A rapid and precise method for the determination of vitamin D3 in rat skin by high-performance liquid chromatography.

Abstract

SummaryIn order to develop the investigations into photobiogenesis of vitamin D3, a rapid and precise method for the determination of the vitamin in rat skin was established by using high-performance liquid chromatography (HPLC). The proposed method included saponification of small pieces of rat skin, extraction of the unsaponifiable matter and application to HPLC using "Zorbax SIL" (straight-phase) as an adsorbent and 0.5% isopropanol in n-hexane as a mobile phase. The applicable lower limit of the method was 2ng of vitamin D3/cm2 of subcutaneous tissue-removed skin and it was possible to assay a concentration higher than 2ng/cm2. The proposed method was applied to determine the content of vitamin D3 in rat skin obtained from in vivo and in vitro irradiation experiments. In the in vitro experiment, the yield of vitamin D3 increased in proportion to the irradiation time. On the other hand, the yield in the in vivo experiment showed a proportional increase similar to the in vitro experiment until 60min irradiation, while a nearly constant value was obtained by irradiation for longer than 60min. When the rat skin obtained from the in vitro experiment was irradiated with monochromatic UV rays in the range 260-350nm, the most effective wavelength for the formation of vitamin D3 was confirmed to be 303nm, which differs from the result obtained from the experiment in a test tube (295nm). Moreover, the yield of vitamin D3 by irradiation with UV rays below 288nm was extremely low, which again differed from the results of a test tube experiment. These differences were thought to be due to the filter effect of the malpighian layer in the epidermis of rat skin. Keywords cholecalciferol, 7-dehydrocholesterol, gas chromatography mass spectrometry, high-performance liquid chromatography, irra diation, pre-vitamin D3, rat, skin, ultraviolet ray, vitamin D3The antirachitic activity of sunlight has long been believed to be due to the photochemical conversion of 7-DHC to vitamin D3 via pre-D3 in skin exposed to UV rays in sunlight (Dorno ray). However, little experimental information on the photobiogenesis had been reported before our previous papers appeared (1-4). In the first report of the series (1), the existence of 7-DHC in rat skin was identified by GC-MS, whereas in vivo conversion of the sterol to vitamin D3 was investigated by HPLC in the second report of the series (2). In the third report (3), a nanogram order of the 25-OH-D3 generated in the blood and liver of vitamin D-deficient rats irradiated by UV light was identified by mass fragmentography and quantified by HPLC. Furthermore, the in vivo-generated pre-D3 and vitamin D3 themselves in the irradiated rat skin were identified by GC-MS in the fourth report (4). On the other hand, HOLICK et al. (5,6) identified the in vivo-generated pre-D3 in UV -irradiated rat skin by using HPLC and mass spectrometry, while in vitro-generated vitamin D3 in skin was also identified by EsVELT et al. (7). Our results agreed in that the endogenous 7-DHC in the rat skin was photochemical...

Investigation of the effect of UV-LED exposure conditions on the production of vitamin D in pig skin

“…(Maughan, 1928) Rachitic rats 296.7 313 no info (Bunker & Harris, 1937) Rachitic rats 280.4-302.4 no info no info (Knudson & Benford, 1938) Ergosterol 295 340 no info (Kobayashi & Yasumura, 1973) Rat skin 303 340 no info (Takada et al, 1979) 7-dehydrocholesterol 295 no info no info (Kobayashi, Hirooka, & Yasumura, 1976) Human skin 297 >320 3 or 5 (MacLaughlin et al,…”

“…The curves of vitamin D against wavelength (usually described as mountain shaped) are also observed when human skin, rat skin, and 7-DHC and ergosterol solutions are exposed to UV light (Kobayashi & Yasumura, 1973;MacLaughlin et al, 1982;Olds, Lucas, & Kimlin, 2010;Takada, Okano, Tamura, Matsui, & Kobayashi, 1979). Estimation of optimal wavelengths has been assessed under a range of test conditions which have been summarised in Table 2. All results, no matter the method, assess the optimal wavelength to be in the range 295-303 nm.…”

Nutrition of Primates in Captivity

“…He named this factor vitamin D. In succeeding years, the photobiogenesis of vitamin D3, cholecalciferol, in cutaneous tissues was described. Provitamin D3, 7-dehydrocholesterol , in the malpighian layer of the epidermis, is converted to pre vitamin D3 by UV irradiation in the range of285-315 nm, with maximum conversion at 297 (Holick et aI., 1982) to 303 nm (Takada et al, 1979). Previtamin D3, in turn, undergoes thermal isomerization to vitamin D3 at a skin temperature of 37°C.…”

Vitamin D3 Is Not Biologically Active for Rora (Labeo rohita) as Vitamin D3.

“…It is well known that vitamin D exists in two major forms, vitamin D3, the animal form and vitamin D2, of plant origin. While vitamin D3 is synthesized by the action of sunlight on 7-dehydrocholesterol (7-DHC) (provitamin D3) in the skin of land animals, vitamin D2 is produced upon ultraviolet (UV) irradiation of the plant sterol, ergosterol (provitamin D2) (1)(2)(3)(4)(5). Vitamin D3 derived either endogenously from photobiogenesis in the skin or exogenously from the diet undergoes two sequential hydroxylations to become biologically active.…”