Identification of tocopherol (vitamin E) in the skin and its histochemical detection

The epidermis upper layer reacts positively to the performic acid-toluidine blue and the performic acid-Schiff; such reactions were inhibited by a previous formalin-HgCl2 fixation. The substance accounted for that reactivity could be extracted from skin histological sections by means of the chloroform-methanol-HCl effect. If this same solvent was used on human skin shaven horny layer powder it was able to extract protein-lipid complex that displays the tocopherol histochemical reactivity. Since this complex was subjected to the KOH hydrolysis, an hexane-soluble lipid moiety could be isolated from the hexane-insoluble protein moiety. The isolated lipid moiety react as does the tocopherol histochemically, in opposite to the protein moiety that react negatively to the performic acid-toluidine blue and the performic acid-Schiff reactions. By using thin layer chromatography it was possible to isolate from the lipid moiety two substances identified as alpha-tocopherol and cholesterol in basis of its chromatographic properties, the first being the sole responsible by the performic acid-toluidine blue and performic acid-Schiff reactivity of the lipid moiety. From the other fractions isolated from the horny layer powder, either the lipid or the protein ones do not show positive performic acid-toluidine blue or performic acid-Schiff reaction. It was deduced, in basis of these findings that the positivity of such reactions on the epidermis upper layer keeps a close correlation with the alpha-tocopherol, contained by the protein-lipid complex. The ethylenic double bonds from lipids and the S-S group from protein could not be taken as responsible by them. On the other hand, the peracetic acid-toluidine blue reaction shows a proper histochemical meaning, since it is able to detect free cholesterol.