Dee., 1921
T H E JOURNAL O F INDUSTRIAL A N D ENGINEERING CHEMISTRY
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FEEDING EXPERIMENTS
Vitamines from the Standpoint of Structural Chemistry'
Although the foregoing constitutes a definite contribution to the chemistry of the hydroxypyridines, no such decisive By R. R. Williams answer is possible as to their resemblance to vitamine B. ENGINEERING DEPARTMENT. WESTERNEI,ECTRICeo., NEW YORK,N. Y. Harden and Zilval have failed to confirm the writer's findings as to the physiological action of a-hydroxypyridine. Their B-HYDROXYPYRIDINE statements are not altogether convincing that the active Previously published papers2 have recorded the fact that form was actually administered or that the birds were well 8- as well as a-hydroxypyridine exists in two crystalline chosen for experiment. However, it must be conceded that modifications. Dr. H. E. Merwin of the Geophysical the positive evidence of antineuritic action is equally unLaboratory kindly examined a preparation and describes convincing, inasmuch as no prophylaxis has been obtained with the substance. A protective feeding experiment is of the two crystal forms as follows: vastly more value than numerous curative tests and such The preparation contained crystals of two very different habits; evidence must be forthcoming if it is to be beyond dispute, crystals having one habit were lath-shaped with parallel extincCurative tests were made with a variety of substances and tion; y, parallel to the long axis, equals or slightly exceeds 1.74; @, parallel to the intermediate axis, equals 1.600; and CY,paralle€ the results of some of them are recorded below. to the short axis, is slightly greater than 1.575, but much less than 1.60. The crystals, therefore, are orthorhombic, with a small positive optic axial angle. Crystals having the other habit were equidimensional and possessed some well-formed faces, but the optical orientation could not be determined. They are optically positive, with moderate optic axial angle. CY = or is slightly < 1.174, y is slightly c 1.735. A few of the lath-shaped crystals were partly changed into ageegates of small crystals having CY and y like those of the equidimensional crystals. By warming the lath-shaped crystals on a slide somewhat below 100" this transformation in the solid state could be brought to completion within a few moments. Although the optical properties of the two kinds of crystals are so similar, there is no doubt that they are different forms.
@-Hydroxypyridinehas heretofore been regarded as enolic, differing sharply from the a and y isomers. This is not the case, for by titration with bromine3 aIl three prove to be non-enolic in neutral solution, though largely enolic in sodium ethylate solution. The a and y isomers have been shown to form both oxygen and nitrogen ethers14but this was held to be impossible in the case of p-hydroxypyridine. As predicted in a previous paper,6 p-hydroxypyridine forms a nitrogen-methyl ether which has the structure
thus establishing the complete parallelism of the p- with the a- and y-hydroxypyridines. This nitrogen ether of phydroxypyridine is prepared from methyl iodide addition products by shaking in aqueous solution with silver oxide. After filtration from silver iodide the solution is saturated with potassium carbonate. The supernatant oil which separates is dried over anhydrous potassium carbonate and distilled in vacuo (b. p. 300" a t 2 mm.). The viscous oil is miscible with water in all proportions; nonvolatile with steam; reduces permanganate instantaneously and gives a white, crystalline precipitate with mercuric chloride; when decomposed in aqueous solution with sodium amalgam it yields copious amounts of primary amine, as shown by mercuric bromide. I n all respects it resembles the cr and y nitrogen-methyl ethers and not the oxygen ethers. Analysis by Kjeldahl-Gunning-Arnold method gave 12.68 per cent nitrogen (calculated 12.85 per cent).
* The work here recorded was largely done at the Bureau of Chemistry. It was interrupted b y the war and as no early opportunity t o resume it can be foreseen the series of papers is closed with this record of some of the incomplete work which seemed most likely t o be useful. * R. R. Williams, J . Bid. Chem., 26 (1916),437; 29 (1917), 495. * K. H.Meyer, A m . , 880 (19111,212. 4 H.v. Pechmann and 0. Baltzer, Ber., 24 (1891), 3144. 6 R.R. Williams, J . B i d . Chem., 29 (1917),495. e Fischer a n d Renouf, Ber., 17 (1884),703, 1896.
SUBSTANCE
BIRDS DOSE I N
TREATEDMG.
. . . . ....... ..... . . . . .......... . .... ...
Anthranil. , , , Benzo-orthoxazinone.. , , 0-Hydroxyp ridine (mixture of 2 formsJj.. 8-Methylpyridone Picolinic acid methylchloride
5 8
10 5 5
1
2-5
.
2 2 2-5
RESULT Evidence of toxicity N o improvement 6 partly relieved 4 unaffected 4 substantially improved N o effect
Protective feeding experiments have been made with a large variety of synthetic substances, including in addition to those used for curative tests the following: vicine, divicine, 6-0xypyrimidine~trimethyluracil, amino-trimethyluracil, dialuric acid, tetramethyl uric acid, methoxy caffeine, l, 3, 7trimethyl uric acid, isocytosine, and 4-phenylisocytosine. Only trimethyluracil and 4-phenylisocytosine gave any suggestion of protective effect. 4-PHENYLISOCYTOSINE The writer's experience with 4-phenylisocytosine is as follows : Following the directions of Johnson and Hill,* an attempt was made to prepare the four modifications described by them. Preparations were obtained which appeared to agree fairly well with three of the four isomers described by Johnson and Hill, but a t no time was the fourth and most unstable form discovered. Its existence is by no means disputed, as experience with the various forms clearly indicated that apparently trivial modifications of method were sufficient to alter the result. The problem appeared bafEting in the extreme. For example, @ and y forms, having the same melting point and identical crystallographic and optical properties as far as they could be measured, still differed from one another in that the p was approximately ten times as soluble in alcohol as the y form. It was found impossible precisely to correlate crystal form with method of preparation, much less with physiological action. A test was made by daily administration by mouth of 5 mg. of freshly prepared 4-phenylisocytosine to eight pigeons fed on a white rice diet. Four birds received preparation B and four preparation D, corresponding in appearance to Johnson's and Hill's p and 6 forms. The birds were picked a t random from the same group and the experiments were carried out simultaneously and side by side. All four birds given preparation B lost weight less rapidly than any of the four receiving preparation D. The result is precisely what would be expected if preparation B had contained a small amount of an antineuritic so unstable that it passed out of existence in the course of a few days. An experiment with the two identical preparations indicated no physiological difference two months later. Whether this result or any of the physiological resurts so far obtained with synthetic substances has any real significance must be left to the reader's judgment. However,
* Biochem. J.,
11 (1917), 173.
* J . A m . Chem. Soc., 86 (19141,1201.
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T H E JOURNAL OF I N D U S T R I A L A N D ENGINEERING CHEMISTRY
the solubilities, chemical reactions, and natural occurrence of vitamine B, so far as known, agree very closely with the pyrimidine bases, a class of substances known to be capable of a very delicate desmotropism. I n view of these facts any suggestion of physiological activity in synthetic preparations of this group or its allies ought not to be lightly dismissed. The writer believes that vitamine B eventually will be found to be a cyclic nitrogen compound with an oxygen substitution in the ring and capable of existence in a betaine configuration. If the work on synthetics offers any useful suggestions as to manipulation in the isolation or identification ofxthe vitamine from natural sources it will have served an adequate purpose.
Vitamines from the Standpoint of Physical Chemistry By Victor I(.LaMer DEPARTMENT OF CHEMISTRY, COLUMBIA UNIVERSITY, NEWYORK, N. Y.
To cover the subject of vitamines in detail from the standpoint of physical chemistry would require more time than has been allotted and for that reason the discussion will be limited to an outline of the subject, dwelling in detail only upon the more important phases. For the same reason the assumption is made that we have a quantitative method for measuring the vitamines in question, a method in which we know approximately at least the errors with which our measurementsare burdened. I Granted that we have such a method, it then becomes feasible to interpret the data in the light of physical chemistry. This procedure promises results of practical value to the manufacturer of food products, who desires information in regulating his process so that he may obtain the maximum preservation of vitamines consistent with the other requirements of the process, and in addition it furnishes data of theoretical value which should aid us in establishing what the chemical structure of the vitamine may be and may not be. The courses which these investigations have taken may be outlined as follows: I-Studies on solubilities in different media. 2-Studies on the adsorption of vitamines under varying conditions. 3-Studies on the size of the particles which carry the vitamine activity, or more specifically studies on ultrafiltration. 4-Studies on the stability of the material under conditions in which time, temperature, hydrogen-ion concentration, radiant energy, and-oxidizing agents are the independent variables.
For the most part studies made with these objects in vicw have appeared in the form of short, disconnected communications, from which it is difficult to interpret the results obtained because of variations in technique, but more often because some one of the variables just mentioned has not been controlled.
SOLUBILITY STUDIES That the science is urgently in need of more uniform and quantitative measurements becomes apparent when we discuss solubility. The early studies on solubility led to the distinction in the vitamines; namely, A, B, and C. Later and more exacting studies have shown that this is but a rough method of classification and that the solubilities given refer only to the more characteristic solubility. The fat-soluble vitamine A in skimmilk is a case in point, Some workers have tacitly considered that diets containing skim milk were free from vitamine A, since this vitamine is soluble in fat solvents. Experiments by McCollum, and later by Sherman, MacLeod and Kramer,l have shown that this assumption is false and that the amount of vitamine A in skim milk is Proc. SOC.ExptE. Biol. Med., IS (1920),41.
Vol. 13, No. 12
roughly equal to that contained in the fat layer. When this result is interpreted in the light of the distribution law (since equilibrium exists between the phases) it will be seen that the ratio of the solubility of the vitamine in the oily phase and the aqueous is about 30 to 1. It is very likely that further studies would reveal a similar state of affairs for the other vitamines. For instance, Meyers and Voegtlin’ have recently reported water-soluble B to be somewhat soluble in fat oil.
ADSORPTION STUDIES Adsorption studies have been confined almost entirely to the type of experiments of Harden and Zilva12who showed that the B vitamine was adsorbed by fuller’s earth and dialyzed iron, while the C vitamine was not appreciably affected under the conditions of their technic. The authors rightly point out that the completeness of the adsorption is especially sensitive to changes such as H-ion concentration, so that it is necessary in reporting experiments of this type that all conditions be expressly stated. The claim of Ellis, Steenbock and Hart3 that blood charcoal removes a measurable amount of the C vitamine from orange juice shows that the separation of B and C by this means is not quantitative.
STUDIES ON ULTRAFILTRATION The same investigators have studied the effects of ultrafiltration and conclude that there is likewise a partial retention of vitamine C when Chamberland candles are used, It is well to emphasize that the nature of the pores and the character of the filtering material are important factors in such procedures. The recent work on colloids indicates more and more that the process of ultrafiltration, although it may give a crude measure of the size of particles, is far from being a simple case of mechanical separation and that instead the relative chemical nature of the material and the filter bag have much to do with the process.
STUDIESON STABILITY Investigations upon the stability of the vitamines has received considerably more attention than have any of the types of investigation just mentioned, due very largely to the more practical aspects of fiuch studies. Strictly speaking14the use of the term stability should not be applied to vitamines at present because this term refers solely to the effect of energy, usually in the form of heat, upon a substance, while the latter is carefully isolated from all other substances. The effects of the other components of the environment should be referred to as separate chemical properties. It is obvious then that we can speak of the stability of the vitamines only when we are careful to define the conditions under which the experiments are carried out. Two years ago the writer, in conjunction with Professor H. C. Sherman and Miss H. L. Campbell of the Laboratory of Food Chemistry at Columbia, began research upon the stability of the antiscorbutic vitamine. At that time it appeared from the work of Holst and Frohlichls Delf,6 and Hess,’ that the destruction of the antiscorbutic vitamine could be expressed as some function of the temperature, the time of heating, storage, and the acidity. Since then it has become very evident that we must consider still other factors, such as the oxidation and reduction potential of the solvent medium, and perhaps the effects of radiant energy. It is the purpose of this paper to show as far as possible how these factors are related to one another. 1 2
J. B i d . Chem., 42 (1920).199. Biochem. J . , 12 (1918),93.
a J . Biol. Chem., 46 (1921). 367. 4
6 0
Alexander Smith, “Inorganic Chemistry” (new edition), p. 149. Z . H y g . Infectionskrankh., 72 (1912),1. Biochqm. J . , 12 (1920),416. Hess and Unger, J . Btol. Chcm., 48 (1918),297.
