STUDIES ON BLOOD CELL METABOLISM.

III. THE EFFECT OF METHYLENE BLUE ON THE OXYGEN CON- SUMPTION OF THE EGGS OF THE SEA URCHIN AND

STARFISH. THE MECHANISM OF THE ACTION OF METHYLENE BLUE ON LIVING CELLS.

BY E. S. GUZMAN BARRON.

(From the Laboratory of Research Medicine, Medical Clinic, the Johns Hopkins University, Baltimore, and the Marine Biological

Laboratory, Woods Hole.)

(Received for publication, December 4, 1928.)

The use of methylene blue in the study of biological oxidations is based on the fundamental researches of Ehrlich (1). The dye seems, as Clark remarks “to have fallen into almost every con- ceivable use, ranging from an indicator in volumetric analysis to a therapeutic agent” (2). Because of the diversity of its applica- tions, the action of methylene blue as a catalyst of biological oxidations has attracted great interest and the mechanism of these reactions, considerable discussion.

It is of interest at this point to examine briefly the changes in metabolism induced by addition of dyes to red blood cells. It was reported by Harrop and Barron (3) that methylene blue added to red blood cells causes an increased respiration, as shown not only by an augmentation of oxygen consumption but also by an in- creased CO2 production. It was shown that the respiratory quo- tient of these cells after the addition of the dye is in the neighbor- hood of 0.76, indicating that the oxidation is complicated, affect- ing not only the carbohydrates, but the fats and proteins as well. In an effort to identify the nature of this action, the carbohydrate metabolism of red blood cells was studied by the same authors (4) with the result that methylene blue was shown to produce an indubitable increase in the carbohydrate oxidation. This was evidenced by a fall of the glycolytic quotient

Millimols lactic acid produced

2 X millimols glucose degraded > 445

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446 Studies on Blood Cell Metabolism. III

Further, it was shown that the oxidative action was not really peculiar to methylene blue but common to the series of reversible dyes possessing definite oxidation-reduction potentials with rH values in the vicinity of that of methylene blue.

Similar experiments with leucocytes, soon to be published, led to the belief that the catalytic action of methylene blue on respira- tion is not confined to blood corpuscles but is common to all living cells. To test this hypothesis a series of experiments has now been performed to determine the effect of this dye on the respiration of sea urchin and starfish eggs. Oxygen consumption alone was measured.

The experiments were performed in Warburg’s micro respira- tion vessels with Barcroft’s manometer, 0.2 cc. of M KOH being used to absorb the COZ produced. The temperature of the bath was kept at 25” f0.03”. The eggs which were collected in the middle of the summer of 1928, were received in flat bottomed dishes, and washed twice with sea water before use. The methyl- ene blue used was the same sample referred to in previous com- munications (purified methylene blue, zinc-free, from Leopold Cassella and Company, Frankfort-on-the-Main) and the solutions were made fresh every day in sea water. The concentration in these experiments, as indicated below, varied from 0.001 to 0.005 per cent.

I. Action of Methylene Blue on Oxygen Consumption of Sea Urchin and Starfish Eggs.

Warburg’s first communication (5) pointed out that unfertilized sea urchin eggs have a definite oxygen consumption which is aug- mented 8- or lo-fold on fertilization. On the other hand, the oxygen consumption of starfish eggs does not rise appreciably after fertilization.

In experiments described in this paper the rate of oxygen con- sumption was first determined for a period of approximately 1 hour; the dye was then added and the oxygen consumption again determined. About 5 minutes only were required to reach tem- perature equilibrium after addition of the dye as the room tem- perature was approximately equal to the thermostat temperature.

As can be seen from Fig. 1, which is an example of a series of similar experiments, the addition of methylene blue to these cells

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E. S. G. Barron 447

definitely increases oxygen consumption. In all the experiments the increase was greater with starfish eggs than with sea urchin eggs. In general the observed increase in the case of sea urchin eggs was from 80 to 100 per cent of the normal rate, while with star- fish eggs it was from 200 to 250 per cent. As was previously found for blood cells, the optimum concentration of methylene blue seems to be about 0.005 per cent. When the concentration is lowered to 0.001 per cent, the increase is considerably dimin-

FIG. 1. The effect of methylene blue on the oxygen consumption of star- fish and sea urchin eggs. Curve 1, oxygen consumption of starfish eggs; Curve 2, oxygen consumption of sea urchin eggs.

ished. As may be seen in Table I, there is a rough proportionality between the dye concentration and the increase of oxygen con- sumption, within the limits of dye concentration employed.

It is noteworthy that the action of methylene blue is not im- mediate. There is a more or less marked latent period which sug- gests the hypothesis that penetration of the dye through the cell membrane is necessary before its action can take place.’

1 The fact maintained by Irwin (6) that chemically pure methylene blue does not penetrate the cell membrane does not interfere with this statement as not even the best preparation of methylene blue available is free from those supposedly less methylized dyes which do penetrate the cell wall.

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448 Studies on Blood Cell Metabolism. III

II. Effect of Cyanides.

The mechanism of the inhibitory action of cyanides in cellular respiration is now well understood. We owe this knowledge to the splendid contributions of Warburg (7). KCN stops the

TABLE I.

Influence of Methylene Blue Concentration. Oxygen Consumption of Unfertilized Sea Urchin Eggs (Arbacia pun&data).

02 consumption per 60 min.

Date. Before addition of After addition of methylene blue. metbylene blue.

c. mm. c. mm.

July 24 11.0 24.2 “ 25 18.6 30.0 “ 26 15.0 20.0

TABLE II.

Concentration of methylene blue.

Increase of Or COnSUlllptiOn

after addition of methylene blue.

per cent per cent

0.005 120 0.002 61 0.001 33

Action of Methylene Blue on the Oxygen Consumption of StarJsh and Sea Urchin Eggs in Presence of 0.01 Y KCN.

I 02 consumption per 60 min.

Time, 60 min. BefoRggdtion of Afterg.vd&tion of After addition of

methylene blue (0.005 per cent).

Starfish eggs. I

II III

c. mm.

38.5 41.0 40.0

Sea urchin eggs. I

II III

14.4 13.3 10.0

C. mm.

0 0 0

0 0 0

c. mm.

20.0 24.2 23.1

14.0 13.2 10.4

respiratory process by forming a complex compound with the iron atom of the respiratory ferment which, according to Warburg’s latest contributions (8), is a hemin compound in which iron is the reactive nucleus of hemin. This inhibitory mechanism can be called a specijk action as it is produced by direct action on the

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E. S. G. Barron 449

catalyst. Warburg correctly designates KCN a “specific nega- tive catalyst” (9).

Great interest has been stimulated by the striking observation that methylene blue produces biological oxidations even in pres- ence of cyanides and it has given a strong argument to the parti- sans of Wieland’s dehydrogenation theory (10) in their criticism of Warburg’s theory. It will be recalled that Thunberg (11) was the first to show that succinic acid in absence of molecular oxygen can be oxidized to fumaric acid by muscle enzymes in presence of methylene blue and that the addition of cyanides has no influence

260

E

FIQ. 2. The effect of methyIene blue on the oxygen consumption of star- fish and sea urchin eggs after addition of KCN (0.01 M). Curve 1, oxygen consumption of starfish eggs; Curve 2, oxygen consumption of sea urchin eggs.

on this reaction. Further Barron and Harrop (4) were able to show that the action of methylene blue on red blood cells is not altered by the addition of KCN and demonstrated that oxidation of carbohydrates produced by methylene blue goes on in the pres- ence of KCN.

In the present experimental series, after determination of the normal rate of oxygen consumption of sea urchin and starfish eggs, KCN was added in sufficient quantity (0.01 M) to produce a com- plete stoppage of oxygen consumption. Half an hour or 1 hour later methylene blue was added to the cells from the side arm of the vessel and the oxygen consumption again determined for 1

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450 Studies on Blood Cell Metabolism. III

hour or more. As can be seen in Table II and Fig. 2, oxygen con- sumption was restored after the addition of the dye. In the case of sea urchin eggs the oxygen consumption was practically re- stored to its normal level; this original level however was never reached in the case of starfish eggs, in which the value reached after addition of the dye was generally 50 per cent below the nor- mal figure. The amount of cyanide added is without influence upon the subsequent restorative power of methylene blue, so long as the cyanide concentration is strong enough to inhibit completely the normal oxygen consumption of the cells. The same observa- tion is reported by Fleisch (12) for the oxidation of succinic acid by muscle tissues in the presence of methylene blue and cyanides. In view of the results it is interesting to note that, as shown by Perlz- weig and Barron (13), sea urchin eggs have a high anaerobic metab- olism demonstrated by the increased lactic acid production of the eggs after addition of KCN.

III. Effect of Narcotics.

That vital oxidations take place only when forces due to the particular structure of the cell, usually referred to as surface forces, are added to chemical forces is very well known. Impair- ment of any of these forces brings inhibition of vital oxidations. The restoration of cellular oxygen consumption by the addition of methylene blue after the normal specific chemical force (War- burg’s respiratory ferment?) has been inhibited by cyanides has just been described. It was thought interesting to study the effect of methylene blue when the other factor, i.e. the general sur- face force, was abolished or its action lowered.

There are two means of destroying the general surface activity: (1) by mechanical means (cytolysis), (2) by addition of strongly adsorbable substances (narcotics). Harrop and Barron have shown that the effect of methylene blue is greatly altered when the cell surface of avian erythrocytes is destroyed by repeated freezing and thawing, as in Warburg’s experiments (14). Thus addition of methylene blue to the bottom layer of hemolyzed goose red blood cells containing cell fragments and nuclei produces an in- crease of oxygen consumption of 21.6 per cent, while the increase is only 11.8 per cent on the top layer containing only a small amount of cellular debris.

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E. S. G. Barron 451

It was therefore decided to use the second method of lowering the surface force in the experiments with marine egg cells. Meyer- hof (15), working with sea urchin eggs (Strongybcentrotus), demon- strated at Naples that urethanes lower the rate of oxygen con- sumption when added to these cells, the inhibiting power being more effective the higher the member of a homologous series. Ethylurethane at different concentrations (3, 6, 8 per cent) and phenylurethane (saturated solutions) were employed as narcotics

TABLE III.

Efect of Narcotics. Action of Methylene Blue (0.006 Per Cent) on Oxygen Consumption of Sea Urchin and Starfish Eggs in Presence of

Narcotics (Ethylurethane and Phenylurethane).

Starfish eggs. I

II III IV V

VI

Sea urchin eggs. I

II

:. mm.

25.4 27.0 27.8 20.7 20.5 16.9

Ethylurethane, 4 per cent. “ 4 “ “ “ 8 “ “ “ (j “ “ I‘ 8 “ “

Phenylurethane, concentrated.

13.7 Ethylurethane, 8 per cent. 18.0 “ 3 “ ‘(

02 consumption per 60 min.

Narcotics wad. I

1

-

A ftar addi- tion of

nar- cotics.

:. mm.

14.7 17.9 12.2 12.5 10.7 5.6

9.1 14.8

-

t

I

_-

E

-

:. mm.

21.8 20.1 17.4 16.8 15.75 12.0

7.3 10.1

in the present experiments. The experiments were conducted in two ways: In the first series, after determining the normal rate of oxygen consumption of the cells (sea urchin and starfish eggs) the narcotics were added, and the oxygen consumption followed for approximately 1 hour. Methylene blue was finally poured into the main vessel from the side arm and the oxygen consump- tion determined. In another series of experiments, methylene blue was first added and then the narcotics.

After addition of urethanes, the oxygen consumption was

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452 Studies on Blood Cell Metabolism. III

lowered in proportion to the concentration of narcotic: the inhibi- tion being more marked, as was expected, with phenylurethane than with ethylurethane. As can be seen in Table III, phenyl- urethane produced 67 per cent inhibition; ethylurethane however, even in such high concentrations as 8 per cent, produced only from 56 to 47 per cent inhibition. In the case of starfish eggs,

FIQ. 3. The effect of narcotics on the oxygen consumption of starfish eggs. Showing the inhibitory effect of narcotics (phenylurethane) on the increased oxygen consumption previously produced by addition of methylene blue.

addition of methylene blue after narcotics increased somewhat (12 to 48 per cent) the oxygen consumption, but this never reached the level of normal eggs. In the case of sea urchin eggs, addition of methylene blue after addition of narcotics produced an even lower oxygen consumption than after narcotic inhibition. A striking result is obtained when oxygen consumption is first in- creased by addition of methylene blue and then the narcotic added

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E. S. G. Barron 453

(Fig. 3). In such an experiment the effect of methylene blue is complet,ely nullified and the cells respire at their normal rate.

Methylene blue, therefore, can be said to exert its catalyt,ic effect as do the general enzymes. It acts at surface inter-

j(‘)

I

Fro. 4. The effect of methylene blue on the oxygen consumption of living cells. Curve 1, mammalian red blood cells; Curve 2, avian (goose) red blood cells; Curve 3, leucocytes (fresh lymph from the thoracic duct uf dog); Curve 4, sea urchin eggs; Curve 5, starfish eggs. The solid line, before addition of methylene blue; the dash line, after addition of methylene blue.

faces, and impairment or destruction of these interfaces lowers or abolishes the activity of the dye.

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454 Studies on Blood Cell Metabolism. III

DISCUSSION.

Some years ago, Meyerhof (16) stated, as a result of his experi- ments on staphylococci, that though the respiration of living microorganisms is inhibited 10 to 40 per cent by methylene blue, the respiration of the same organisms, damaged or destroyed by acetone and heat, rises in the presence of methylene blue. He ad- vanced the hypothesis that the dye may be able to replace a nor- mal factor in an oxidizing mechanism when this factor has been inhibited or destroyed. The experiments reported elsewhere (3,4) and those presented here do not confirm Meyerhof’s hypoth- esis since the increased oxygen consumption produced by methyl- ene blue has been observed on living cells, showing definite oxygen consumption previous to the addition of the dye.

Explanation of the mechanism of the oxidative action of methyl- ene blue on living cells can best be made by recalling the essential facts to be drawn from the experiments of Harrop and Barron and those reported in this paper.

It has been established by experiments on cells with high anaero- bic metabolism (mammalian red blood cells) and cells with high aerobic metabolism (avian red blood cells) that the action of methylene blue is stronger the higher the level of the anaerobic metabolism of the cell. This can be clearly seen in Fig. 4 which shows graphically both the normal oxygen consumption and t.he methylene blue effect on (1) mammalian red blood cells, (2) avian red blood cells, (3) fresh leucocytes from the lymph of the dog’s thoracic duct, (4) sea urchin and (5) starfish egg cells. Further- more, this action is not disturbed by total cyanide inhibition of the aerobic metabolism. In addition to this it has been demonstrated (Barron and Harrop) that methylene blue effects its oxidative action on the carbohydrates of blood, only when these substances have been activated; i.e., transformed into more easily oxidizable compounds by the glycolytic ferment. Thus inhibition of gly- colysh in red blood cells either by destruction of the glycolytic enzyme (action of temperature) or by destruction of the cell sur- face (hemolysis in red blood cells possessing low glycolytic power) always stops the oxidative action of methylene blue. This rela- tionship appears more striking when the dye is added to chicken blood in which there is no glycolysis and the material incubated at 37’ for some hours. The action of methylene blue here is nil, as

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E. S. G. Barron 455

the concentration of sugar and lactic acid remain unchanged. It can be concluded therefore that inhibition of the respiratory fer- ment by cyanide affects in no way the oxidative power of methyl- ene blue, but that inhibition of the fermenting enzyme (glycolysis) prevents the dye action.

The increased oxygen consumption and the correlated increase of carbohydrate oxidation produced by methylene blue are in accordance with the well established fact that cells during respira- tion oxidize chiefly carbohydrates; methylene blue acts only dur- ing the first process of carbohydrate metabolism, i.e. during the anaerobic phase, its action starting when the carbohydrates have been “activated,” or broken down into easily oxidizable compounds by the fermenting enzymes. As a reversible dye and a strong

Fro. 5.

hydrogen acceptor, methylene blue accepts 1 H molecule from these compounds to be reduced to methylene white, which in turn is oxidized by the atmospheric oxygen. Thus there is established a cycle by which small amounts of the dye can carry on the oxida- tive process. This process can be expressed diagrammatically in the accompanying scheme (Fig. 5) and the action of methylene blue on living cells can be classified as a type of oxidative dehydro- genation, following in its mechanism the same principles as those expressed by Thunberg for the oxidation of succinic acid by muscle tissues in the presence of methylene blue, and based on Wieland’s oxidation theory.

So much evidence has been accumulated during the last few years in support of Warburg’s theory of the cellular respiratory mechanism that it can safely be .assumed that iron-containing

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456 Studies on Blood Cell Metabolism. III

compounds play the Ale of catalysts, even though it is not yet entirely clear whether iron activates the compounds to be oxidized or whether it activates molecular oxygen. From the experiments described above it is clear that oxidative dehydrogenations pro- duced on living cells by addition of methylene blue can take their place along with thenormalrespiratoryprocessesof the cells. Even though it is accepted that there is no relation between methylene blue oxidation and the normal respiratory process, there is a strik- ing relationship between the fermenting enzymes and methylene blue action as the latter does not take place when the action of the enzyme is inhibited. It is possible, but not certain, that some of the oxidative processes of living cells are normally carried out by a mechanism equivalent to the methylene blue mechanism. Har- vey’s important contributions to the mechanism of biolumines- cence (17) seem to prove that such oxidations can exist. He con- siders that the oxidation of luciferin to h&erase belongs to the type of oxidative dehydrogenations.2

CONCLUSIONS.

1. The addition of methylene blue to living cells (sea urchin, starfish eggs) produces an increased oxygen consumption.

2. Cyanides added in sufficient concentration to produce com- plete inhibition of cellular respiration do not affect the oxygen consumption produced by methylene blue.

3. The addition of narcotics inhibits the action of methylene blue.

4. The increase of the oxygen consumption produced by methyl- ene blue in a living cell is proportional to the level of its anaerobic metabolism. While marked in cells possessing high fermenting power (mammalian red blood cells), it is less marked or even nil in cells with low anaerobic metabolism (avian red blood cells, leu- cocytes) .

5. The oxidative action of methylene blue on living cells be- longs to the type of oxidative dehydrogenations; the dye plays

2 Quastel’s observations (18), Fleisch’s experiments (12), and Stephenson’s latest contribution (19) along the line of Thunberg’s fundamental re- searches, also bring strong evidence to support the likelihood of normal oxidative dehydrogenations in the living cell.

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E. S. G. Barron 457

its catalytic r6le on account of its reversibility and spontaneous oxidability by molecular oxygen without a catalyst.

BIBLIOGRAPHY.

1. Ehrlich, P., Centr. med. Wissensch., 23, 113 (1885). 2. Clark, W. M., Cohen, B., and Gibbs, H. D., Pub. Health Rep., U. S. P.

H. S., 40,113l (1925). 3. Harrop, G. A., Jr., and Barron, E. S. G., J. Exp. Med., 46, 207 (1928). 4. Barron, E. 5. G., and Harrop, G. A., Jr., J. Biol. Chem., 79,65 (1928). 6. Warburg, O., 2. physio’. Chem., 66, 305 (1910). 6. Irwin, M., J. Gen. Physiol., 12,147 (1928). 7. Warburg, O., 2. physiol. Chem., 76,331 (1912); 92,231 (1914). 8. Warburg, O., Natuwissenschaften, 16, 345 (1918). 9. Warburg, O., Science, 61, 575 (1925).

10. Wieland, H., Ergebn. Physiol., 20, 477 (1922). 11. Thunberg, T., Skand. Arch. Physiol., 33,233 (1916); Zentr. Physiol., 31,

91 (1917). 12. Fleisch, A., Biochem. J., 16,294 (1924). 13. Perlzweig, W. A., and Barron, E. S. G., J. BioZ. Chem., 79, 19 (1928). 14. Warburg, O., 2. physio2. Chem., 70, 413 (1911); Ergebn. Physiol., 14,

253 (1914). 15. Meyerhof, O., Arch. Physiol., 167, 2.51 (1914). ges. 18. Meyerhof, O., Arch. Physiol., 169, 87 (1917); 170, 367 (1918). ges. 17. Harvey, N. E., Bull. Nat. Research Council, No. 59, 50 (1928). 18. Quastel, J. H., Biochem. J., 20, 166 (1926). 19. Stephenson, M., Biochem. J., 22,605 (1928).

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E. S. Guzman BarronMETHYLENE BLUE ON LIVING CELLS

MECHANISM OF THE ACTION OF SEA URCHIN AND STARFISH. THE

CONSUMPTION OF THE EGGS OF THE METHYLENE BLUE ON THE OXYGEN

METABOLISM: III. THE EFFECT OF STUDIES ON BLOOD CELL

1929, 81:445-457.J. Biol. Chem. 

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