Abstract
Aim: A commercially available light emitting diode (LED) that transmitted narrow band ultraviolet B (UVB) radiation was evaluated for its efficacy and efficiency to produce vitamin D3 in human skin. Materials and Methods: Human skin samples were obtained from surgical procedures. The LED had peak emission wavelength of 295 nm. Skin samples were exposed to the UVB-LED for varying times and then were analyzed by high-pressure liquid chromatography (HPLC) to determine the vitamin D3 content. Results: There was a statistically significant time- and dose-dependent increase in the percent of 7-dehydrocholesterol that was converted to vitamin D3 in the skin type II samples; 1.3%±0.5, 2.3%±0.6 and 4.5%±1.67 after exposure to 0.75 (11.7 mJ/cm2), 1.5 (23.4 mJ/cm2) and 3 (46.8 mJ/cm2) minimal erythemal doses (MEDs), respectively. Conclusion: The UVB-LED was effective and efficient in generating vitamin D3 in human skin, in vitro. The amount of vitamin D3 production increased in a dose-dependent fashion with increased UVB energy. UVB-LEDs can be developed for devices that can efficiently produce vitamin D3 in human skin.
- Previtamin D3
- ultraviolet radiation light emitting diode (LED)
- vitamin D3
- human skin
- ultraviolet B radiation
Vitamin D is mainly obtained from sun exposure, as well as from few dietary sources (1, 2). Specifically, during sun exposure, epidermal 7-dehydrocholesterol (7-DHC or provitamin D3) absorbs solar ultraviolet (UV) B radiation, which results in the thermodynamically unstable molecule, previtamin D3. Once formed, the triene system in previtamin D3 rearranges to form the more thermodynamically stable product, vitamin D3 (1-4). After its formation, vitamin D3 enters the circulation from the skin and is transported to the liver to be metabolized into 25-hydroxyvitamin D3 (25(OH)D), and to the kidneys to undergo additional metabolism to 1,25-dihydroxyvitamin D3 (1, 3, 5-7).
The Sperti lamp, which contained a mercury arc lamp, was produced in the 1940s in the United States, where it was available in pharmacies to treat and prevent the bone disorder known as rickets (1, 6, 7). Since then, there has been an evolution of improved, more user-friendly vitamin D-producing devices for the treatment and prevention of vitamin D deficiency. In particular, the modern version of the Sperti lamp, Sperti D/UV-Fluorescent lamp (KBD, Inc., Las Vegas, NV, USA), was designed with UVB emitting fluorescent bulbs, which have the benefit of a lower heat emission than the previously mercury arc lamps. Additionally, these new UVB-emitting bulbs allow for a larger area of the user's skin to be exposed (8-10). Indeed, the Sperti D/UV-Fluorescent lamp has been shown to be effective in raising blood levels of 25(OH)D in healthy adults, as well as in patients with fat malabsorption syndromes who may not benefit from oral vitamin D supplementation (8, 9).
The modern version of the Sperti lamp includes improved gallium nitride-based UV light-emitting diode (LED), and is commercially available for use in clinical application (11, 12). These LEDs can also be designed to emit specific UV narrow band in order to be utilized therapeutically to convert 7-DHC to previtamin D3 cutaneously in humans (13). The purpose of this study was to evaluate the capability of human skin to produce vitamin D3 after exposure to a commercially available LED with a peak emission at 295 nm.
Materials and Methods
Equipment and sample exposure to UVB radiation. The UVB-LED was obtained from RayVio Corp. (Hayward, CA, USA) and spectral characteristics of the LED are shown in Figure 1. The peak wavelength of the LED was 295 nm (Figure 1). A digital UV Solarmeter (Solar Light Company Inc., Glenside, PA, USA) was used to measure radiation in minimal erythemal doses (MEDs) in which 1 MED is equivalent to 15.6 mJ/cm2. Human skin tissue samples of Fitzpatrick skin type II were collected from five healthy individuals during plastic surgeries at the Department of Surgery of Boston Medical Center, and the tissue sample retrievals were approved by the Institutional Review Board (IRB) at the Boston University Medical Center (BUMC). The skin samples were cut into 1 cm2 pieces. Duplicate skin samples were exposed to UVB-LED radiation for different times and the percent production of vitamin D3 from 7-DHC was evaluated as previously described (14) (Figure 2). Borosilicate ampoules (Wheaton. Millville, NJ, USA) containing 50 μg of 7-DHC dissolved in 1 ml of ethanol were exposed to the same amount of UVB radiation as the human skin samples and served as the positive controls, as previously described (14).
Three ampoules containing 7-DHC and the duplicate human skin type II samples were placed in a quartz dish. The quartz dish was placed on top of a plastic apparatus containing a 1-cm2 opening at its center at a distance focused 10.0 mm±1.0 mm from the top of the LED. The 1-cm2 pieces of skin were placed over the 1-cm2 opening, and were exposed to UVB-LED irradiation for varying times equivalent to 0.75 (4 min 12 s or 11.7 mJ/cm2), 1.5 (8 min 30 s or 23.4 mJ/cm2), and 3 MEDs (9 min or 46.8 mJ/cm2). The ampoules were exposed for the same time as were the skin samples as previously described (14). Ampoules in triplicate and a skin sample that were not exposed to the UVB-LED served as negative controls.
Vitamin D3 and previtamin D3 content analyses. After the exposure to UV-LED, each skin sample was placed in water at 60°C for 1 min to separate the epidermal from the dermal layer. This epidermal layer was then completely removed using a clean scalpel. The dermis was discarded and the epidermis was kept for further analysis. Each epidermal sample was then submerged in a test tube containing 4 ml of methanol, sonicated for 20 s, and immediately incubated overnight at a temperature of 50°C to facilitate the conversion of previtamin D3 to vitamin D3. After the incubation period, the supernatant, which contained the lipid extract, was dried down under nitrogen gas, resuspended in 1ml of 0.8% isopropyl alcohol (IPA) in hexane and centrifuged. The lipid extract was dried under nitrogen gas and resuspended in 130 μl of 0.8% IPA in hexane. The mixture was then transferred to vials for analysis by straight-phase high-performance liquid chromatography (HPLC) with a flow rate of 1.5 ml/min to determine the amount of previtamin D3 and vitamin D3 that was produced as previously described (14). The same procedure was followed for the skin sample that was not exposed to UVB-LED radiation.
Statistical analysis. The SPSS version 25 software for Mac (SPSS, Chicago, IL, USA) was used to perform statistical analysis. Analysis of variance (ANOVA) test was used to compare the mean vitamin D3 production between skin samples exposed to three different energy levels, 11.7 mJ/cm2, 23.4 mJ/cm2, and 46.8 mJ/cm2, to determine if there were statistical differences. A p<0.05 was considered to be statistically significant.
Results
The HPLC analysis of the content of 7-DHC, previtamin D3 and vitamin D3 is shown in Figure 2. Ampoules that were not exposed to the UVB-LED did not demonstrate any production of previtamin D3 (Figure 2B). In the ampoules exposed to varying doses of UVB radiation previtamin D3 was produced (Figure 2C). More specifically, a significant time- and dose-dependent increase in the percent conversion of 7-DHC to previtamin D3 was observed (p=0.02) (Table I). The HPLC analysis of the lipid extract of the negative control skin sample did not show any vitamin D3 content (Figure 2D). On the other hand, in the 5 duplicate skin type II samples (10 lipid extract samples) that were exposed to varying doses of UVB radiation vitamin D3 content was observed (Figure 2E). In these samples, the percent of vitamin D3 produced from the epidermal 7-DHC was significantly increased (p=0.04) in as time- and dose-dependent manner, after exposure to UVB radiation (Table I).
Discussion
Patients suffering from fat malabsorption syndromes, including patients with cystic fibrosis, inflammatory bowel disease, as well as those who have undergone a gastric bypass surgery, are at a high risk for developing vitamin D3 deficiency (9). These patients would greatly benefit from a simple and convenient device that could enhance the cutaneous production of vitamin D3. Gallium nitride LEDs, which are capable of emitting the specific narrow band UVB radiation that converts 7-DHC to previtamin D3, are commercially available. However, the capability of commercially available UVB-LEDs to produce vitamin D3 in human skin has not been studied. The current study aimed to determine the production of vitamin D3 in surgically obtained human skin samples after exposure to the UVB-LED from RayVio Corp., which emitted a peak wavelength of 295 nm.
Previous in vitro studies (3, 14) and the Comite International de l'Eclairage (CIE) (15) as well have reported that the narrow band of UV light is able to convert 7-DHC into previtamin D3 in human skin. Specifically, UV narrow band between 290 and 300 nm was reported to be the most efficient for production of vitamin D3 in human skin samples (3). In addition, Morita et al. (2) compared different wavelengths of radiation and reported that although 316 nm was less effective in producing vitamin D in mice than wavelengths between 268-305 nm, the serum levels increased in the wavelengths in this range as compared to a control group.
The major advantage of the present study was that the specific UV narrow band of 295 nm was shown to efficiently increase the production of previtamin D3 in ampoules and vitamin D3 in human skin in a dose-dependent manner (Figure 3). Therefore, the commercially available UVB-LED that was tested may be useful for the treatment and prevention of chronic vitamin D deficiency in patients who suffer from fat malabsorption syndromes, since these patients are not able to easily absorb orally ingested vitamin D.
Acknowledgements
The Authors are grateful to RayVio for supplying us with the LED used in this study. They are also grateful to Dr. Jaromir Slama and Dr. Nilton Medina of the Boston Medical Center plastic surgery department, who so generously provided them with the skin samples used in this study. The PI (MFH) dedicates this publication to his former mentor Dr. Hector DeLuca in honor of his 90th Birthday celebration.
Footnotes
Authors' Contributions
A.J. Veronikis, M.B. Cevik, and M.F. Holick designed the experiments. R. Allen helped design the LED device. A.J. Veronikis, M.B. Cevik, A. Sun and K. Persons conducted the studies. A.J. Veronikis, M.B. Cevik, and M.F. Holick analyzed the data. A.J. Veronikis, M.B. Cevik, A. Shirvani, and M.F. Holick drafted the manuscript. All authors contributed to and reviewed the manuscript. M.F. Holick provided final approval.
Presented a the Joint International Symposium “Vitamin D in Prevention and Therapy” and “Biologic Effects of Light, 5-7 June, 2019, Homburg/Saar, Germany.
Conflicts of Interest
All Authors have no conflicts of interest.
- Received November 21, 2019.
- Revision received December 30, 2019.
- Accepted January 10, 2020.
- Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved