THE TERM “CHROMAFFIN SYSTEM” AND THE NATURE OF THE “CHROMAFFIN REACTION” B. F. KINGSBURY Cornell University Medical College, Ithaca, N . Y . The general use by morphologists of the terms, chromaffin system, chromaffin cells, chromaffin tissue, chromaffin reaction, etc., suggests this critical note as timely. Kohn (’98) first introduced this term as a name for the cellgroups found by him in the sympathetic system, the medulla of the suprarenal gland, the carotid gland, etc., which assume a brown color when treated with chromic acid or dichromates. Observation of this interesting color reaction is older than Kohn’s extensive and systematic study, going back, I believe, to Henle (’65). Stilling (’98) preferred the term “chromophile,” while Poll (’02) has more recently suggested the term phaeochrome (brown color reaction with chrome salts). The query is but natural, therefore, as to the cause and nature of the chromaffin reaction and the attempt to determine this was precipitated by the peculiar way the medulla of the suprarenal gland behaved in different fixing solutions. Naturally, the effect of reagents on the medulla of this organ has already received considerable attention. The medulla blackens with osmic acid (Schultze and Rudneff, ’65, Bonnamour, ’05); turns green to blue with iron chlorid (Vulpian, ’56, Fusari, ’91, Giacomini, ’02, Mulon, ’03, Stoerck and Haberer, ’08); reduces gold chlorid (Mayer, ’72, Loewenthal, ’94) stains intravitam with neutral red (Mayer, ’96); stains with basic stains (many authors). 12 B. F. KINGSBURY Whether or not the reaction is associated with, and if so limited to, granules in the cytoplasm has been a matter of discussion which does not particularly concern us now. Hultgren and Anderson ('99) Grynfeltt ('03) Kohn ('00) Wiesel ('02) Poll and Sommer ('03) find that the reaction takes place in granules; Rabl ('91) Stoerk and Haberer ('08), on the other hand, found the chromaffi reaction to be a diffuse one. It seems to have been shown however,-and this is a more pertinent point,-that after treatment with potassium dichromate or Muller's fluid, the reaction is diffuse, while potassium dichromate and formaldehyde limits it t o granules in the cells of the medulla, (Kohn, '04). Lastly, Neubauer and Langstein ('02) ,pointed out that adrenalin blackens osmic acid solutions as does the medulla, and Ellio t t and Tuckett ('06) state that the intensity of the chromaffin reaction corresponds to the amount of adrenalin present. This brings me to the comparison of the reaction of the "chromaffin cells" of the suprarenal and those of adrenalin. It is a simple matter to test these reactions as I have done by taking free hand sections of the living suprarenal gland and placing them in the reagents in watch glasses, carrying out the corresponding test of adrenalin as is customary in test tubes. The results are arranged in tabular form below: REAQ~NT Potassium dichromate BECTIONS OF BUPRARINAL (MEDULLA) uickly color SOLUTION OF ADRENALIN 1:lOoo assumes brown Precipitate, reddish brown, formed a t once Potassium dichromate in Quickly assumes brown Precipitate, reddish color brown, formed a t once alkaline solution Potassium dichromate and Quickly assumes brown Precipitate, reddish color brown, formed a t once formalin Potassium dichromate and acetic acid Assumption of color which quently fades brown subse- Precipitate formed by potassium dichromate, soluble on addition of acetic acid THE NATURE OF THE “CHROMAFFIN REACTION” REAGENT SECTIONS OF SUPRARENAL (MEDULLA) 13 0OLUTION OF A D R E N U N 1:1000 Zenker’s fluid Color variable; usually present, but disappears Not tested on standing Silver nitrate (ammoniacal solution) Medulla (and zona reticularis) turns a slaty Grayish-black precipitate (gray) black Osmium tetroxid (Osmic acid) Medulla turns immediately black; subsequent Black precipitate blackening of cortcx Flemming’s fluid. (Osmic acid, chromic acid and acet.ic acid) Medulla assumes a black Color turns t o brownish color, which subse- red and subsequently quently fades more or blackens; no definite precipitate less completely Gold chlorid Medulla takes on a brownish red, becoming more of a purple on standing Ferric chlorid color, turning Medulla a blue-green; Green brownish red on heatturns brown on adding ing or adding ammonia ammonia Potassium ferricyanide Brownish red color appears on warming Fehling’s solution dedulla turns immediatelj Reduction in t h e cold a brick red; cortex a (green-yellow-red ppt.) bluish purple Turns brownish red with a tinge of pur>le, which becomes stronger on standing Grecn color, turning reddish brown on heating Benedict’s solution (ont Medulla turns gray; in. On heating, reduction (whitish-yellow ppt.) tensified on heating fluid formula) The parallelism shown in the simple if rather crude series of tests, leaves little doubt that the st’ainingreactions of the suprarenal medulla, with osmic acid, chromic acid, ferric chlorid, etc., are due to the adrenalin present although of course there may be other reducing substances present as well. The so-called chromaffin reaction therefore depends not upon any special affinity 14 B. F. KINGSBURY of the cells of the suprarenal for chromium salts, but simply and solely is due to a reduction of the chromate to a chromium trioxid (?) combination by strong reducing substances present. The grouping of organs or cell groups based upon the occurrence of reducing reactions has little significance unless it can be shown that the reducing substances are identical. The animal body is rich in reducing substances; most cell bodies show a moreorless well marked “chromaffi reaction,” due to reduction of chromates. The nervous system shows in the assumption of the wellknown brown color when it is “mordanted” in dichromate for the Weigert hematoxlyin method, a chromaffin reaction” proceeding more slowly, it is true, due in this case to a reduction by the lipoids present. The cortex of the suprarenal, to go no further,-shows, particularly in the eona reticularis, a marked brown color with chromates which has, however, somewhat different reaction conditions. If it is desirable to have the reduction of chromates represented in the name of the “system,” phaeochrome seems to the writer preferable to chromaffin or chromophile. The peculiar behavior of the suprarenal in different fixing fluids becomes clear in the light of the chemical reactions of adrenalin.’ When placed in Flemming’s fluid, the medulla rapidly turns black, but on standing, the black coloration disappears, the black being transferred, so to speak (that is, in a suprarenal whose cortex is rich in lipoids as in the dog or cat or guinea pig), to the cortex whose rich lipoid content blackens more slowly, so that after twenty-four hours, the medulla is grayish white, the cortex black. In osmic acid solution alone the blackening of the medulla is persistent, the difference in the Flemming’s fluid reaction being due undoubtedly to the solvent action of the acetic acid probably assisted by the presence of the oxidizing chromic acid. In potassium dichromate acetic (Tellyesniecky’s fluid), the brown color of the medulla largely disappears, which is not the case in neutral dichroqfite solutions or dichromate combined with formalin (a reducing substance) as in Orth’s fluid. In tissue fixed in Zen(‘ 1 Cf. also: Oppenheimer: Handbuch der Biochemie, vol.1, pp.768-9. THE NATURE OF THE “CHROMAFFIN REACTION” 15 ker’s fluid the brown color which appears a t first gradually fades, the color residue depending apparently on the duration of the fixation. Acid fixing fluids are not adapted for the fixation of the medulla of the suprarena1,-from this side at least. The mixture employed by Harvey (’06) for the fixation of gastric glands (equal parts water, formalin, 3 per cent dichromate and saturated solution of mercuric chlorid), or a formula of similar composition such as Helly’s fluid recommended by Poll therefore give excellent results with the medulla. 16 B. F. KINGBBURY BIBLIOGRAPHY BONNAMOUR, S. 1905 Thesis. Lyon. 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