P. T. Donaldson

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

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 α-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.

USE OF THE DIAZOSULFANILIC ACID, pH 1 AZURE A SEQUENCE ON ADRENAL MEDULLA AND THE EFFECTS OF PRIOR OXIDATION AND REDUCTION ON THE REACTION IN SEVERAL SPECIES

With the diazosulfanilic acid, pH 1 azure A sequence adrenal medulla exhibits areas of pale green cells with green nuclei alternating with other areas grading from green to dark blue. Adrenal cortex and capsular collagen color greenish yellow, nuclei and elastin green, smooth muscle yellowish green and erythrocytes greenish brown to greenish black. The reaction is obtained best after 5% glutaraldehyde, then 10% formol then glyoxal, quite well with formol-free sublimate and lead acetate fixations and fails after Zenker and Kose bichromate formol fixations. Staining is prevented by prior oxidation (quinonization) with 10% iodine/methanol (1-2 hr 3°C), 0.1 M FeCl3 (2 hr), 3-5% K2Cr2O7 (2-3 hr) and 1% H5IO6 (10 min) and after these oxidations is restored by 4 hr 5% dithionite (N2S2O4), metabisulfite (Na2S2O5) or thiosulfate (Na2S2O3 · 5H2O) reductions. The reaction has been demonstrated in man (Homo sapiens), monkey (Macaca mulatta), hog (Sus scrofa dom.), dog (Canis familiaris), cat (Felis cattus), guinea pig (Cavia cobaya), rat (Rattus norvegicus albinus), mouse (Mus musculus albinus), gerbil (Meriones unguiculatus) and opossum (Didelphis virginiana). In the hog most of the adrenal medulla gave the dark blue reaction, fairly large areas with some segregation into islets of dark blue occurred in man, monkey, cat, dog and opossum and islets are seen in mouse, smaller in rat and inconstantly in the guinea pig and gerbil. It is thought that the reaction is showing noradrenaline. The method is not specific for this substance; certain proteins as hemoglobin and keratohyalin as well as enterochromaffin cells react strongly.

THE pH RANGE OF THE DIAZOSAFRANIN REACTION OF RAT AND OTHER MAST CELLS

5-Hydroxytryptophan and 5-hydroxytryptamine (5-HT) give strong red azo coupling colors with fresh p-nitrodiazobenzene (fast red GG) at coupling pH levels of 3-9. Tyrosine, histidine, histamine, adrenaline, noradrenaline, dopa, dopamine and tryptophan give strong red colors at pH 7-9, weakening to orange and yellow at pH 5-6 and negative below. Tryptophan gives weak orange-yellow to about pH 4. Rat mast cells color deep red with diazosafranin at pH 3-8. Protein colors pink to red at pH 7-8, weaker at 5-6, and remains almost uncolored below that. Preoxidation with 10 min 1% H5IO6, 2 hr 0.1 M FeCl3, 3 hr 5% K2Cr2O7 or 1 hr 3°C 10% I2/CH3OH does not prevent the diazosafranin reaction of rat mast cells. Periodic acid does not inhibit in vitro azo coupling of 5-HT or 1-naphthol, that of noradrenaline is prevented, and can be restored by reduction with Na2S2O5 or Na2S2O3. Azo coupling of enterochromaffin and adrenal medulla is prevented by these oxidations and restored by Na2S2O4 reduction. Diazosafranin staining of mast cells is not extracted by 24 hr 0.24 N HCl/70% alcohol. Safranin and other cationic dye staining of rat mast cells resists aqueous 0.1 N HCl some hours, largely disappearing at 24 hr, and is removed by 5-15 min in 0.12 N HCl/7O% alcohol. At pH 1 0.1% toluidine blue colors rat mast cells deep violet; when superimposed after acid diazosafranin the red mast cells assume a deep purple, intermediate color. Since in extended use of the method pH 3 diazosafranin colors only bilirubin casts, hematoidin and dog, rat, mouse and gerbil mast cells, and not monkey, most human or lead and mercury fixed guinea pig mast cells, it is suggested that the method is showing 5-HT in rat mast cells. The occasional reaction of human mast cells may be due to pathologic presence of that substance in these cells.

Difficulties in ferrous iron mordant hematoxylin staining and some hematoxylin substitutes.

A gradual deterioration of intensity of sequence ferrous sulfate hematoxylin staining was traced, after elimination of hematoxylin quality as a cause, to a deterioration of the metal salt, associated with caking of the crystals. Fresh samples were also partly caked and ineffective. Ferrous ammonium sulfate was found also subject to the same deterioration. Ferrous chloride freshly prepared as a 1 M solution from iron wire under anaerobic conditions at biweekly intervals proved to be satisfactory as a mordant source. Of several other mordant dyes tested: gallein, brazilin and chromoxane pure blue B were the best, but none was equal to good hematoxylin.

The influence of formaldehyde fixation time on the rate of nitrous acid deamination of erythrocytes and spinal cord proteins

SummaryAlcohol fixed blood films and fresh blocks of spinal cord were immersed in phosphate buffered neutral 10% formol for graded intervals, the films for 10, 30 min, 1, 2, 4, 8, 24 hr; the blocks for 2, 4, 6, 24 hr at 3 and 24° C; 1, 3, 7, 14, 21, 28, 42, 56 da, 3 and 14 mo at 24–26°. Graded deaminations in 2 N NaNO2/HAc at 3° C were applied: 1, 2, 5, 10, 20, 30 min; 1, 2, 4, 6, 8, 12, 18, 24, 36 hr. Blood films were stained at pH 6 and 6.5, tissue at pH 4.5 and 5.0, both in azure A eosin B. The point at which erythrocytes reached a slightly bluish green was taken as the end point, since no further color change occurred on further exposure and erythrocytes were the last of usually deamination susceptible tissue elements to lose their oxyphilia on deamination. Deamination of alcohol fixed blood films is completed in about 2 min, of sublimate fixed spinal cord in about 1 hr. Progressive formaldehyde exposure increased deamination time of blood films to 10–20 min in 1 hr, to 6–8 hr in 4 hr and to 12 hr in 24 hr. The tissue deamination showed similar progressive increase of deamination time, slower with 3° C fixation than with 24–26°, reaching 18–36 hr by about 3 days formol, and remaining about the same thereafter.

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

Letter: Direct carbonyl reaction in mouse mast cells.

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