A cytophotometric and electron-microscopical study on catalase activity in serial cryostat sections of rat liver.

The validity of the histochemical procedure for demonstrating catalase activity in cryostat sections of rat liver at the light- and electron-microscopical level was studied cytophotometrically. Incubations in the presence of 5 mM diaminobenzidine, 44 mM hydrogen peroxide and 2% polyvinyl alcohol performed on fixed cryostat sections resulted in the highest amounts of final reaction product precipitated in a fine granular form which was specific for catalase activity. Serial sections processed for electron microscopy indicated that the osmiophilic final reaction product was exclusively localized in the matrix and core of peroxisomes. The relationship between incubation time and the amounts of final reaction product generated by catalase activity as measured at 460 nm in mid-zonal areas of liver lobules showed non-linearity for the test-minus-control reaction because first-order inactivation of the enzyme occurred during incubation. Linearity of the test-minus-control reaction and section thickness was observed up to 8 microns. Catalase in rat liver showed a Km value of 2.0 mM for its substrate hydrogen peroxide when the diaminobenzidine concentration was 5 mM. It is concluded that the procedure for demonstrating catalase activity in serial cryostat sections of rat liver at the light- and electron-microscopical level is specific and can be applied to quantitative purposes. This approach may be useful in pathology, when only small biopsies are available, when the tissue is heterogeneous, and when other histochemical markers also need to be studied in the same material.

Histochemistry of peroxisomal enzyme activities: a tool in the diagnosis of Zellweger syndrome.

The localization of the activity of the peroxisomal enzymes D-amino acid oxidase and hydroxyacid oxidase was studied at the light-microscopical level in livers and kidneys of control subjects and patients with Zellweger syndrome, an inherited disease characterized by a lack of intact peroxisomes. D-Amino acid oxidase and hydroxyacid oxidase activities were demonstrated in unfixed cryostat sections with the cerium-diaminobenzidine-cobalt-hydrogen peroxide procedure, in which cerium ions capture hydrogen peroxide, the product of both enzymes. In a second step reaction decomposition of cerium perhydroxide gives rise to a diaminobenzidine polymer complexed with cobalt ions. D-Amino acid oxidase and hydroxyacid oxidase activities were found in peroxisomes of liver parenchymal cells, and only D-amino acid oxidase in peroxisomes of proximal tubular cells of kidneys of control humans. The activities of these enzymes were not detectable in livers and kidneys of Zellweger patients. It is concluded that the cerium-diaminobenzidine-cobalt-hydrogen peroxide procedure enables the demonstration of peroxisomal enzyme activities in human tissues at the light-microscopical level and is an important tool in detecting patients with Zellweger syndrome.