HISTOCHEMICAL AZO COUPLING REACTIONS A CATECHOLAMINE IN ENTEROCHROMAFFIN CELLS IN PLACE OF OR IN ADDITION TO 5-HYDROXYTRYPTAMINE

R. D. LILLIE ¹, P. PIZZOLATO ¹, L. L. VACCA ¹, R. A. CATALANO ¹, and P. T. DONALDSON ¹

¹ Departments of Pathology, Louisiana State University Medical Center and U.S. Veterans Administration Hospital, New Orleans, Louisiana 70112

Appreciable azo coupling of enterochromaffin cells (EC) and of adrenal medulla (AM) is restricted to the alkaline range, fading out around pH 6, negative at 3-5. Low pH (3.0) azo coupling of rat and mouse mast cells (MC) with diazosafranin correlates with their known content of 5-hydroxytryptamine (5-HT) and with the pH 3-9 range of deep red azo coupling color reaction of p-nitrodiazobenzene with 5-hydroxytryptophan and 5-HT. The MC reaction is covalent azo bonding and not cationic dye salt bonding. The MC reaction is not influenced by prior oxidation by HIO₄, FeCl₃, K₂Cr₂O₇ or I₂/CH₃OH exposures which prevent azo coupling of EC and of AM. The oxidation blockades of the azo coupling of EC and AM are reversed by Na₂S₂O₅, N₂S₂O₄ or Na₂S₂O₃ reductions. These reductions after oxidation do not affect the MC diazosafranin reaction. In vitro the azo coupling of 5-HT is only slightly retarded but not weakened by HIO₄ oxidation and then slightly enhanced by Na₂S₂O₃, while noradrenaline gives red before oxidation, light yellow after, and after Na₂S₂O₃, again deep red, the control -naphthol giving deep red with p-nitrodiazobenzine at all three phases, before HIO₄, between HIO₄ and after Na₂S₂O₃. Indole reactions for EC have been reported on glutaraldehyde (G), acetaldehyde and acrolein fixed but not formaldehyde (F) fixed guinea pig duodenum by Solcia and Sampietro (63), Geyer (27) (G), Barter and Pearse (5, 6) and Lillie and Greco-Henson (44). Repetition of the Lillie and Greco-Henson test of combining in the same preparation a blue indole reaction (postcoupled benzylidene) and a red azo coupling reaction with p-nitrodiazobenzene in both sequences disclosed numerous red EC and blue Paneth cells and eosinophil leukocytes, alike on F and G tissue. Direct observation during the second reaction and color photography of the same field after each of the two reactions showed that no red stained cell was replaced by a blue or purple one, and that no blue stained cell altered its color to purple or red. Moreover, azo positive cells were seen in the epithelium of the tips of the villi and in the sides of the gastric glands of the guinea pig pylorus and fundus. Indole positive cells did not occur in villus tip or pyloric gland epithelium and were restricted to fundus chief cells.

It is concluded that the azo reactive substance in EC is a catechol and not 5-HT. The fluorescence studies have indicated the presence of 5-HT in EC, but the quantity present is insufficient for the acid azo coupling reaction shown by rat and mouse MC with diazosafranin.

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