一种通过高效液相色谱法快速精确测定大鼠皮肤中维生素D3的方法。

Takada K, Okano T, Tamura Y, Matsui S, Kobayashi T

J Nutr Sci Vitaminol (Tokyo). 1979;25(5):385-98. doi: 10.3177/jnsv.25.385.

In 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 2 ng/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 60 min irradiation, while a nearly constant value was obtained by irradiation for longer than 60 min. When the rat skin obtained from the in vitro experiment was irradiated with monochromatic UV rays in the range s60-350 nm, the most effective wavelength for the formation of vitamin D3 was confirmed to be 303 nm, which differs from the result obtained from the experiment in a test tube (295 nm). Moreover, the yield of vitamin D3 by irradiation with UV rays below 288 nm 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.

为了开展维生素D3光生物合成的研究，采用高效液相色谱法（HPLC）建立了一种快速、精确测定大鼠皮肤中维生素的方法。该方法包括对大鼠小块皮肤进行皂化、提取不皂化物，并使用“Zorbax SIL”（正相）作为吸附剂，以正己烷中0.5%的异丙醇作为流动相应用于HPLC分析。该方法的适用下限为去除皮下组织的皮肤每平方厘米2 ng维生素D3，且能够测定高于2 ng/cm2的浓度。所提出的方法应用于测定体内和体外照射实验获得的大鼠皮肤中维生素D3的含量。在体外实验中，维生素D3的产量与照射时间成正比增加。另一方面，体内实验的产量在照射60分钟之前与体外实验相似呈比例增加，而照射超过60分钟则得到接近恒定的值。当对体外实验获得的大鼠皮肤用60 - 350 nm范围内的单色紫外线照射时，证实形成维生素D3的最有效波长为303 nm，这与试管实验的结果（295 nm）不同。此外，用低于288 nm的紫外线照射时维生素D3的产量极低，这也再次与试管实验结果不同。这些差异被认为是由于大鼠皮肤表皮中马尔皮基层的滤过作用。