1969 年 21 巻 1 号 p. 11-31
It has been regarded that all the histochemical methods so far reported to detect adrenal ketosteroids are of little practical value. The main reason for this invalidity seems to be that the ketosteroid distribution is obscured by the similar positive reaction presented by aliphatic aldehydes that always co-exist with ketosteroids. It is noticeable that formalin appears to increase the atmospheric oxidization effect which will cause or participate in the production of aliphatic aldehydes in formalin fixed tissues as well as frozen sections. Moreover, no suitable carbonyl reaction to distinguish the ketone group of ketosteroids from aliphatic aldehydes in frozen sections, has yet been discovered.
In respect to the reducing reaction methods, even when fresh frozen s e ctions are used, the presence of various reducing substances in those sections to seems obscure the relatively faint reducing reaction of ketosteroids. When carbonyl reaction methods are applied to fresh frozen sections, positive results can be hardly obtained, even with the Ashbel-Seligman method indicating the most clear positive color among several carbonyl reaction methods, because it is supposed that these methods are not sensitive enough to detect so small amount of ketosteroid contained in sections.
Therefore, the author and others have contrived the iron hematoxylin-sudan III staining method which is regarded to detect ketosteroids indirectly by showing the localization of lipoids or proteins combind with ketosteroids in tissues fixed in dichromate solution containing no formalin. Thus in order to confirm the histochemical validity of this method, we investigated the nature of sudanophilic granules and iron hematoxylinophilic granules found in paraffin sections to which this method is applied.
The result was that the sudanophilic granules were found to be a rsudanophilic polymer in paraffin sections where a type of ketosteroid-combining phospholipid was rendered insoluble in fat-solvents through the peculiar oxydizing action of potassium dichromate solution, and the "iron hematoxylinophilic" granules were found to be composed of a nonsudanophilic protein-combining phospholipid detectable in paraffin sections due to the strong "iron hematoxylinophilic"nature of its protein portion fixed through the protein-precipitating action of the potassium dichromate acetic acid solution.
The distributional peculiarities of these two sorts of granules in the glomerular, fasticular, and reticular zones of the adrenal cortex suggested that these granules would be closely related to adrenocortical endocrine function.
In addition, the author could obtain extr act containing sudanophilic substances from the sudanophilic granules by means of a peracetic acid treatment followed by methylation, so that the author could messure a certain amount of ketosteroid content in the extract containing sudanophilic substances, using the colorimetric method based on the reducing reaction such as the technique of Nakao-Aizawa.
These results as well as the above morphological findings, will support the conclusion that the iron hematoxylin-sudan III staining method will play a great part in the histochemical investigation of ketosteroids, as various histochemical test methods employed up to this time have been found valueless for the ketosteroids-detection.