Preparation and structure-activity relationship of reagents for cytochrome oxidase activity: potential for light and electron microscopy

Summary The syntheses of a series of p-substituted aromatic diamines and some representative thioureido- and mercapto-naphthols are described. Their cytochemical behavior in the Nadi reaction using fresh frozen sections of rat heart was studied in an attempt to learn more about the structural requirements of the Nadi reaction for cytochrome oxidase.The lipid solubility of the dyes formed from either N,N-dimethyl-p-phenylenediamine or ADN (4-amino-1-N,N-dimethylnaphthylamine) with 1-naphthol can be decreased by substituting hydrophilic hydroxyethyl or dihydroxypropyl side chains for one or more of the methyl groups in these reagents. However, these dyes diffuse readily, fade rapidly and are too soluble in water as well as lipid, which renders them unsuitable for light and electron microscopy.Burstone's reagent PPD (N-phenyl-p-phenylenediamine) and ADN (4-amino-1-N,N-dimethylnaphthylamine) are capable of undergoing self condensation either alone or in the presence of an unreactive naphthol to give colored indamines which osmicate readily. Blocking either the 4-position of N-phenyl-p-phenylenediamine, the secondary amino group, or both of these positions, with a methyl group did not prevent indamine formation.N-Benzyl-, N-4-methylbenzyl- and N-4-methoxybenzyl-p-phenylenediamine are good reagents for the demonstration of cytochrome oxidase activity in the Nadi reaction with light microscopy. They produce blue indoaniline dyes with 1-naphthols and do not self condense to form highly colored pigments. These indoanilines are osmiophilic eliminating the necessity for introducing osmiophilic groups for electron microscopic studies. Their drawback in electron microscopy is in the droplet nature of the deposit.Incorporating a carbonyl or carboxyl function into the 2-position or a bulky substituent into the 5-position of 1-naphthol prevented indoaniline formation.A new method for the cytochemical demonstration of cytochrome oxidase is presented utilizing a new reagent N,N-bis(p-aminophenyl)-1,3 xylylenediamine (XIII) (BAXD). This reagent is polymerized to an insoluble osmiophilic polymer distributed in non-droplet form, thus providing a useful method for demonstrating cytochrome oxidase activity in light and electron microscopy.This investigation was supported by a research grant (CA-02478) from the National Cancer Institute, U.S. Public Health Service, Bethesda, Maryland. Presented (in part) as a Presidential Address (AMS) at the Third International Congress of Histo- and Cytochemistry on August 19, 1968 in New York, N.Y. Acknowledgement is due Lionel, Katzoff for preparation of the thiolnaphthols XXXV and XXXVI, and to Norberto I. Schinitman for the osmium analysis.