On the Inhibitory Action of Potassium Cyanide Upon the Cytochemical Peroxidase Reaction

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On the Inhibitory Action of Potassium Cyanide Upon the Cytochemical Peroxidase Reaction. The 14th Report of Histochemical Study of Peroxidase

Tadao Mitsui, Shoji Maruyama and Shizue Kamezawa

Department of Anatomy, School of Dentistry, Nihon University, Surugadai, Tokyo.

Many biochemical studies have been made on the inhibitory influence of cyanide upon the peroxidase in plants and animal tissues, especially Agner (1941)" reported its influence upon the myeloperoxidase in the leukocyte. However, from the standpoint of hemato- morphology, we cannot fully accept these works without considering the state of the reacting substance as well as the substrate used. In other words, it is not absolutely determined whether both biochemical and cytochemical peroxidase reactions are similarly inhibited by cyanide, and whether the same experimental results can always be obtained by a variety of the substrates for the reaction. In order to answer these questions, the present study was undertaken, four different potassium salts and four different substrates being used. The Dopa oxidase reaction was employed in parallel with the peroxidase reaction.

Materials and Methods.

Human blood films, within 24 hours after preparation, were fixed for 30 seconds with ethano-formol solution, which consists of 14 parts ethanol and 1 part formol. This fixative was better than the formol- alcohol solution commonly used, although L ill ie2) recommends the immersion in 75% ethanol for 10 minutes. In order to compare the inhibitory action of potassium cyanide with that of other potassium salts, KNO3, KC1, K2SO4 were also tested, and from each a series of 178 T. Mitsui, S. Maruyama and S . Kamezawa decimal dilutions were made, ranging from 1 to 10-5M (mole) . Molar solution of KCN is 6.51%, of KNO3 10.11%, of KCI 7.46%, and of K2SO4 17.43%., The ,saturated solution of K2SO4 being 10.04% at 25°C, its molar solution is saturated . Benzidine, guaiacol, orthotolidine and orthophenylenediamine, were utilized for peroxidase reaction , and Dopa for oxidase reaction as shown in table 1. The NAS-Benzidine solution (Mitsui & Iked a, 1951)3) was used because of the simple technique and of color variation from blue to yellow according to the reaction intensity. The blood film was counterstained with eosin solution for several seconds in the o-phenylenediamine reaction which occurs predominantly in the nuclei of the myeloid leukocytes ; in the other peroxidase reactions, counterstained with the Pf e if f er's solution for twenty seconds, which is preferable to prolonged Wright's stain - ing, since this prolonged staining is frequently " epistatic " to the peroxidase staining. The peroxidase reactions were generally carried out for 2 minutes at 24°C, while the Dopa reaction for 30 minutes at 37°C. Exposure time to the inhibitors in these experiments was 2 minutes. The peroxidase reacting substance has a double specificity , which we shall call hydrogen donor and hydrogen acceptor specificity , whereas some authors" speak of hydrogen peroxide as the substrate and of the hydrogen donors as " acceptors ".

BenzidineH 2N--/\/\—NH9 (diaminodiphenyl)

G /\ —OCH3 uaiacol (brenzcatechin-monomethylether)

\/ CH3 CH3 Orthotolidine (dimethylbenzidine) H "/\/ 2N— /\ ›—NH2 NH2

Orthophenylenediamine

\/ OH D opa 1 NH2 (3, 4 dihydroxyphenylalanine) HO —/ )—CH2—CH—COOH

_ On th Inhibitory Action of Potassium Cyanide Upon the Cytochemical 179

According to Bloch, B. (1916, 1917, 1929, 1932)', Dopa is attacked by two different enzymes : (1) an absolutely specific Dopa oxidase which does not act on any other substrate, is easily inactivated by chemical and physical agents, and is present only in cells concerned with the elaboration of melanin (basal layer of epidermis, chromato- phores, cells of melanoma, etc.), and (2) a non-specific phenol oxidase which is relatively resistant to various chemical and physical agents and is present in a number of structures, especially in myeloid leukocytes.

Table 1. Special reagents used for the peroxidaseand Dopa reactions.

* The peroxidase has a double substrate, which we shall call hydrogen donor and hydrogen acceptor specificity. (ref. 12)) ** Parachromasia, by which we mean the property of a colorless reagent to stain cells or tissues a certain color.

Observations

Experiment 1. Influence of various potassium salts on the benzidine reaction. Of four different salts, KNO3, KC1, and K2SO4 couldn't inhibit the benzidine reaction even at concentration of 1 M, while KCN could inhibit the reaction strongly at 10-1 M, weakly at 10-3 M. However, 180 T. Mitsui, S. Maruyama and S. Kamezawa it should be noted that the eosinophils did not become entirely peroxidase negative even by two minute exposure to M KCN. This is one of the proofs that the peroxidase reacting substance of the eosinophils is more stable than that of the neutrophils. With increase of the influence of KCN, the neutrophils loose the peroxidase reacting granules in the perinuclear zone at first, and only the narrow ecto- plasmatic zone remains peroxidase positive, until all the cytoplasm turns in negative. The results are presented in table 2. Experiment 2. Influence of potassium salts on the guaiacol, o- tolidine and o-phenylenediamine reactions. KCN exerted the strongest influence on these three reactions among potassium salts, too. However, M solution of KNO3, KC1 and K2SO4 (saturated) also influenced to some extent on these peroxidase reactions of the neutrophils, especially the o-phenylenediamine reaction became entirely negative as in the case of M KCN. The results are presented in table 3. This table indicates, too, that the benzidine solution reacted best. Experiment 3. Influence of potassium salts on the Dopa reaction. Decimolar solution of KCN entirely abolished the Dopa reaction of the neutrophils, while slightly retarded that of the eosinophils. On the other hand, M solution of KNO3, KCl and KSO4 (saturated), could not entirely abolish the reactions of the neutrophils and the eosinophils. Almost similar results to the peroxidase reactions are presented in table 3, 4. Experiment 4. Relationship between the inhibitory actions of potassium cyanide and the substrates used. There is no doubt that the velocity as well as the color of products of the peroxidase reaction are varied according to the substrates used. Furthermore, it was confirmed by us that some substrate reacted only on the cytoplasmatic components, while others predominantly on the nuclei of the myeloid series. As shown in table 4, KCN solution inhibited the four different peroxidase reactions in various degrees, and this can be summarized as under :— In the neutrophils, the benzidine reaction was inhibited by 10-3M solution, the guaiacol and o-phenylenediamine reactions by 10-2 M solution, and the o-tolidine reaction by 10-' M solution of KCN. On the other hand, in the eosinophils, the benzidine reaction was slightly inhibited by 10-3 M solution, the guaiacol and o-tolidine reactions by M solution, while the o-phenylenediamine reaction was not influenced even by M solution of KCN. 181

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indicates an intense alkalinity. KCN + H20 -+ KOH+ HCN Then, a new problem arose here whether its alkalinity played a certain role in the inhibitory action mentioned above. In order to solve this problem, further experiments were carried out by the use of the benzidine solution. First, M solutions of potassium cyanide and potassium hydroxide were prepared, next, from each a series of decimal

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dilutions were made, ranging from 10-' to 10-5 M. To our surprise, the results of the experiments showed that there was no remarkable difference between inhibitory actions on the peroxidase reaction in potassium cyanide and potassium hydroxide solutions. Furthermore,

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Table 5. Inhibitory actions of potassium hydroxide and potassium cyanide upon the peroxidase reaction. Blood specimen used is different from that in table 2-4.

* measured with the glass electrode-potentiometer.

it was confirmed, too, that these two solutions at various concentrations indicated almost similar alkalinity, and that their inhibitory actions on the peroxidase reaction disappeared when the solutions lost a strong alkalinity, diminishing their pH value under 10.0. The results are presented in table 5. Experiment 6. Comparison between inhibitory actions of potassium cyanide and methanol. It has already been reported by us3.6'7.8) that methanol exerts a stronger inhibitory influence on the peroxidase reaction than other fixatives such as ethanol, acetone or 10% formalin. With respect to this strong inhibitory action, both methanol and potassium cyanide seem to be a similar reactant,- however, it should be noted here that methanol is able to fix blood cells, while potassium cyanide rather destroys them in a high degree. In other words, methanol destroys On the Inhibitory Action of Potassium Cyanide Upon the Cytochemical 185 only the peroxidase reacting substance in blood cells, while potassium cyanide dissolves whole cell components including nucleus. This is made clear by comparing the findings of Wright's staining of blood cells after exposure to potassium cyanide and to methanol. The results are presented in table 6. As already reported by T s u k a mote, the peroxidase reaction of erythrocyte is activated by methanol, and the hematological granules of leukocytes are often water-soluble even after fixation with methanol—this is the reason why the blood film wasn't washed in water before the Wright's staining as shown in the table.

Table 6. Comparison between influences upon the blood cells of potassium cyanide and methanol.

* (with dissolved nucleus), ** (with normal nucleus), — (negative), + (faint), 1+ (normal), +I+(intensely positive).

Discussion.

This experimental study was designed to examine the influence of KCN on the cytochemical perOxidase and Dopa reactions. As a preliminary step, the special inhibitory influence of KCN on the peroxidase reaction was investigated by comparing the results with those of other potassium salts such as KNO3, KC1 and K2SO4 using benzidine as a substrate (NAS-Benzidine solution). Next step was to examine at what concentration KCN can inhibit the peroxidase reactions with four different substrates, namely, benzidine, guaiacol, o-tolidine and o-phenylenediamine. And the Dopa reaction was employed in parallel with these peroxidase reactions. Finally, an attempt was made to examine the essential nature of the inhibitory action of KCN by comparing the results with those obtained using KOH as well as 186 T. Mitsui, S. Maruyamaand S. Kamezawa methanol. According to T he o r ell'', the biochemical peroxidase activity is reversibly inhibited by cyanide and sulfide in concentration of 1O- 10-6 M, while fluoride, azide, and hydroxylamine inhibit in somewhat higher concentrations , around 10-3 M. Agnee) reported in vitro (inhibition by Mr' M KCN (inhibition rate 100%), 10-3 M sodium azide 66%), 10-2 M hydroxylamine (95%), and sodium fluoride (49.5%), furthermore, only slight inactivation was caused by 10% formalin in 70 % alcohol. L em berg & Legge") fully accept Agner's work and state that the myeloperoxidase is inhibited by 10-4 M cyanide (86%) 10-3 M h , ydroxylamine (30% ), and 10-3 M azide (66%) ; it is not inhibited by carbon monoxide. It is now evident that the biochemical peroxidase activity is inhibited by cyanide in concentrations of 10-3-10-6 M. On the other hand, Lillie & Burtner2) recently reported that the cytochemical peroxidase reaction of human neutrophil leukocytes was inhibited in a high degree (but not entirely abolished) by one hour exposure to 10-' M KCN at 25°C ; the blood film, however, was stained with benzidine- sodium-nitroprusside solution for as long as ten minutes—this prolonged staining time should be criticized in order to compare with the results of our experiment. It is obvious from the present study that the cytochemical peroxidase reaction of human leukocytes is inhibited by two minute exposure to 10-1-10-3 M KCN at 24°C in various degrees dependent on the substrates used. These concentrations of 10-1 to 10-3 M in the cytochemical technique are not always in accord with those of 10-3-10-6 M in the biochemical technique mentioned above. From these facts, someone may infer that so-called peroxidase reaction in hematology is not of the same nature as the biochemical one. As regards this inference, however, we should precisely discuss the sorts of the substrates used, the reaction time, the reaction temperature, the exposure time to cyanide, furthermore about the state in which the peroxidase or the peroxidase reacting substance is contained . In blood film technique, the substances in blood cells which are expected to react positively, are present in an entirely dry condition, and the positive reaction is identified only when the colored reaction products are intensely adsorbed to the cell bodies, as many hematological peroxidase techniques indicate. On the contrary, the biochemical peroxidase reaction, where pyrogallol, leucomalachite green or ascorbic acid are commonly utilized for the substrate, is based on the color- imetric treatment of the colored oxidation products which is homogene- On the Inhibitory Action of Potassium Cyanide Upon the Cytochemical 187 ously dissolved in aqueous media. Therefore, it seems now impossible to regard the cytochemical peroxidase reaction as a false enzyme reaction from the results of these experiments with KCN alone . It is worthy of special comment that alkalinity of KCN ran parallel to the inhibitory action on the cytochemical peroxidase reaction. It may be said, too, that alkalinity of KCN played the main role in this inhibitory action, because the influence of KCN obviously diminished with decrease of its pH value. On the other hand, the toxity of KCN to the living body is due to the action of HCN produced by hydrolysis in the body as has been indicated by a number of pharmacological works. However, the relationship between the inhibitory action of KCN on the cytochemical peroxidase reaction and its toxity to the living body is unknown. Besides us€.7.8', Lillie & Burtner2 identified the inhibitory action of methanol, reporting that both the cytochemical peroxidase and W inkler-Sc h u I tze oxidase reactions were entirely abolished by 1 hour exposure to pure methanol at 25°C. Methanol exerts much the same influence on the cytochemical peroxidase reaction of leukocytes as M KCN solution, and is also extremely poisonous to the living body, but methanol differs from KCN in capacity of fixing blood cells. As far as we know, the correlation between the alkalinity of KCN solution and the peroxidase reaction, and the specific inhibitory influence of methanol on the reaction, have not sufficiently been reported from the standpoint of biochemistry.

Conclusion.

Four different potassium salts at varying concentrations as well as four different substrates for the peroxidase reaction were utilized for the present study. It was shown by comparison of potassium salts that KCN exerted the strongest influence upon the peroxidase reactions, namely, the benzidine reaction of human neutrophils was inhibited by 10-3 M, the guaiacol and o-phenylenediamine reactions by 10-2 M and the o-tolidine reaction by 10-1 M of KCN, and that the experiment with the Dopa reaction indicated almost similar results to the o-tolidine reaction. The eosinophils were always more stable in all the experiments than the neutrophils. It was also shown that the inhibitory influence of KCN on the reaction was of a different nature from methanol, while of a similar nature to KOH, in other words, the inhibitory influence of KCN appeared based on its strong alkalinity. 188 T. Mitsui, S. Maruyama and S. Kamezawa

The difference between cytochemical and biochemical peroxidase reactions was discussed.

References. 1) A g ner, K. (1941), Acta Physiol. Scand., 2, Suppl. 8, cited from ref. 10) P. 432. 2) Lillie, R. D. & Burt ne r, H. J. (1953), Stable sudanophilia of human neutrophil leucocytes in relation to peroxidase and oxidase. Journ. of Histochemistry & Cyto- chemistry. 1 (1): 8-26. 3) Mitsu i, T. & Ik ed a, S. (1951), NAS-Benzidine reaction of blood cells. Fol. anat. jap. 23 (4-5) : 331-336. 4) (1951), On the four phases of peroxidase reaction of blood cells using benzidine and salts. Fol. anat. jap. 23 (4-5): 323-329. 5) Theorell, H. (1951), The iron-containing enzymes, in Sumner & Myrb5ck, The Enzymes, New York, Acad. Pr. Inc., Publishers, p. 426. 6) Sasaki, M. (1952), Influence of fixatives upon the peroxidase staining. Fol. anat. jap. 24 (3) : 193-197. 7) Tsukamoto, K. (1952), Experimental study on the peroxidase reacting granules in the blood cells. Fol. anat. jap. 24 (5-6): 347-369. 8) Mitsui, T., Sasaki, M., Tsukamoto, K. & Morita, F. (1952), On the difference between peroxidase reaction positive substances in leukocytes and erythrocytes . V. Medicine & Biology. 25 (6): 295-298. (Japanese). 9) cited from ref. 2), p. 11-12. 10) Lemberg, R. & Legge, J. W. (1949), Hematin compounds and bile pigments. New York, lnterscience Publishers, Inc., p. 432. 11) W a sh burn (1928), Journ. Lab. & Clin. Med. 14: 246, cited from Lillie's Histo- pathologic Technic, Philadelphia, Blakiston Co. (1948), p. 115. 12) Everett, M. R. (1948), Medical Biochemistry, New York , Paul B. Hoeber, Inc., p. 94. 13) cited from Gomor i, G. (1952), Microscopic Histochemistry, Chicago, The Uni- versity of Chicago Press, p. 159-160.