II. Histochemical Study of Copper of Liver and Brain in Wilson's Disease

reported that we could confirm the definite increase of copper excretion in the urine and could demonstrate that dimercaprol (BAL) treatment gave rise to the marked increase of copper excretion. In this paper we report histochemical findings in the liver and brain in Wilson's disease (hepatolenticular degeneration). Recently, case reports of Wilson's disease are appearing rather frequently in Japan, and we were able to obtain five livers and two brains from autopsy materials of the School of Medicine of Tokyo, Tohoku Imperial University, Osaka University, and Saint Luke's Hospital of Tokyo. Copper granules were demonstrated histochemically in four of these five livers and in the putamen of one brain. MATERIAL AND METHOD Livers were obtained at autopsy and fixed in 10% formalin before examination. Histochemical demonstration of copper was performed by the method of Okamoto and Utamura,2 in which rubeanic acid and p-dimethylaminobenzylidenerhodanine are used as reagents. Rubeanic acid is considered to be more specific and sensitive. With the rubeanic acid method copper is demon¬ strated as green granules against alum-carmine counterstaining, and with the rhodanine method, as red-brown granules against hematoxylin staining. Gomori stated that, contrary to the findings of Okamoto, positive results were never obtained in human tissues by the rubeanic method.3 However, we observed always the marked parallelism of results with the rubeanic acid and rhodanine methods in the liver in Wilson's disease, and demonstrated that both methods are excellent for the demonstration of copper in human tissue. Fixation in alcohol may be preferable, but materials fixed in 10% formalin gave satisfactory results. The quantitative determination of copper was performed according to the method of Kimura and Murakami,4 introduced by us in the previous report.5 Tissues are ignited by the wet-ashing method with sulfuric and perchloric acid, and copper is isolated from other metals, using dithizonecarbon tetrachloride solution. The separated copper becomes golden-yellow when diethyldithiocarbamate is added. The colored substance is extracted with carbon tetrachloride and is measured colorimetrically.