(Received 18 September 1957) Andersson, 1899).

Transcription

1 193 J. Physiol. (1958) I4I, 193-I97 OBSERVATIONS ON THE NATURE OF THE CHROMAFFIN REACTION BY A. J. HALE* From the Department of Medical Physics, Karolinska Institutet, Stockholm (Received 18 September 1957) In the chromaffin reaction a brown colour is produced in fresh tissue by the action of fixatives containing bichromates (Henle, 1865). In the adrenal medulla the reaction is considered to be produced by adrenaline (Hultgren & Andersson, 1899). Borberg (1913) thought that the reaction was caused by the reduction of bichromate to chrome yellow by adrenaline and Ogata & Ogata (1923) have attributed to this reduction product the formula (CrO2)f. Verne (1923) pointed out that not only adrenaline but all those substances which contained polyphenols, aminophenols or polyamines with the hydroxyl group in ortho or para positions would give the reaction. Gerard, Cordier & Lison (1930) have suggested that the reaction is due to the appearance of an oxidation product of the reacting substance rather than a reduction product of the bichromate. They support this argument with evidence that a brownish colour, similar to that which is produced by bichromate, can be produced by the action of iodates. They suggest that the brown colour is caused by the oxidation of the reacting substance to a quinone or quinoneimine and the combination of such an oxidation product with one of the unaltered polyphenols of the reacting substance, to give a quinhydrone. Verne (1923) points out that a chromaffin reaction, produced by oxidizing agents other than bichromate, has a less stable colour complex which is not identical to that produced by bichromate. Since it is possible that a precipitate of chromium dioxide might be produced during the oxidation reaction (Gerard et al. 1930; Lison, 1953), it is possible that chromium might be deposited in the tissues irrespective of the mode of formation or the coloured substance. Coupland (1954) has, however, shown that chromium is deposited in the adrenal medulla and that it remains there even when the colour of the chromaffin reaction is removed by exposure * Present address: Institute of Physiology, University of Glasgow. 13 PHYSIO. CXLI

2 194 A. J. HALE to bromine water. But he does not show the localization of the chromium within the medulla. Chromium has a mass absorption coefficient for soft and ultra-soft X-rays which is approximately double that of the proteins, lipids and other components of a normal cell (Text-fig. 1). Thus, if chromium is present in a tissue, one would expect to get a heavy absorption of soft X-rays there. Utilizing the X-ray micro-technique of Engstrom (1946) and Engstr6m & Lindstrom (1950), I have therefore examined tissues stained by the chromaffin reaction to see if chromium is present in them and if so, where it is located. METHODS Adrenals were removed from rats which had been killed by a sharp blow on the head. Some were fixed in 10% neutral formalin (24 hr) and some in 3-5% potassium bichromate (24 hr) followed by 10% neutral formalin (24 hr). After fixation the tissues were washed (24 hr in running water), dehydrated, cleared, embedded and blocked in paraffin wax. The tissues were arranged so that each block contained at least one adrenal fixed by each method. Sections 4 and 20 ui in thickness were cut and mounted on Kodak maximum resolution plate in the dark room, according to the technique of Greulich & Engstrom (1956). Some sections were also mounted and counterstained by conventional methods. The plates, with sections, were exposed in a miniature X-ray tube fitted with an aluminium window (filter) 1000 A thick (Engstr6m & Lundberg, 1957). The sections 4y in thickness were exposed for 40 min at 1-0 kv and 1.0 ma and for 3 hr at 0-8 kv and 1.0 ma. The sections 20,u in thickness were exposed for 40 min at 1-5 kv and 1-0 ma. These voltages in this tube provide polychromatic X-rays which have a short wave-length limit of about 12 A (1-0 kv), 15 A (0-8 kv) and 8 A (1-5 kv). Some of the sections 20, in thickness were also exposed in a sealed-off X-ray tube (Machlett OEG-50) for 8 min at 7-8 kv and 8 ma. This provides filtered X-rays having a maximum intensity around the wave-length 2-0 A but with a short wave-length limit of about 1-5 A. This wave-length is hardly absorbed by protoplasm but is considerably absorbed by chromium. After exposure the sections were removed by immersing the plate in acetone and the plates were developed in Kodak 19B developer for 5 min. The relative amounts of absorption produced by the images of the different tissues on the X-ray were measured in a microdensitometer. RESULTS In adrenals fixed in bichromate there is a strong chromaffin reaction in all the cells of the medulla (P]. 1, figs. 1, 2) except in ganglion cells seen in some sections. This chromaffin tissue shows a very heavy absorption of X-rays at all the wave-lengths at which it is examined (P1. 1, fig. 3). This high absorption is markedly different from the very low absorption of the medullary cells seen in the tissue fixed in neutral formalin (P1. 1, fig. 4). Examination of the heavily absorbing chromaffin cells at high magnification shows that the increased X-ray absorption is marked in the nuclei and also in the cytoplasm of many of the medullary cells (P1. 2, fig. 1). This distribution of highly absorbing areas corresponds to that of the chromaffin reaction. The ganglion cells show a very low absorption similar to that seen in ganglion cells fixed in fornmalin (P1. 2, fig. 2).

3 NATURE OF CHROMAFFIN REACTION ,000 / I b,uul E -._ U) 1,000 - I 500._ U) k- rii 50 Si1 01~ II - Text-fig Wave-length (A) The mass absorption coefficient of chromium and protein as a function of wave-length; log.-log. scale. DISCUSSION Coupland (1954), using autoradiography of tissue fixed in potassium bichromate prepared from radioactive chromium, has shown that the chromaffin reaction is associated with the deposition of chromium in the medulla. He could not, however, localize the deposition of the chromium within the medulla because of the lack of resolution in the autoradiographic technique. Using the diphenylcarbazide method he measured the amount of chromium present (per dry weight: personal communication) in isolated medullary and cortical tissues and found that there was approximately four times as much chromium present in the medulla as in the cortex after conventional histological treatment. Lindstr6m (1955) found that the medullary to cortical ratio of relative X-ray absorptions in frozen-dried rat adrenal was 1: 4. In the present investigation the ratio is approximately 2: 3 for tissues fixed in formalin. In adrenals fixed in bichromate the ratio is altered to approximately 4:3. This marked increase in the ratio of the absorptions indicates that a large amount of chromium is deposited in the medullary tissue. The increase in absorption occurs in the nucleus and the cytoplasm and is marked in those edges of the cells which border on the venous sinuses. I have used relatively high voltages for some sections since, at lower voltages, absorption might have been due to some cause other than the deposition of chromium, e.g. cell shrinkage and thus increase in dry mass per 13-2

4 196 A. J. HALE 196 A.H L unit volume. At higher voltages very little of the X-rays which have a short wave-length is absorbed by the tissues. Chromium, however, has a marked absorption maximum around the wave-length produced at the higher voltages (K-edge of chromium is at 2-07 A) (Text-fig. 1): thus the heavy absorption pattern in the medulla of the chromaffin-fixed tissue at this wave-length can be attributed with some confidence to the deposition of chromium. It is still not possible to say if the colour of the chromaffin reaction is due to the production of an organic oxidation product of adrenaline, or to the deposition of chromium, or to the formation of an adrenochrome-chromium complex: but chromium is certainly deposited and firmly bound at the site of the colour reaction. The parallelism between the intensity of the colour reaction and the heavy absorption of soft X-rays shows that the chromium is actually present where the colour is produced and is not just deposited in the medulla during the reaction. Since the increase in X-ray absorption can be measured accurately, it might be possible to develop a method for the measurement of the amount of reacting material in individual medullary and other chromaffin cells. SUMMARY 1. Sections of adrenals fixed to demonstrate the chromaffin reaction were examined by ultra-soft X-ray methods to determine if chromium was deposited at the site of the reaction. 2. It was found that chromium was deposited in the cytoplasm and nuclei of the medullary cells. I should like to thank Professor Arne Engstrom of the Department of Medical Physics, Karolinska Institutet, Stockholm, for letting me use his laboratory facilities, and the Wellcome Trust for a travel and maintenance grant which allowed me to carry out the investigation. REFERENCES BORBERG, N. C. (1913). Das chromaffine Gewebe. Skand. Arch. Physiol. 28, COUPLAND, R. (1954). Observations on the chromaffin reaction. J. Anat., Lond., 88, ENGSTR6M, A. (1946). Quantitative micro- and histocheinical elementary analysis by roentgen absorption spectrography. Acta radiol., Stockh., Suppl. 63. ENGSTR6M, A. & LINDSTROM, B. (1950). A method for the determination of the mass of extremely small biological objects. Biochim. biophys. acta, 4, ENGSTR6M, A. & LUNDBERG, B. (1957). A simple midget X-ray tube for high resolution microradiography. Exp. Cell Res. 12, GERARD, P., CORDIER, R. & LisON, L. (1930). Sur la nature de la reaction chromaffine. Bull. Histol. Tech. micr. 7, GREULICH, R. C. & ENGSTR6M, A. (1956). A new approach to high resolution microradiography using extremely soft X-rays. Exp. Cell Res. 10, HENLE, J. (1865). Ueber das Gewebe dernebenniere und der Hypophyse. Z. rat. Med. 24, HULTGREN, E. 0. & ANDERSSON, 0. A. (1899). Studien uber die Physiologie und Anatomie der Nebennieren. Skand. Arch. Physiol. 9, LINDSTROM, B. (1955). Roentgen absorption spectrophotometry in quantitative cytochemistry. Acta radiol., Stockh., Suppl. 125.

5 THE JOURNAL OF PHYSIOLOGY, VOL. 141, No. 2 PLATE 1 (Facing p. 196)

6 THE JOURNAL OF PHYSIOLOGY, VOL. 141, No. 2 PLATE 2 II0~ qe.. -xta.s * P- * ---W- - r A-.i l" 61, ' '.4s'M - 4#~ ~ W * _~~~~~~~~~~~lk oṫ::.',' :.# 91 S {

7 NATURE OF CHROMAFFIN REACTION 197 LIsoN, L. (1953). Hi8tochemie et Cytochemie AnimaleB, 2nd ed. Paris: Gauthier-Villars. OGATA, T. & OGATA, A. (1923). tber die Henle'sche Chromreaktion der sogenannten chromaffinen Zellen und den mikrochemischen Nachweis des Adrenalins. Beitr. path. Anat. 71, VERNE, J. (1923). La reaction chromaffine en histologie et sa signification. Bull. Soc. Chim. biol., Pari8, 5, EXPLANATION OF PLATES PLATE 1 Fig. 1. Section 20 IL in thickness of adrenal fixed in 3-5% potassium bichromate. Counter-stained with haematoxylin. Fig. 2. Section 20u in thickness of adrenal fixed in 10% neutral formalin. Stained with haematoxylin. Fig. 3. Microradiograph of 4 IL section of adrenal fixed in 3.5% potassium bichromate. Exposed for 3 hr at 0-8 kv and 1.0 ma. Areas of high absorption are light. Fig. 4. Microradiography of 4ju section of adrenal fixed in 10% neutral formalin. Exposed for 3 hr at 0.8 kv and 1.0 ma. Areas of high absorption are light.. These semi-serial sections were mounted under dark-room conditions where it was difficult to avoid overspreading or folding of the tissue. PLATE 2 Fig. 1. This is the preparation, shown in P1. 1, fig. 3, at a higher magnification. The large dark areas to the left are ganglion cells. Light areas are heavily absorbing. Fig. 2. This is the preparation, shown in P1. 1, fig. 4, at a higher magnification. The light mass in the centre consists of densely packed blood corpuscles and plasma. The large cells to the left of it are ganglion cells. Cortical tissue lies to the right. Light areas are heavily absorbing.