and approved in April 2015 and April 2018, respectively. Volunteers provided written, informed consent before participation.

#### *2.1. Participants*

Young male and female volunteers (19–39 years of age) and healthy community-dwelling older adults (50–70 years) with skin type II or III were recruited from the local community in the spring (April–June) of 2015 (younger cohort) and spring of 2018 (older cohort) through advertisements posted on a university campus and in the local community. To be eligible, participants had to have skin type II or III and must not have a personal or family history of skin cancer or melanoma. Skin type was self-identified using the Fitzpatrick skin typing scale [35], which was read to potential participants. Those who self-identified as skin types II (white, fair; blond or red hair; blue, green or hazel eyes; usually burns, tans minimally) or III (cream white, fair with any hair or eye color; sometimes mild burn, tans uniformly) were permitted into the study. Exclusion criterion included: current vitamin D supplementation or supplementation one month before the study, current use of medications that have the potential to alter vitamin D status or photosensitivity, travel within three months to a sunny location close to the equator, appearing visibly tanned or the inability to either refrain from sunlight or artificial UV exposure from screening until the scheduled sun exposure session and for 3 days following this session or fully participate in the study due to work, school or personal scheduling constrains.

A total of 51 volunteers in the younger-aged cohort and 45 in the older-aged cohort responded to the advertisement. Interested volunteers were initially screened by phone and asked a series of questions about their general health, personal and family risk of skin cancer, use of supplements and perceived skin type. In the younger age cohort, five were unable to complete screening and 15 were unable to participate due to schedule conflicts. Thirteen were deemed ineligible for participation because they reported a family history of skin cancer, were taking vitamin D supplements, did not meet skin type guidelines or had travelled to a sunny location and/or spent a generous time outside without sunscreen in the previous three months. In the older-aged cohort, the majority of interested participants were not eligible due to either a personal or family history of melanoma or pre-cancerous skin lesions. Additional reasons for ineligibility included skin type, tanning bed use, recent travel to a sunny location or use of medications that might influence vitamin D status. Of the 51 and 45 individuals interviewed in the younger and older-age groups, 18 and 12 were both eligible and available to participate in the study.

#### *2.2. Baseline Measurements*

Baseline anthropometric measurements, which included height, weight, and body composition measured by dual energy x-ray absorptiometry (DXA) (Lunar Prodigy, GE Healthcare, Fairfield, CT, USA), were obtained on all participants. Height and weight were measured without shoes and in minimal clothing using a standing digital scale (Tanita, Tokyo, Japan) and stadiometer (Invicta Plastics, Leicester, England). Body mass index (BMI) was calculated as weight divided by height in meters squared and body surface area (BSA) was calculated using the Mosteller equation [36]. Participants also completed a vitamin D-specific food frequency and lifestyle questionnaire (FFLQ) [37]. The FFLQ addresses questions about the frequency of consumption of vitamin D-containing foods, vitamin D supplementation, sun exposure and tanning bed usage.

### *2.3. Sunlight Exposure*

Following the baseline visit, volunteers were scheduled to participate in one of several sunlight exposure sessions or test days. The exposure days had to meet certain criteria (cloudless sunny day with a minimum ambient temperature of 16 ◦C and a maximum of 32 ◦C) and were selected based on weather patterns, the university calendar and participant/research staff availability for the exposure day and for the 72 h (all participants) to 168 h period (older group only) following exposure. The period of April through June was selected to ensure that exposure occurred to early-season naive skin (i.e., skin that had not been recently tanned). On each test day, a sun exposure station was set up in a mostly sheltered outdoor environment at a local park (younger group) or landscaped courtyard (older group). Volunteers had pre-sun exposure (baseline) blood drawn for analysis of serum vitamin D3 (cholecalciferol), vitamin D2 (ergocalciferol) and 25-hydroxyvitamin D (25(OH)D) immediately before undergoing sunlight exposure that consisted of exactly 15 min of exposure to both the front and back sides of the body (30 min total) between 11:30 and 13:00 h standard time (12:30 to 14:00 h daylight saving time) while lying supine and prone, respectively, in shorts (men) or shorts and sports bra (women). The shorts worn had a 2- to 3-inch inseam or were cuffed to a similar length. This amount of skin exposure was estimated to be approximately 43% of BSA for men and 41% for women. A member of the research team closely monitored the sessions to ensure compliance with time and protocol and to watch for signs of notable erythema. Close to solar noon was selected because UVB is most intense during this time, which allowed for more efficient vitamin D synthesis and reduced risk for skin cancer [38,39]. Sunscreen (Equate Broad Spectrum SPF, Bentonville, AR) was applied to the face and sunglasses were provided to all participants who did not bring their own protective eyewear. Following carefully timed exposure, volunteers took immediate cover in a designated area indoors and were served a light lunch. Participants were asked to completely avoid outdoor sunlight for the next 72 h; if outdoor daytime exposure was unavoidable, participants were asked to cover up with full-body clothing and/or the provided sunscreen. 72 h was extended to 168 h in the older participants. To prevent the chance that some newly synthesized vitamin D was sloughed off with skin cells [40], volunteers were asked not to bath, shower or swim until after their 24-h blood draw.

#### *2.4. Blood Draws and Analyses*

Blood was drawn at baseline and approximately 24-, 48- and 72-h after initial exposure for the analysis of serum vitamin D3, vitamin D2 and 25(OH)D concentration. Blood was also obtained at 168 h post-exposure in the older group. Blood was allowed to coagulate at room temperature for 30-min and then centrifuged at 5800 rpm for 15 min (VWR Clinical 100, VWR International, Woodbridge, NJ). Serum was frozen until analysis. Vitamin D3/D2 was analyzed via liquid chromatography-mass spectrometry (LC/MS) by a commercial laboratory (Heartland Assays, Ames, IA). The intra- and inter assay coefficient for these assays is <3% and <6%, respectively. 25(OH)D concentration was analyzed via Diasorin 25(OH)D RIA (B.W. Hollis, Charleston, SC). The intra- and inter-assay coefficient for 25(OH) D assay is less than 10%.

#### *2.5. Estimates of UV-Irradiance*

UV irradiance was measured during sun exposure using a meteorological grade UV instrument (Total Ultraviolet Radiometer model 27901, The Eppley Laboratory, Inc. Newport, RI) and a logging data acquisition system (National Instruments, Austin, TX). UV data were measured in one-second intervals for all exposure days in the younger participants and for a single representative day in the older participants due to computer malfunction. Total UV exposure was calculated per participant from actual recorded start and stop times on the test day or the representative test day. Data from the Eppley radiometer is recorded in native units of volts and were then converted to irradiances using the device calibration coefficient of 1 mv = 2.28 mW/cm2. The UVA/UVB ratio of 20:1 or 5% of the total UV irradiance was used to estimate UVB [41]. The sum of irradiances for 30 min (1800 s) were then calculated and subsequently converted to a standard erythemal dose (SED) using the conversion: 1 SED = 100 J/m2 [32]. As participants were supine and prone during exposure, total exposure was divided by two to represent the exposure to each body side. Prior to data collection, baseline data (or calibration data) were measured by taking running averages of one-minute data segments with the radiometer shielded from any incident light; these data indicated that there was no significant offset to the total UV data collected.

#### *2.6. Statistical Procedures*

Statistical analyses were performed using IBM SPSS version 26.0 (Chicago, IL, USA) and Minitab version 19.0 (State College, Pennsylvania, USA). Independent Samples t-tests were used to test for differences between older and younger groups for baseline physical characteristics, baseline serum vitamin D metabolites, and sun exposure variables. To address our primary aim, Repeated Measures Analysis of Variance (ANOVA) was initially used to evaluate the effect of group (older vs. younger) by time (baseline, 24, 48 and 72 h) on serum D3 concentration following sun exposure. Paired T-Tests with Bonferroni correction were then used post hoc to test for differences in serum vitamin D3 from baseline to 24, 48 and 72 h, and to test for differences between serum 25(OH)D at baseline and 72 h (and baseline and 168 in the older group). A Fishers Exact Test was used to evaluate differences in timing of the peak serum D3 concentration following sun exposure. ANOVA was also employed to evaluate sun exposure and UVB irradiance variables between and within studies on peak vitamin D3 concentration following sun exposure including sun exposure day/year, temperature during sun exposure and estimated UVB dose (SED). The best subsets method was utilized to identify predictors of D3 production from baseline to peak concentration that included important inter-subject characteristics and confounders (age, baseline serum vitamin D3 concentration, fat mass, lean mass, reported baseline vitamin D intake and sun exposure history). A simple linear regression model for D3 production was subsequently constructed to evaluate the relationship between age and D3 production. Log transformation was performed to best quantify the relative relationship.

#### **3. Results**

#### *3.1. Subject Characteristics*

All participants (*n* = 30) were Caucasian who identified with having skin type II or III. The characteristics of the participants, including estimated vitamin D intake and reported sun exposure are summarized in Table 1. The older group was significantly shorter than the younger group but no other differences (other than age) were observed. There were no differences in average daily vitamin D intake over the past 3 months and average daily sun exposure between the older and younger groups (Table 1).

#### *3.2. Sun Exposure and Estimates of UVB Irradiance*

Weather conditions and participant availability allowed for three mostly cloudless, sun exposure days in the spring of 2015 and four similar days in the Spring of 2018. In 2015, days were 68, 19 and 4 days prior to the summer solstice (21 June) and had recorded temperatures (typically at 12:53 h) of 19.4◦, 25◦ and 23.3 ◦C, respectively. In 2018, days were 35, 29, 17 and 9 days prior to the summer solstice, with recorded temperatures of 21.6◦, 21.1◦, 25.6◦ and 25.6 ◦C, respectively. Average body surface area exposed was estimated to be 0.78 m<sup>2</sup> <sup>±</sup> 0.02 in the younger cohort and 0.73 m2 <sup>±</sup> 0.03 in the older cohort and average UVB radiant exposure estimated via the radiometer was 386.3 <sup>±</sup> 9.2 mJ/cm2

and the SED was 38.6 ± 0.92 for whole body (19.3 for the frontal and dorsal sides) for the younger group and was 377.3 <sup>±</sup> 1.6 mJ/cm2 with an average total SED of 37.7 <sup>±</sup> 0.16 (18.85 for the frontal and dorsal sides). Neither BSA exposed nor SED differed between groups (*p* > 0.10).



Data are mean ± SD with ranges shown in parentheses. Bl, blond; Br, brown; R, red BMI, body mass index. \* Difference between older and younger cohorts via Independent Samples t-tests.
