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PUBLIC HEALTH REPORTS
July 27, 1923
STUDIES ON OXIDATION-REDUCTION.
IV. ELECTRODE POTENTIALS OF INDIGO SULPHONATES, EACH IN
EQUILIBRIUM WITH ITS REDUCTION PRODUCT.
By M. X. SULLIVAN, Binchemist, BARNETT COHEN, Chemist, and W. MANSFIELD CLARK, Chief of Division
of Chemistry, Hygienic Laboratory, United States Public Health Service.
From ancient India, through the trade routes of the world, spread the first knowledge of indigo. The distribution of this commodity was not always accompanied by an appreciation of its best use. Pliny (cf. Thorpe and Ingold), for instance, writes of indigo as a pig
But the Indian yers had early discovered that the substance is transformed to soluble indigo-white in an alkaline fermentation Vat;' that indigo-white penetrates cloth; and that it is then fixed as the blue, insoluble dye on exposure to air. Thus the principle of vat dyeing was introduced. At various times and places the vat fermentation was replaced, as it is to-day, by inorganic reducing agents. Consequently, when processes of reduction and of oxidation were systematically formulated in the early part of the last century, the reversible transformation indigo = indigo-white was already in mind to be made familiar as an example of oxidation-reduction. When the biological nature of fermentation was revealed the bacterial formation of indigo-white became one of the first classified instances of biological reduction.
To the reversible system indigo = indigo-white, investigators have turned again and again as an aid in the study of biological oxidation reduction. When Gunning (1878) brought the culmination of his researches to the attention of the French Academy of Sciences and faced Pasteur with the contention that bacteria can not grow without free oxygen, Pasteur (1878) replied by resting his case upon then recent experiments with indigo. He had frequently shown that bacterial growth may occur in culture media exhausted of oxygen, and now he shows that this oxygen exhaustion is sufficient to prevent coloration of indigo-white. His conception of the extent of deoxy
The comparatively simple reduction of indigo itself should not be confused with the complex fermentative preparation of indigo from the glucoside of the indigo plant.
? Where there is no occasion to make a distinction we shall let this system represent any one of those to
genation attained may be judged by his frequent use, in other papers on this subject, of the adjective “absolute.”
A different concept-a concept of degree in oxygen requirementis the basis of Ehrlich's (1885) work in which he made use of differential reduction of dyes. These investigations of Ehrlich revived that research on bacterial reduction which dates at least from Helmholtz (1843), and led to the study of differential reactions between various bacteria and various dyes. Among the dyes so studied, indigo carmine was employed by Spina (1887), Cahen (1887), Müller (1889), Wolff (1902), and others. An interesting study of the conduct of indigo carmine in anaerobic cultures was made by Kitasato and Weyl (1890).
It is generally conceded that the origin of the well-known methylene blue reduction test of the bacterial quality of milk is found in the opening page of Le Lait, where Duclaux (1894) cites the rapid reduction of indigo carmine as evidence of bacterial activity. Vaudin (1897) correlated this test with bacterial numbers. This test is, in principle, identical with the putrescibility test of sewage; and here indigo carmine has again played a part in the development. (H. W. Clark and Adams (1908). Compare Clark and Cohen (1921).)
The conduct of indigo carmine in various tissues has not been very clearly defined. Gautier (1891) observed reduction and considered it evidence of a primary anaerobic phase of metabolism in all living cells. Štolc (1912) claimed that indigo blue remained in the protoplasm of Pelomyra and was eliminated unreduced. Schutzenberger and Risler (1873) claimed that indigo-white, produced by hydrosulphite, takes up more oxygen from blood in vitro than can be extracted by the mercury pump. Lambling (1888) verified this and from spectroscopic studies concluded that indigo-white does not reduce oxyhemoglobin further than to hemoglobin. On the other hand, Shafer (1908) maintained that leuco-indigo carmine may circulate as such in the blood of the living rabbit, may be held unoxidized by tissues, and is oxidized after passing into the lumens of the convoluted tubules. He also states that “there is no direct proof that indigo carmine is reduced in the living animal body.” Indigo carmine was early used by Heidenhain (1874) in a study of kidney secretion. His observation that the cells of the tubules stain blue, while the capsules of Bowman remain colorless, was considered an impressive argument in favor of Bowman's (1842) theory of secretion; but Nussbaum (1878) suggested that the absence of stain in Bowman's capsule is due either to the high dilution of the passing indigo carmine or to its reduction to the colorless form. Shafer (1908) supports Heidenhair in finding no staining of the capsule by indigo carmine; but with Dreser (1885) he believes that indigo carmine is not reduced in the kidney. Höber and Kempner (1908) placed indigo carmine in the lymph sac
of the frog and studied its excretion. They found the excretion to be chiefly by the second segment of the tubules and that the indigo carmine became apparent only after oxidation with hydrogen peroxide. In commenting on the work of Heidenhain, Nussbaum (1878) cites an experiment showing that the liver may take up and reduce indigo. The absorption was made evident only on oxidizing the reduced indigo, a precaution to be remembered wherever indigo is used as a stain. Indigo carmine (indigo disulphonate), which is now a permissible food color (Pugsley, 1922), has recently been employed by Thomas (1914, 1922) and Thomas and Min Hsu (1922) as a kidneyfunction test. Hatiegan (1922) used it in a test for hepatic hyperpermeability.
It goes without saying that a substance so well known and so common as indigo should have been employed both with and without reason. A review in the American Journal of Medical Science, 20, 483 (1837), cites the use of indigo as a therapeutic agent in epilepsy, hysteria, amenorrhea, St. Vitus's dance, and infantile convulsions. It has been used by the chemist as a test for urinary sugar, for oxygen, for chlorine, for nitrates, for tannin, and as an indicator for caustic alkali.
Popular writers on the romance of synthetic indigo have emphasized those achievements of the chemist which have led to the artificial production of a pleasing color. But it should be noted that the researches which led to the final synthesis revealed the nucleus of indigo, indole. This group is now found in an essential nutrient, tryptophane, and in an essential hormone, thyroxin. In the metabolism of tryptophane the indole group persists, appearing as indolethylamine, a stimulant of smooth muscle, as indole, a bacterial product, and as indican, an end product found in mammalian urine. In plants the occurrence of the indigo-yielding glucoside in the genus Indigofera and of free indole in certain odorous flowers is but an indication of the wide distribution of the indole group, a distribution curiously brought to general attention through the discovery that the “royal purple of the ancients,” the product of a snail, is brominated indigo. Wherever found, one method of detecting the indole group is to convert it to indigo, and thus the study of the system indigo = indigo-white is a first, although minor, step toward an understanding of processes of oxidation or reduction by which the indole group in nature is carried through a wide variety of transformations.
In many of the investigations briefly mentioned above, indigo has been used under circumstances or for definite purposes which will hardly bear critical examination. Of these instances we shall later refer to one and shall now mention another, a brief discussion of
which will reveal both the nature and the limitations of our own contribution.
The coloration of indigo-white has been used as a test for free oxygen, and, conversely, the decoloration of indigo blue has been used as an end-point indication of oxygen exhaustion. We have already emphasized, in the first article of this series, the fact that many reversible oxidation-reductions can be accomplished by a variety of reactions and can be formulated by a variety of schemes. We shall slow in this paper that whatever the manner in which a given state of the system indigo sulphonate = leuco compound is established, that state may be expressed in units of universal applicability in essential accord with the formulations of our second paper. The significance may be illustrated by means of an analogy.
Everyone is familiar with the fact that a certain state of colorchange or virage of phenolphthalein indicates only that its aqueous solution has a certain definite hydrion concentration. If now phenolphthalein is to be used as an end-point indicator in the titration of an acid by a base, the acid and base chosen must be such that equivalent quantities mixed in the given concentration furnish a hydrion concentration within the range of color change of phenolphthalein. Otherwise, or if there be present material tending to buffer the solution at or above the zone of hydrion concentration within which phenolphthalein changes color, this indicator will be quite useless for end-point work. The failure to recognize this has led to gross mistakes both in theory and in practice. The first and fundamental task in the development of a rational theory of acidimetric titration was the establishment of the acid-base equilibrium-constant or at least the pH zone of color change of each indicator.
The reader will at once perceive the analogy. Indigo, according to the circumstances of its use, may or may not be valuable as an end-point indicator of free oxygen. Most certainly it has sometimes been employed in solutions poised 3 by material active between the true end point of oxygen removal and the potential at which indigo is reduced; but for the present we need not discuss this. We are now concerned in establishing universally applicable data of the oxidation-reduction intensities of different states of the systems. This is a first and fundamental step. Without this information it is easy to misjudge results; and were it worth while, instances of mistakes could be cited from the chemical and medical literature. The establishment of the reduction potentials of each system should make possible the use of that system in the colorimetric estimation of reduction intensity. The possible applications of this fundamental knowledge are numerous.
3 See the first article of this series.