The goal of this research program is to focus on the skin as the organ responsible for the endogenous production of vitamin D. During the last 5 years, the investigators have not only accomplished the major Specific Aims that they have proposed in their last granting period, but also established five specific methodologies in their laboratory that will be critical for the continuation of their studies on the photobiology of vitamin D and the biologic activity of provitamin D, previtamin D and vitamin D photoproducts using cultured human keratinocytes and a keratinocyte-fibroblast co-cultured skin equivalent as models. These techniques include: a) a direct analysis of solar irradiated samples for tritiated-previtamin D and tritiated-vitamin D by the use of an HPLC linked to an in-line radioactive detector; b) the method to detect the production of previtamin D, vitamin D and their photoproducts in human skin biopsies previously exposed to UV irradiation using an in-line photodiode detector coupled to an HPLC system; (c) a method to monitor intracellular calcium movement and phospholipid metabolism in cultured keratinocytes; d) development of a human skin equivalent keratinocyte-fibroblast co-culture model that will be used to explore the integrated interaction between dermal fibroblasts and keratinocytes in response to UVB irradiation and exposure to photoproducts; and e) the development of a liposomal model to evaluate the mechanism for the membrane enhanced isomerization of previtamin D to vitamin D. The goals of this grant application are: 1) determining the kinetics for the thermal isomerization of previtamin D to vitamin D in vivo and in vitro and determining the translocation of previtamin D and vitamin D3 from the epidermis into the circulation in vivo; 2) conducting in vitro and in vivo studies to evaluate the metabolism and potential biologic activity of photoproducts of previtamin D3 and vitamin D including lumisterol, tachysterol, suprasterol I, suprasterol II and 5,6-trans-vitamin D; and 3) evaluating the effect of previtamin D and vitamin D photoproducts, on genomic and non-genomic biologic responses in cultured human keratinocytes and fibroblasts and in a human skin equivalent keratinocyte-fibroblast co-culture model.
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