The Effects of Regular Tanning Bed Use and Increased Vitamin D Status on Serum Markers of Bone Turnover in Healthy Adult Women

Background: Vitamin D is a key nutrient in bone health and the vitamin D status of individuals with regular exposure to solar or artifi cial ultraviolet B (UVB) radiation is generally superior to those with limited exposure. Objective: By means of a cross-sectional, observational design, explore the association of serum 25-hydroxy vitamin D (25(OH)D) concentrations and biochemical markers of bone turnover across a spectrum of vitamin D status by comparing women who regularly use tanning beds with women of minimal UVB exposure. Methods: A total of 69 healthy women, ages 25–82 y, were recruited. Serum concentrations of 25(OH)D, intact parathyroid hormone (iPTH), leptin, bone-specifi c alkaline phosphatase (BAP), osteocalcin (OC), and C-terminal telopeptides of Type I collagen (CTx) were measured. Results: There were no signifi cant differences in age, height, weight, BMI and dietary intakes between groups. Serum 25(OH)D concentrations were signifi cantly higher in tanners (n = 20) compared with non-tanners (n = 49) (p 0.0001). Serum iPTH concentrations were lower in tanners than in non-tanners (p 0.0001) and were negatively correlated with serum 25(OH)D concentrations (r = –4571, p 0.0001). Of the bone turnover markers, only serum OC concentrations were lower in tanners compared with non-tanners (p = 0.0002). After adjusting for age and menopausal status, osteocalcin was negatively correlated (r = –0.0178; p = 0.04) with 25(OH)D and positively correlated with iPTH (r = 0.035; p = 0.05). Conclusions: Our results show healthy women with regular UVB exposure via tanning beds have signifi cantly greater vitamin D status and lower serum osteocalcin concentrations than those without and that there is a signifi cant inverse relationship between serum serum 25(OH)D and osteocalcin concentrations which appears to be PTH-dependent.


Introduction
Vitamin D is classically known for its biological function in maintaining calcium homeostasis through its actions on the intestine and bone.1,25-dihydroxyvitamin D (1,25(OH) 2 D), the active metabolite of vitamin D, increases intestinal calcium absorption and serum calcium concentration via the induction of both genomic and non-genomic mechanisms of calcium transport across the enterocyte. 1 In the bone, 1,25(OH) 2 D enhances the mobilization of calcium and phosphorus from stores during times of calcium deprivation by inducing stem cell monocytes to become mature osteoclasts, thus stimulating bone breakdown and the subsequent release of minerals into circulation. 2A lack of vitamin D leads to secondary hyperparathyroidism which too accelerates bone breakdown. 1here is also evidence for a direct anabolic effect of 1,25(OH) 2 D on bone.In animal models, both acute and chronic vitamin D treatment resulted in increased bone formation and an increase in the number of osteoblasts and osteoblast precursor cells. 3,4In vitro studies using osteoblast-like cells have demonstrated the effects of 1,25(OH) 2 D on mRNA, protein expression and enzyme activities; including effects on the regulation of collagen type I (major bone matrix protein), osteocalcin (most abundant noncollageneous bone matrix protein) and alkaline phosphatase activity (indicator of bone formation). 5lthough there is clear evidence that adequate vitamin D status has a positive effect on bone mineral density (BMD) in humans, [6][7][8] results from studies exploring the relationship between vitamin D status and measures specifi c to bone formation in humans are more ambiguous.Presumably this is because the mineral homeostasis effects of vitamin D are intertwined with any direct role the vitamin has on bone metabolism. 5][11][12][13] Circulating concentrations of 25-hydroxyvitamin D (25(OH)D), the standard measurement of vitamin D status, are typically higher in individuals with increased exposure to ultraviolet B (UVB) radiation due to the cutaneous production of vitamin D from its precursor, 7-dehydrocholesterol. [14][15][16] One investigation recently demonstrated that healthy individuals routinely exposed to artifi cial UVB light via tanning beds have significantly greater serum 25(OH)D concentrations and hip BMD z scores than those who do not. 17While serum parathyroid hormone concentrations were assessed and found to be signifi cantly lower in tanners, no other biochemical markers of bone turnover were reported.
Bone biochemical markers offer a dynamic assessment of the skeleton.They can be used to assess the balance between bone formation and resorption and can measure changes over short periods of time. 18Additionally, bone markers can be used to predict response to therapy.][21][22] In the current study, our objective was to explore the association of serum 25(OH)D concentrations and biochemical markers of bone turnover across a spectrum of vitamin D status.Towards this end, we compared the vitamin D status and serum bone marker concentrations of healthy tanning and nontanning women.We included women from the age of skeletal maturity (∼25 years) through older adulthood.

Subject recruitment and testing procedures
The study described herein is part of a larger project to explore the relationship between vitamin D status and markers of general health in women. 23ll procedures involving human subjects received approval from the University of Missouri Health Sciences Institutional Review Board.
A total of 69 female subjects, ages 25-82 years, were recruited from the Columbia, Missouri, with advertisements posted via University of Missouri campus email and to local tanning salons and gyms/health clubs as previously described to be included in the study, participants had to be Caucasian females who were at least 25 years of age.Additionally, to qualify as a "Tanner," women had to regularly use a broad spectrum (UVA and UVB) tanning bed at least once per week for a minimum of 4 months."Non-Tanners" needed to have minimal daily sunlight exposure, as assessed by a screening questionnaire, and no tanning bed use.Exclusion criteria included exclusive use of high-pressure tanning beds, use of tobacco, use of vitamin D supplements other than regular multivitamins (Ն400 IU); current or previous medical condition or medication affecting vitamin D status, bone health, or immune function; current use of hormone replacement therapy; use of ultraviolet radiation as medical therapy; regular exercise Ͼ7 hours per week; and pregnancy.
All study visits were conducted between late January and early June to obtain serum samples during the seasonal nadir for 25(OH)D. 16Test visits of all qualifi ed, consented subjects were scheduled between 7 am and 11 am to control for diurnal variations; they were also instructed to refrain from exercise and fast for 8 to 10 hours prior to testing.On the day of the visit, all subjects of childbearing age took a urine pregnancy test to confi rm non-pregnant status.

Anthropometric measurements and questionnaire data
Each subject's weight was determined to the nearest pound and height was measured to the nearest 0.5 inch.With this data, Body Mass Index (BMI) was calculated for each subject.
All subjects completed several questionnaires including a one-page medical history form to obtain data on previous health conditions, menopausal status, use of medications and exercise habits; a brief sun exposure questionnaire developed for this study that assessed incidental and intentional sun exposure; and a Fitzpatrick skin typing survey, a well-established method of determining skin pigmentation and sun sensitivity. 24 Registered Dietitian also conducted a diet history interview which included the Harvard Food Frequency Questionnaire (88GP), a validated tool to assess habitual dietary intakes and a 24-hour dietary recall. 25

Statistical analysis
Unpaired, two-tailed Students' t-tests were used to determine differences in subject characteristics and bone and serum measurements between tanning and non-tanning groups.For data not normally distributed or of unequal variance, a rank-sum test was performed.Multiple linear regression analysis was used to determine the relationships between serum 25(OH)D and serum measurements adjusting for age and menopausal status.The sample size was chosen to detect a difference of 50% in 25(OH)D concentrations and of Ͼ30% in serum bone markers.All statistics were performed using SigmaStat for Windows version 3.01a (Systat Software, Inc., San Jose, California).A p-value less than 0.05 was considered signifi cant.

Results
Forty-nine of the women were classifi ed as Non-Tanners and 20 women were classifi ed as Tanners.There were no signifi cant differences in age, BMI, serum estradiol or cortisol concentrations, or hormonal contraceptive use between groups (Table 1).The Fitzpatrick skin type score of the Tanners was significantly higher than that of the Non-Tanners.The mean serum 25(OH)D concentration of the Tanners was signifi cantly greater than the Non-Tanners.Conversely, the mean serum iPTH concentration of the Tanners was signifi cantly less than the Non-Tanners.There were no differences between groups for selected bone-related nutrient intakes, including total energy, protein, fat, calcium, phosphorus, magnesium, and vitamin D. There were also no differences between groups for intakes of alcohol or caffeine which are agents known to affect bone.
Using the vitamin D status classifications proposed by Grant and Holick, 26 26.5% of Non-Tanners had defi cient levels of serum 25(OH)D, 35.7% were insufficient, and only 38.8% had suffi cient serum concentrations (Fig. 1).Of the Tanners, no one was classifi ed as defi cient, 15% had insuffi cient levels, and 85% had suffi cient serum 25(OH)D concentrations.
Serum bone marker data are shown in Table 2.Only serum osteocalcin concentration was signifi cantly less in the Tanners compared with the Non-Tanners.There were no differences between groups for any of the other serum bone markers.Table 3 shows the results of the multiple linear regression analysis of the serum bone markers adjusting for age and menopausal status.There was a signifi cant inverse relationship between serum 25(OH)D and iPTH concentrations.There was also a signifi cant inverse relationship between serum 25(OH)D concentrations and serum osteocalcin concentrations and a signifi cant positive relationship between serum iPTH and serum osteocalcin.

Discussion
8][29] Moreover, in the only other study to examine the 25(OH)D concentrations of regular users of tanning beds, Tangpricha et al. noted in their subjects a similar distribution across vitamin D status classifi cations. 17ndoubtedly, vitamin D plays an important role in achieving and maintaining a healthy skeleton through its function in ensuring an adequate supply of calcium to actively mineralizing bone.A defi cit of vitamin D would be expected to impair calcium absorption effi ciency and a lead to a rise in PTH production. 30][32][33][34] What's less understood, however, is the role that vitamin D plays in directly affecting bone remodeling and turnover.The measurement of serum biochemical markers can be employed to explore this function noninvasively in humans. 35n our study, we assessed three bone markers: BAP and OC, markers of bone formation; and CTx, a marker of bone resorption.In addition we measured serum leptin, a hormone produced by adipocytes, shown to play an important role in bone metabolism, 36 including stimulating bone formation through its action on osteoblasts. 37Categories: defi cient ( Ͻ20 ng/ml), insuffi cient (20-32 ng/ml), or suffi cient (32-100 ng/ml) as suggested by Grant and Holick. 26lthough there were not signifi cant differences in all of bone markers measured between groups, results from our study of healthy women ages 25-82 years of age, show that those with regular UVB exposure via tanning beds have signifi cantly lower serum osteocalcin concentrations than those without and that there is a signifi cant inverse relationship between serum osteocalcin and serum 25(OH)D.Furthermore, this relationship appears to be PTH-dependent.
In the published literature, data on the association between vitamin D status and serum osteocalcin concentrations is inconsistent.For example, studies of postmenopausal women without estrogen replacement therapy, show either no signifi cant relationship 10,31 or a signifi cant inverse relationship between serum 25(OH)D and osteocalcin 13 while investigations including subjects of premenopausal status have reported both a significant weak positive 31,38 and significant weak negative relationship. 39steocalcin is a noncollageneous bonespecifi c protein produced by osteoblasts and incorported into the bone matix. 35In vitro studies of human osteoblast cultures show 1,25(OH) 2 D to be a strong inducer of osteocalcin production, but it has also been described that the 1,25(OH) 2 D-induced stimulation of osteocalcin decreases toward the mineralization stage because of concurrent accumulation of osteocalcin in the bone matrix. 5This timing of mineralization phenomenon observed in cell culture research, may in part explain the disparate results on vitamin D status and circulating osteocalcin concentrations.The process of bone remodeling and turnover during adulthood is continuous and is characterized by two distinct phases: bone resorption which results in the disintegration and release of bone mineral and bone matrix fragments; and bone formation which follows resorption and involves the construction of matrix followed by its mineralization.Our fi ndings on PTH and bone marker concentrations indicate that at the time of sample collection, the bones of those with low serum 25(OH)D concentrations (Ͻ30ng/ml or so) were undergoing greater active bone formation than those with higher vitamin D status, mostly likely related to an overall increase in total bone turnover to maintain calcium homeostasis. 40nother confounder in the interpretation of results reported in the literature is the varying ranges of vitamin D status studied, as well as the defi nition of hypovitaminosis D used.For example, Sahota et al. in comparing serum bone markers between two levels of vitamin D status used a   13 most of the subjects categorized as "suffi cient" would be categorized as "insuffi cient" in later studies. 26In the report of Gannage-Yared et al. all of the subjects studied would be classifi ed as "insuffi cient". 38In contrast, the subjects studied in the work described herein spanned the range of current vitamin D status classifications, from "defi cient", "insuffi cient" and "suffi cient". 26he purpose of this study was to further explore the relationship between vitamin D status and serum markers of bone turnover in healthy women.To do so, we exploited the known superior vitamin D status of individuals who are regularly exposed to UVB radiation through the use of commercial tanning beds compared with controls. 17Other than tanning bed use, there were no signifi cant differences in dietary intakes or other lifestyle factors (i.e.smoking, alcohol or caffeine consumption, physical activity, medications) known to affect bone or vitamin D status between the tanning and non-tanning groups.So while it has been reported that women who tan regularly also engage in more high risk behaviors than non-tanners, 41 the present study was designed to control or account for these behaviors through well-defi ned subject inclusion/exclusion criteria and the collection of pertinent questionnaire data for analysis.We are reasonably confident, therefore that our results may be extended to the general population of healthy women older than 25 years.
Vitamin D insuffi ciency is a serious problem worldwide. 29Its prevalence is attributed to few natural dietary sources compounded by a lack of sunlight exposure.Our fi ndings, along with those of the only other published report examining the effects of chronic exposure to artificial UVB radiation on vitamin D status and bone health 17 indicate that regular tanning bed use results in higher 25(OH)D concentrations and more favorable bone measures including bone density and now, bone marker profi les.However, making recommendations to use tanning as a means to improve vitamin D status would not be prudent due to concerns about skin cancer [42][43][44][45] nor should the results of this study be used to support or defend tanning.Rather, the results presented here should be used to further our understanding of the direct effects of vitamin D status, across a spectrum, on bone turnover.

Table 1 .
Subject characteristics and vitamin D status.Data are expressed as means ± SEM.
*Means are signifi cantly different from Non-Tanners, P Ͻ 0.05.

Table 2 .
Serum bone marker concentrations in Non-Tanning and Tanning women.Data are expressed as means ± SEM.

Table 3 .
The relationship between serum 25(OH)D and serum iPTH concentrations and serum bone biomarkers in Non-Tanning and Tanning women adjusted for age and menopausal status.