COMMENT ON SOLCIA AND BUFFA'S "AZO COUPLING OF INDOLE-REACTIVE ENTEROCHROMAFFIN CELLS"

Solcia and Buffa (16) in a Letter to the Editor reiterate their claim (17) that glutaraldehyde fixation permits benzy! idene and xanthydrol reactions of 5hydroxytryptamine (5-HT). They report experiments in which mucosal epithelial cells were first reacted with xanthydrol, photographed and then hydrated, during which process they largely lost their staining, and then reacted with fast garnet GBC. In 1957 Lillie (7) reported a positive xanthydrol reaction not only with indoles, tryptamines and tryptophans but also with tetrahydronorharman prepared by condensation of formaldehyde and tryptamine in Witkop’s laboratory. Nevertheless we and others have been unable to demonstrate enterochromaffin cells (EC) in formaldehyde-fixed tissue with xanthydro! or any of the p-dimethylaminobenzaldehyde methods. In 1960 Lillie and Greco-Henson (9) reported what was at first thought to be a successful demonstration in formol sucrose-fixed guinea pig tissue with the postcoupled benzylidene reaction. However, when after application of fast garnet GBC and photography and then performing the postcoup! ed benzy! idene (PCB) reaction, all of the previously photographed enterochromaffin cells were still red and many blue stained cells appeared, at least a part of which closely resembled the blue colored cells previously thought to be enterochromaffin cells, the concept that they had shown enterochromaffin cells by the PCB reaction had to be abandoned. In our 1973 report (14) we went a step further. The postcoupled benzy! idene and p-nitrodiazobenzene reactions were combined in both directions. Fields were photographed after the first reaction. Then, without moving the section, the second reagent was applied under direct microscopic visual observation. The blue indole reaction was not lost during the following azo reaction. The positive red azo reaction was not lost during application of p-dimethy! aminobenza! dehyde in HC1-HAc mixture. No red stained cell was replaced by an indole-reactive cell. No indo! e-reactive cell was replaced by an azo-reactive one. Solcia and Buffa (16) report no such direct visual observation. Rather they relied on morphologic resemblance. Geyer’s (4) claim that acetaldehyde fixation simi! arly would permit an indo! e reaction of 5-HT presumably by leaving the critical a-indole position open is contradicted by the known synthesis of harman (2methyl-fl-carboline) from trypt amine or tryptophan and acetaldehyde. Harvey and Robson (6) reported formation of the tetrahvdro-carboline derivative from the simple addition of a stoichiometric amount of freshly distilled acetaldehyde to a cold saturated aqueous solution of L-tryptophan. They cited also the preparat ion of tetrahydroharman from tryptamine and acetaldehyde by Akabori and Saito (2). Snyder, Parmertes and Katz (15) also prepared harman by a simple aceta! dehyde condensation with DL-tryptophan followed by dichromate oxidation. We were able to discern no difference whatever between formaldehydeand g! utaraldehyde-fixed guinea pig duodenum with respect to their reactivity to the indo! e or azo coupling reactions. It is true that glutaraldehyde inhibits the Clara hematoxylin reaction of enterochromaffin (11) but that reaction apparently depends on an entirely different mechanism. In the recent study we used freshly diazotized p-nitroaniline rather than fast garnet GBC, because in our 1961 study (10) it demonstrated practically all of the ferric ferricyanide reactive enterochromaffin cells at 2-10-mm exposures, and 1 mM, whereas fast garnet GBC only reacted 90% at 10 mM and 62% at 1 mM. The evidence of formaldehyde fluorescence indicates the presence of 5-HT both in EC (5) and rat and mouse mast cells (1, 3) and the latter has been established by isolation and chemical assay. The latter cells give the pH 3 diazosafranin reaction in agreement with the in vitro pH 3 p-nitrodiazobenzene reactivity of 5-HT and 5-hydroxytryptophan reported by us (12) in 1973. Carcinoid tumors have rarely given the histochemica! postcoupled benzylidene react ion (8). We are now occasionally finding a positive pH 3