The Control of Methemoglobinemia with Methylene Blue

THE CONTROL OF METHEMOGLOBINEMIA WITH METHYLENE BLUE

William B. Wendel

J Clin Invest. 1939;18(2):179-185. https://doi.org/10.1172/JCI101033.

Research Article

Find the latest version: https://jci.me/101033/pdf THE CONTROL OF METHEMOGLOBINEMIA WITH METHYLENE BLUE"12 By WILLIAM B. WENDEL (From the Department of Internal Medicine, Washington University School of Medicine, St. Louis; and the Department of Chemistry, University of Tennessee, College of Medicine, Memphis) (Received for publication October 13, 1938) The cyanosis seen in a high percentage of pa- the concentration of methemoglobin from levels tients receiving sulfanilamide and related drugs as high as 25 to 40 per cent of the total pigment has been attributed variously to methemoglo- to less than 4 per cent in 30 to 40 minutes. binemia (1, 2), sulfhemoglobinemia (1), an un- In one case of sulfhemoglobinemia methylene usual degree of unsaturation of the venous blood blue was without effect upon the abnormal (3), and to the presence of aniline black (4) and pigment. other colored derivatives of sulfanilamide (5) ANIMAL OBSERVATIONS in the red blood corpuscles. Marshall and Walzl Since, in our experience, sulfanilamide does not (4) and Chesley (6) consider methemoglobin to produce methemoglobin in dogs, rabbits, rats, and be of no importance except perhaps in an occa- mice, nitrite was used to produce experimental sional patient. methemoglobinemia. Figure 1 illustrates the The present paper summarizes the results of course of blood pigment changes in seven control determinations of methemoglobin in the blood of dogs which received intravenous injections of 30 more than one hundred patients who were receiv- mgm. of sodium nitrite per kilogram of body ing sulfanilamide and showing cyanosis. Methe- weight. Following injection of nitrite, methemo- moglobin was found, at least in traces, in over 90 globin 8 rapidly accumulates and after 60 to 100 per cent of these. In thirty-five cases the blood minutes reaches a maximum concentration corre- contained methemoglobin to the extent of 15 per sponding to a loss of 60 to 70 per cent of the cent or more of the total pigment. In one in- normal oxygen capacity. It then progressively stance 40 per cent of the total blood pigment was decreases, due to reconversion to hemoglobin, and in the form of methemoglobin after 8 to 9 hours has entirely disappeared. The Animal experiments are described which con- average maximum rate of regeneration of hemo- firm and extend the observations of Williams and globin from methemoglobin (indicated in Figure Challis (7), Steele and Spink (8), and Hauschild 1 by a broken line) in these seven animals was (9) that methylene blue hastens disappearance of 0.03 volumes per cent per minute. This is essen- methemoglobin from the blood. Clinical applica- tially the same rate as observed when blood con- tion of these experiments to patients showing taining methemoglobin from these animals is in- cyanosis from sulfanilamide has shown that intra- cubated at body temperature in zitro, and may be venous injection of 0.1 to 0.2 cc. of one per cent looked upon as the rate at which the enzyme methylene blue per kilogram body weight reduces systems in the erythrocytes are able to reduce methemoglobin (10). 1 A preliminary report of this work was given before the Middle Section of the American Laryngological, 8 Methemoglobin concentration is expressed in this Rhinological, and Otological Society, St. Louis, January paper in two ways, as per cent of the total pigment and 26, 1938, and the American Society of Biological Chem- as volumes per cent, one volume per cent of methemo- ists, Baltimore, March 30, 1938 (J. Biol. Chem., 1938, globin being that concentration which results from loss 123, cxxiv), and was contained in a letter to the Editor of one volume per cent oxygen capacity. of the J. A. M. A., 1937, 109, 1216. In most experiments methemoglobin was determined 2This work was supported at both schools by grants by a spectroscopic method which will appear in a forth- from the Upjohn Company, Kalamazoo, Michigan. The coming number of the J. Lab. and Clin. Med. Occa- Upjohn Company has supplied the writer with 10 cc. sionally, methemoglobin was determined also by differ- ampoules of methylene blue which were used in the ence between total pigment and oxygen capacity. The clinical studies. two methods usually gave identical results. 179 180 WILLIAM B. WENDEL

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200 300 MINUTES AFTER NITRITE INJECTION FIG. 1. AccumuLMAoN AND DISAPPEARANCE OF METHEMOGLO}IN IN THE BLOOD OF DOGS FOLLOWING THE INTRAVENOUS INJECTION OF SODIUm NimTE (30 MGM. PER KILOGRAM OF BODY WEIGHT)

Having determined the physiological rate of obtained with a number of dogs, shows that in- hemoglobin regeneration from methemoglobin jection of 2 mgm. of methylene blue (in the form various substances were tested for possible accel- of a one per cent aqueous solution) per kilogram erating action upon this process. Sodium formal- of body weight increases the rate of disappearance dehyde sulfoxylate, which reduces methemoglobin of methemoglobin 'from the blood four or five- in aqueous solution, was found to be without ef- fold. Table I summarizes the results of a series fect in trvo. Sodium formaldehyde sulfoxylate, of similar experiments in which the effect of vari- however, reduces methylene blue and since re- ous quantities of wnethylene blue upon the rate of duced (leuco) methylene blue converts methe- disappearance of methemoglobin from the blood moglobin to hemoglobin very rapidly, it seemed was determined. A measurable effect is evident possible that simultaneous injection of these two even with the smallest amount of dye injected, substances might accelerate conversion of methe- namely, 0.1 mgm. per kilogram of body weight. moglobin to hemoglobin. A cycle between sul- Oxygen capacity figures in Table I indicate that foxylate in the plasma and methemoglobin in the the methemoglobin which disappears rapidly fol- red cells mediated by methylene blue was pictured. lowing injection of methylene blue is converted When tested it was found that simultaneous intra- to functionally active hemoglobin. The duration venous injection of methylene blue and sulfoxy- of the anti-methemoglobin action of 5 mgm. of late caused methemoglobin to disappear from the methylene blue per kilogram in dogs is at most blood very rapidly. Control experiments, how- about 2 hours. ever, showed that methylene blue alone was equally Methylene blue accelerates the disappearance of effective. Figure 2, which illustrates the results methemoglobin also from the blood of rabbits. METHEMOGLOBINEMIA AND METHYLENE BLUE 181 Methylene blue is effective as an anti-methemo- DOG 22 globin agent also when given by mouth. As com- HbO2, INITIAL, 16.4 VOL.X N AFTER 95 MIN. 6.3 VOL L.X pared with intravenous injection the response to o t 155 15.4 administration by mouth is slow, and more of the 10- 2 MG. MB./KG. dye is required. Observations to date indicate that SLOPE 0.2 VOL- /MIN. accumulation of methemoglobin can be prevented in adults by oral administration of 0.5 to 1.0 gram i-j of methylene blue per day even when the patient is receiving large doses of sulfanilamide. z Before methylene blue was injected into pa- 0 -i tients receiving sulfanilamide, compatibility of 0 these two substances was tested in dogs and rab- Lai s bits. Several times as much methylene blue was Lai injected into experimental animals as is required in humans. Also the dye was injected at a time when the animals were severely toxic from large MB doses of sulfanilamide (1 gram per kilogram per I I No increase in was evident follow- 0 100 200 300 day). toxicity MINUTES AFTER NITRITE I NJECTION ing intravenous injection of the dye. In order to elicit cumulative effects of administra- FIG. 2. EmFCr OF INTRAVENOUS INJECTION OF [ETHY- possible LENE BLUE, 2 MGM. PER KILOGRLAM OF BODY WEIG:ET ON tion of the combination of drugs one dog was RATE OF CONVERSION OF METHEMOGLOBIN TO HEMO*GLOBIN given 0.6 gram sulfanilamide per kilogram per IN THE DoG day and 0.27 gram of methylene blue per day HbO, means oxygen capacity. MB over arrovv mdi- by mouth for 4 weeks. No outward evidence of cates injection of methylene blue. toxicity or abnormal blood rhanges was observed. Preliminary experiments with Dr. Anna Dula- CLINICAL APPLICATION ney of the University of Tennessee Medical In collaboration with Dr. Alexis F. Harn tmann School on mice injected with highly virulent of Washington University Medical School, where strains of beta hemolytic streptococci indicate that the writer began and completed an importanit part of this work, these observations receivedI first TABLE I clinical application in the summer of 1937. Since Effect of various amounts of methykne blue on rate of these early observations many others have been conversion of methemoglobin to hemoglobin in dogs made with the cooperation of physicians c n the staff of the University of Tennessee Mledical Amount of Rate of OXYen capacity Dog methylene disappear- School. In summary, intravenous injection of number blue ance of After After icted methemoglobin Before 0.1 to 0.2 cc. per kilogram of body weighit of a nitrite i methlee bluet Mm. per otumes per cent soluma otusm solume one per cent aqueous solution of methylene blue kilegram per minute per cent per cet per cent converts in the course of about 45 minutes all of 14 10 0.77 17.3 (50 minutes) 14 10 0.33t 12.8 4.4 13.2 (30 minutes) in the ocytes 21 10 0.31t the methemoglobin circulating erythr 16 5 0.19 functionally active hemoglobin, even when 18 5 0.25 17.7 7.3 17.0 (65 minutes) into 17 2 0.23 the concentration representss initi- 22 2 0.20 16.4 6.3 15.4 (60 minutes) methemoglobin 19 1 0.076 12.7 5.5 10.6 (95 minutes) as much as 25 to 40 per cent of the totsal pig- 20 1 0.14 ally 6 0.5 0.12 ment. rate at which reap- 17 0.5 0.082 The methemoglobin 22 0.1 0.049 of the varie.s 20 0.1 0.051 pears following injection dye with Nine control different individuals, but as a general rule the animals 0 Average=0.028 concentration of methemoglobin will have re: turned * Determined at peak of methemoglobin formation. to its pre-methylene blue level nfot sooner tlhan 12 t Figures in parentheses denote interval between inject- hours and not later than 24 hours after injjection ing methylene blue and determining oxygen capacity. $ Rate was actually faster than this. Method did not of 2 mgm. of dye per kilogram of body vveight. permit accurate determination. 182 WILLIAM B. WENDEL methylene blue does not interfere with the thera- TABLE II peutic action of sulfanilamide in these animals. Lack of relationship bettween concentration of sulfanilamide in the blood and the degree of methemoglobinemia Sulfanilamide methemoglobinemia Patient suBfnlide Methemoglobin Total pigment The frequency of methemoglobinemia in patients receiving sulfanilamide has not been deter- per cent of volumes mgm.mgm.PerpffentrCitttotal pigment per cent mined. We have, however, examined for the L. F. 4 22 7.8 presence of methemoglobin the blood of more than 4 16 7.8 one hundred patients who were showing cyanosis H. L. 34 33 14.6 from this drug. In this group of patients where 50 25 27 27 the blood was examined within 30 minutes after 20 22 being drawn and the sulfanilamide concentration 17 20 was greater than 4 mgm. per cent, methemoglobin E. B. 8 29 13.4 was found at least in traces (more than 3 per L. M. 14 17 18.1 cent of the total pigment) in every blood except two. Table II summarizes data obtained on W. R. S. 14 29 15.1 samples of blood containing more than 15 per M. A. 5 21 16.5 cent of methemoglobin and whose sulfanilamide 6 29 concentration also was determined (11). Most of B. D. 20 25 6.4 these samples of blood were from patients who L. McM. 7 22 11.9 were receiving 0.1 to 0.2 gram of sulfanilamide 5 17 12.4 per kilogram of body weight per day in divided E. W. 26 30 14.4 doses at 4-hour intervals. In each case the dose 10 30 of sulfanilamide had been constant 24 hours prior 12 23 to collection; in most, it had been constant 48 E. J. 6 22 22.0 hours. Where several sets of data are given on 5 25 one patient, they represent analyses of samples of T. 5 23 13.9 blood taken on different days, sometimes at widely Y. 20 25 separated intervals and following change in dos- 9 25 age. No correlation between concentration of sulfanilamide in the blood and intensity of methe- H. I. 5 25 11.0 moglobinemia is evident in these J. T. 9 33 data. 8 33 Evidence that methemoglobin is the principal abnormal pigment in the blood of patients showing C. B. 6 20 cyanosis from sulfanilamide will be published in B. T. 3 13 detail elsewhere but may be as 10 30 summarized fol- 8 33 lows. (a) When a freshly drawn sample of blood 6 25 from such a patient is laked with 4 or 5 volumes M.L. M. 5 28 of water in a tube of about one inch in diameter and is examined with a hand spectroscope before G. 0. 8 27 18.1 a 60 to 100 watt frosted light bulb, it usually R. L. L. 14 29 14.0 shows an absorption band in the red region of B.S. M. 3 26 the spectrum (at A 630 mu). In a given sample of blood the intensity of this band is approxi- O. B.S. 7 22 mately the same as that calculated for methemo- B. 12 27 14.2 globin from the difference between total pigment 7 30 and oxygen capacity, this difference being as- sumed to be owing to methemoglobin alone. Fol- cyanide or carbonate dissipates it entirely. In an lowing addition of a buffer solution of pH 6.5 occasional sample of blood there is an absorption this band is intensified. Addition of sodium band in the red portion of the spectrum (some- METHEMOGLOBINEMIA AND METHYLENE BLUE 183 what nearer the yellow) which is unaffected by erythrocytes of a colored derivative of sulfanila- alkali or cyanide and which is caused by the pres- mide, formed by irradiation of dilute solutions ence of sulfhemoglobin. The following state- of the latter. It is interesting to note, however, ments, therefore, are intended to apply only to that the colored derivatives of sulfanilamide de- those cases in which sulfhemoglobin is absent. scribed by these workers convert hemoglobin to (b) The absorption band in the red region of the methemoglobin in zitro. spectrum is greatly weakened or is entirely dis- sipated by injection of methylene blue. At the DISCUSSION same time the color of the blood changes from In 1933, Williams and Challis (7) and Steele reddish brown to a more normal red. Also fol- and Spink (8) reported that intravenous injec- lowilng injection of methylene blue the oxygen tion of methylene blue into three patients showing capacity of the blood increases and usually be- severe poisoning and methemoglobinemia result- comes equal to the total pigment concentration ing from aniline and aniline derivatives brought (determined spectrophotometrically as cyanmethe- about rapid recovery of the patients. Both moglobin). This important point is illustrated groups of workers stated that the methemoglo- by the data in Table III. (c) Spectrophotometric binemia shown by their patients rapidly subsided TABLE III following injection of the dye. These observa- Increase in oxygen capacity of blood of patients following tions did not receive the attention they deserved, intravenous injection of methylene blue probably because of the emphasis which was placed at that time upon the reverse transforma- Differ- ence be- Methe- tion which accounts for the antidotal action of Blood Total Og ton mobin methylene blue in cyanide poisoning (12). Also, Patient aulfanil- Meoa p,ttlyRtmatatyto- amide Imn aaiypgmnad scopecpcr- Bnri the spectroscopic data of Williams and Challis oxygen method) capacty (7) were not altogether convincing. Neverthe- men. uonea wtinu oluma olum. less, their conclusions were correct, and it is p ct prcad per cent pr cent pr cent H. L... 23 13.4 10.3 3.1 3.2 Before MB necessary to explain how methylene blue may 12.5 12.4 0.1 0.3 1 hour after 13 oc. 1 per cent MB either convert hemoglobin to methemoglobin and 23 12.6 3.7 Before MB thus act as an antidote for cyanide, or hasten the 12.6 0 80 minutes after 10 cc. ______ipercent MB reverse transformation and act as an antidote for L M.... 14 20.5 15.9 5.4 3.6 Before MB methemoglobin-forming substances. 16.6 25 minutes after 15 c. 1 per cent MB Methylene blue forms methemoglobin by cata- W. R. S. 14 15.1 10.4 4.7 4.4 Before MB lytic oxidation ( 10). The dye reacts directly 13.6 0.7 45 minutes after 2 cc 1 per cent MB with hemoglobin to form methemoglobin, and the IL A.... 6 16.5 12.8 3.7 4.8 Before MB reduced (leuco) form of methylene blue formed 165 16.0 0.5 0.5 40 minutes after 10 c. 1 per cent MB by this reaction reacts with oxygen to regenerate H. I 5 11.0 9.0 2.0 2.7 Before MB methylene blue. It is not equally clear how 11.0 10.6 0.4 0.3 75 minutes afte 10 cc. 1 per cent MB methylene blue accomplishes reduction of methe- R. L L. 14 13.0 9.4 3.6 3.8 Before MB moglobin to hemoglobin. Since each molecule of 11.7 11.1 0.6 0.4 60 minutes after 5 cc. 1 per et MB dye injected effects conversion of many molecules of methemoglobin to hemoglobin, this reaction, *Concentration previous day. t MB =methylene blue. too, is catalytic. Here, however, the catalysis is one of reduction, and leuco methylene blue would measurement of the visible absorption spectrum appear to be the effective reductant. Two possi- indicates that oxyhemoglobin and methemoglobin ble sources of the leuco methylene blue in the are the only colored substances present in sig- body are reduction of methylene blue in the er- nificant amounts in the blood of these patients. ythrocytes by enzyme systems present there and Such observations lend no support to the sugges- reduction of methylene blue in other tissues. tion of Ottenberg and Fox (5) that the dark Preliminary experiments suggest that the rate of color of the blood from patients treated with sul- formation of leuco methylene blue in the erythro- fanilamide is owing per se to adsorption by the cytes may not be sufficiently rapid to account for 184 WILLIAM B. WENDEL all the methemoglobin reduced. Thus it would The extent to which methemoglobin accumu- appear that leuco methylene blue formed in the lates is not proportional to the concentration of more actively metabolizing tissues and returned sulfanilamide in the blood. as such to the erythrocytes may play a r6le in Following intravenous injection of small reducing methemoglobin to hemoglobin. Experi- amounts of methylene blue, methemoglobin rap- ments designed to test this possibility are in prog- idly disappears from the blood and is replaced ress. by an equivalent amount of hemoglobin. Owing to the limited time during which methyl- Observations on animals which preceded the ene blue has been used with sulfanilamide, its clinical ones are described. A possible mechanism clinical value as an adjunct to sulfanilamide ther- by which methylene blue accomplishes conversion apy can not as yet be assessed. It seems possible of methemoglobin to hemoglobin is outlined. that the use of sulfanilamide might be less haz- The valuable assistance of Charles Anderson, Jane ardous in many patients, especially those having Erganian, and Nanette Miller Wendel and the coopera- respiratory and cardiac involvements, if the action of Dr. J. B. McElroy, Dr. Gilbert Levy, and the cumulation of methemoglobin were prevented. Staff of the Isolation Division of the John Gaston Hos- Also, the usefulness of sulfanilamide might be pital, Memphis, is acknowledged. extended by giving larger doses, the methemoglobinemia being controlled by methylene blue. BIBLIOGRAPHY In conclusion it should be borne in mind that 1. Paton, J. P. J., and Eaton, J. C., Sulfhemoglobinemia intravenous injection of methylene blue may not and methemoglobinemia following administration be entirely without ill effects. Gregoire (13) has of p-aminobenzenesulfonamide. Lancet, 1937, 1, 1159. reported experiments on rats which suggest that 2. Bensley, E. H., and Ross, J. B., Methemoglobinemia patients kept in oxygen tents should be given due to sulfanilamide therapy. Canad. M. A. J., methylene blue intravenously only with caution. 1937, 37, 62. Nadler, Green, and Rosenbaum (14), on the basis 3. Mull, J. W., and Smith, J. T., Effect of sulfanilamide of electrocardiographic studies, state that methyl- on the oxygen capacity of the blood. J. A. M. A., ene the 1938, 110, 439. blue depresses ventricular musculature 4. Marshall, E. K., Jr., and Walzl, E. M., On the and warn against its indiscriminate use. Also, cyanosis from sulfanilamide. Bull. Johns Hopkins hemolytic anemia has been observed in dogs fol- Hosp., 1937, 61, 140. lowing intravenous injection of this dye (15). 5. Ottenberg, R., and Fox, C. L., Jr., Explanation for These reports of toxic effects, however, were as- the cyanosis of sulfanilamide therapy. Proc. Soc. sociated with much greater amounts of Exper. Biol. and Med., 1938, 38, 479. methylene 6. Chesley, L. C., Cyanosis without sulf- or methemo- blue (20 to 50 mgm. per kilogram of body weight) globinemia in patients receiving sulfanilamide treat- than is required to control sulfanilamide methemo- ment. J. Gin. Invest., 1938, 17, 445. globinemia. Nevertheless, even small amounts of 7. Williams, J. R., and Challis, F. E., Methylene blue the dye should be injected carefully and slowly. as an antidote for aniline dye poisoning. J. Lab. Leakage of the dye around the vein may and Cin. Med., 1933, 19, 166. produce 8. Steele, C. W., and Spink, W. W., Methylene blue a painful infiltration, and too rapid injection may in the treatment of poisonings associated with produce hives and a severe burning sensation of methemoglobinemia. New England J. Med., 1933, the lips. 208, 1152. 9. Hauschild, F., Die Wirkung des Katalysins (Thionin) SUMMARY bei der Methamoglobinvergiftung. Arch. f. exper. Path. u. Pharmakol., 1937, 184, 458. The principal abnormal pigment in the blood 10. Warburg, O., Kubowitz, F., and Christian, W., tYber of patients showing cyanosis from sulfanilamide die katalytische Wirkung von Methylenblau in appears to be methemoglobin. lebenden Zellen. Biochem. Ztschr., 1930, 227, 245. Patients whose functionally active blood pig- 11. Marshall, E. K., Jr., Determination of sulfanilamide ment is decreased 15 to 30 per cent by formation in blood and urine. Proc. Soc. Exper. Biol. and Med., 1937, 36, 422. and accumulation of methemoglobin are not un- 12. Wendel, W. B., Methylene blue, methemoglobin, and common. Higher concerntrations of methemo- cyanide poisoning. J. Pharmacol. and Exper. globin are occasionally encountered. Therap., 1935, 54, 283. METHEMOGLOBINEMIA AND METHYLENE BLUE 185

Chen, K. K., Rose, C. L., and Clowes, G. H. A., Wendel, W. B., and Hefley, M. L., Methylene blue Comparative values of several antidotes in cyanide as an agent for reducing red blood cell count. poisoning. Am. J. M. Sc., 1934, 188, 767. Proc. Soc. Exper. Biol. and Med., 1934, 31, 973. 13. Gregoire, P. E., Action of methylene blue on body temperature and metabolism. J. Exper. Med., 1931, 54, 827. ADDENDUM 14. Nadler, J. E., Green, H., and Rosenbaum, A., In- Since this paper was accepted for publication the travenous injection of methylene blue in man, with method used for methemoglobin determination has been reference to its toxic symptoms and effect on the described (J. Lab. and Clin. Med., 1938, 24, 96). electrocardiogram. Am. J. M. Sc., 1934, 188, 15. Also Hartmann, Perley, and Barnett (J. Clin. Invest., 15. Huyghebaert, E., Action hemolytique du bleu de 1938, 17, 699) have published results of their observations methylene. Arch. Internat. de pharmocodyn. et de on methemoglobinemia resulting from sulfanilamide, and therap., 1924, 29, 405. the effect of methylene blue upon this.