Evaluation of a Ultraviolet B Light Emitting Diode (LED) for Producing Vitamin D₃ in Human Skin

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.

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Figure 1.

The spectral output of the light emitting diode (LED) with a peak at 295 nm.

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.