y-amino-acids, phy- droxy-acids and a-ketonic

THE INTERCONVERSION OF (Y-AMINO-ACIDS, PHY- DROXY-ACIDS AND a-KETONIC ALDEHYDES.

PART II.

BY H. D. DAKIN AND H. W. DUDLEY.

(From the Herter Laboratory, New York.)

(Received for publication, May 26, 1913.)

1. Introduction. COKTENTS.

2. The formation of methyl glyoxal from lactic acid. 3. The formation of methyl glyoxal and ammonia from alanine. 4. The formation of methyl glyoxal from glucose. 5. The formation of other a-ketonic aldehydes from a-hydroxy acids and

a-amino-acids. 6. The fate of methyl glyoxal and phenyl glyoxal on perfusion through the

liver. Formation of phenyl glyoxylic acid. i. The fate of methyl glyoxal and of Z-lactic acid in the glycosuric or-

ganism.

1. Introduction.

The object of the following paper is to present the detailed experiments upon which we have based a hypothesis concerning the intermediary metabolism of amino- and hydroxy-acids, and in particular the mechanism concerning the mutual interconversion of alanine, lactic acid and glucose.1 For the sake of clearness, R-e may reproduce the essential features of the types of reactions which we believe to be operative in the changes concerned.

By making use of a substance capable of forming extremely insoluble derivatives with a-ketonic aldehydes, namely, para- nitrophenylhydrazine, we have been able to show, by experiments in vitro, that amino-acids and hydroxy-acids, such as alanine and lactic acid, readily undergo decomposition in faintly acid solution in conformity with the following equations:

CH~.CHOH.COOH--+ CHI.CO.CHO + Hz0 CHS.CHNH~.COOH+ CHB.CO.CHO + NH?

i This JowmzZ, xiv, p. 555, 1913.

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128 Metabolism of Carbohydrates and Proteins

Amino-acids have been commonly regarded as extremely stable substances, at least in vitro, but’ our observations tend to show that under suitable conditions, in aqueous solution, when due provision is made for the prompt removal of the products of their decomposition, both the a-amino-acids and oc-hydroxy-acids are in a state of unstable equilibriume2 Furthermore, we have been able to show that the decomposition of amino-acids with formation of ketonic aldehydes is not due to a complicated reaction dependent upon the presence of the nitrophenylhydrazine, for it has been possible to demonstrate ammonia formation from amino-acids under similar conditions, but in the absence of the hydrazine. t

The production of oc-ketonic aldehydes from or-amino- and cu-hydroxy-acids is of biochemical significance, partly on account of the existence of enzymes which we have named “glyoxalases” capable of converting the former substances into hydroxy-acids3

R.CO.CHO + H,O = R.CHOH.COOH

Moreover, we have been able to gather a considerable amount of indirect, evidence indicating that a!-ketonic aldehydes may play a part in ihtermediary metabolism. Thus we ‘find that methyl glyoxal yields glucose in the glycosuric organism just as do alanine and lactic acid, from which methyl glyoxal may be derived in vitro (section 7).

In addition, it is possible to demonstrate, in vitro, the reverse change, ‘namely, the conversion of glucose into methyl glyoxal under conditions which, apart from temperature, are comparable with those existing in the animal body (section 4).

From the foregoing evidence and other that has been referred to in our previous paper, it appeared justifiable to construct a scheme which may crudely represent the interconversion of alanine, lactic acid, methyl glyoxal and glucose by a series of reversible reactions involving the addition or subtraction of water or ammonia. Of the various reactions indicated, the direct formation of alanine from methyl glyoxal is the only one that thus far has

2 It is of interest to note that the fl-amino-acids, such as p-alanine and fi-phenylalanine, which do not occur in nature, do not yield ketonic aldehydes, at any rate under the above conditions.

3 This Journal, xiv, p. 423, 1913.


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H. D. Dakin and H. W. Dudley 129

not been demonstrated although the analogous synthesis of glycine from glyoxal has been effected.

Glucose GH12Os

1-r Lactic acid Methyl glyoxal Alanine

CHz. CHOH . COOH ~2 CH3. CO. CHO @ CH3. CHNH2. COOH

The lactic acid which we have obtained by the action of glyox- alase upon methyl glyoxal is a mixture of the two forms in which the laevo acid is in excess. The production of glucose from d-lactic acid and from the inactive acid is well established through Mandel and Lusk’s experiments, but it appeared very desirable to determine whether the pure laevo acid might also yield glucose. Accordingly, we have prepared pure l-lactic acid from morphine Z-lactate and find that it also yields glucose freely in the glycosuric animal.

This result appears to us to be of considerable significance, for the almost quantitative conversion of both d- and Z-lactic acids, substances possessing asymmetric carbon atoms enantio- morphously related, into the same d-glucose apparently necessitates a loss of asymmetry in the lactic acid molecule in the process of glucose synthesis. The intermediate formation of methyl- glyoxal, such as we have suggested, would furnish a satisfactory explanation of such a change, and in addition, the conversion of methyl glyoxal into glucose, possibly with intermediate formation of glyceric aldehyde, would give an opportunity for the introduction of new asymmetric groups.

CHzOH

I CH3 CH3 C& CHzOH HCOH

I I HOCH HCOH + CO -+ HCOH -+ HCOH

I I I I I COOH COOH CHO CHO HOCH

I

d-and I-Lartic acid H&OH

Finally, reference may be made to the relation of the cy-ketonic acids to amino-acids and a-ketonic aldehydes. Neubauer and


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Knoop have clearly demonstrated the interconversion of amino- and ketonic acids. A clue to the mechanism of this reaction may be furnished by our observations (section 6) on the formation of phenyl glyoxylic, acid as well as I-mandelic acid on perfusing a liver with blood containing phenyl glyoxal.

C&H&. CO. CHO

/ I( / L \

C6H5. CO. COOH ct C,~HS. CHOH COOH

It is possible that the phenyl glyoxylic acid originates as a secondary product of the oxidation of mandelic acid rather than by the direct oxidation of phenyl glyoxal.4 But in any case, the result is of interest since it serves to bring the cr-ketonic aldehydes in close biochemical relation with the amino- as well as the hydroxy-acids.

In the experimental part of this paper it will be shown that the nitrophenylhydrazones of glyoxylic and pyruvic acids may be obtained by the direct action of nitrophenylhydrazine upon glycollic and lactic acids. Hydrazino-acids appear to be first formed and are readily oxidized to the ketonic acid derivatives in the presence of air. Although ordinary hydrazines are not known to occur in the animal body many substances such as arginine and creatine contain the -NH.NH, group and it is conceivable that substances of this type may be concerned in the biochemical oxidation of hydroxy to ketonic acids.

2. The formation of methyl glyoxal from lactic acid.

It is an extremely easy matter to demonstrate the formation of methyl glyoxal from lactic acid by simply allowing a filtered 5 or 10 per cent aqueous solution of lactic acid (500 cc.) containing a little nitrophenylhydrazine (l-2 grams) to stand at room temperature or in the incubator. After two or three hours, a flocculent red precipitate begins to appear, which in the earlier

4 It is of interest to note that Evans (Amer. Chem. JOUTTL., xxxv, p. 128, 1906) oxidized phenyl glyoxal to phenyl glyoxylic acid by means of alkaline ‘permanganate. In the absence of alkali benzoic acid is obtained.


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H. D. Dakin and H. W. Dudley

stages of the reaction, is composed of almost pure methyl glyoxal dinitrophenylhydrazone.

The precipitate gradually increases in amount during the SUC-

ceeding three or four days, when it will be found that all the nitrophenylhydrazine has disappeared, although the amount of precipitate does not account for nearly all of the base added.5 When this stage has been reached, it is well to filter off the precipitate on a small funnel and to dissolve an additional quantity of nitrophenylhydrazine (l-2 grams) in the filtrate by warming and subsequently cooling and filtering from any trace of insoluble matter. The clear filtrate, on standing, soon begins to deposit more methyl glyoxal dinitrophenylhydrazone and the process may be repeated as often as desired. After a time it will be noticed, however, that the character of the precipitate begins to change and that a yellowish-brown crystalline substance begins to deposit in addition to the amorphous methyl glyoxal derivative. The crystalline deposit is a mixture of nitrophenylhydrazinopropionic acid and the nitrophenylhydrazone of pyruvic acid, the latter being formed from the former by oxidation. The changes may be represented as follows:

CH3

CO

7 I CH3 / CHO

I / hIethyl glyoxal CHOH I

\ bOOH \\ CH3

----+ C:S.NH.C6H4N0z

I CH : N NH . C6H~N0z Methyl glyoxal dinitrophenylhydrazonc

CH3

Lactic h I I

acid CH NH. NH. CeH4N02 --+ C : N NH. C6H4N0z

I I COOH COOH

cu-Nitrophenylhydrazino- Pyruvic acid propionic acid nitrophenylhydrazone

The separation of the constituents of the red precipitate may be conveniently carried out as follows:

The mixture is first of all washed with hot 10 per cent sodium

6 Unchanged nitrophenylhydrazine is conveniently tested for by adding a drop of acetone or benzaldehyde to a few cubic centimeters of the solution.


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carbonate solution which dissolves the nitrophenylhydrazinopropionic acid and the pyruvic acid nitrophenylhydrazone. The residue is then washed with warm alcohol and finally dissolved in a small amount of boiling nitrobenzene, filtered hot and toluene added to the filtrate. The pure methyl glyoxal dinitrophenylhydrazone separates out almost immediately in the form of glistening crimson needles which are filtered off, washed with toluene and dried at 140”. The substance was ident,ical in every way with the hydrazone prepared directly from methyl glyoxal and melted with decomposition at about 302”304” after darken- ing slightly above 290”. On warming the merest trace of the substance with caustic soda solution, best with the addition of a few drops of alcohol, there develops a magnificent deep blue color slowly changing to purple, then violet and finally a dull brown- red. The reaction is extremely sensitive.

ANALYSIS : 0.1218 gram dried at 150” gave 0.0299 gram N = 24.6 per cent N. C&H,,O.,NF, requires 24.6 per cent N.

For purposes of comparison, the nitrophenylhydrazone of methyl glyoxal was prepared directly from methyl glyoxal obtained by the hydrolysis of its acetal (Meisenheimer). A slight, excess of the hydrazine (2.2 mols.) dissolved in 30 per cent acetic acid was added to methyl glyoxal (1 mol.). The hydrazone is at once precipitated in practically theoretical amount and may be washed with alcohol and then recrystallized as above from nitrobenzene and toluene. The substance is very sparingly soluble in almost all solvents with the exception of nitrobenzene and bases such as pyridine. It should be noted that nitrobenzene and especially pyridine, com- bine with the substance very tenaciously and are only given off in vacua at 100” with extreme slowness. Heating at 140”-150” is much more efficient in driving off the solvents. Pyridine seems to be a somewhat more objectionable solvent than nitrobenzene on account of the ease with which it unites with the hydrazone forming dark red-brown solutions. The pure substance melts at 302” -304”, the exact temperature varying slightly with the speed of heating. On analysis the substance was found to contain 25.1 per cent nitrogen (theory = .24.6). The same substance has been described by Neuberg,6 who obtained it from a-aminopropionic

6 Ber. d. deutsch. them. Gesellsch., xli, p. 956, 1908.


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H. D. Dakin and H. W. Dudley I33

aldehyde. The melting point is given as 277” with discoloration at 255”. We believe this melting point to be much too low. The reaction with caustic soda was not described.

The cl-nitrophenylhydrazinopropionic acid and pyruvic acid nitrophenylhydrazone contained in the sodium carbonate washings from the original precipitate were recovered on acidifying with acetic acid. On repeated recrystallization from boiling water, the first substance is slowly oxidized to the pyruvic acid derivative, so that probably the former was not obtained perfectly pure. It is fairly soluble in alcohol, melts above 250” and gives an intense red coloration with caustic soda.

ANALYSIS: 0.0919 gram substance gave 0.0169 grain N = 18.3 per cent N. C&HllOaN3 requires 18.6 per cent N.

By repeated crystallization from water of the mixture of the hydrazino acid and the pyruvic acid nitrophenylhydrazone, sev- eral grams of the latter were readily obtained as a bright yellow crystalline substance melting at 223”-225”. It is also formed by oxidizing nitrophenylhydrazinopropionic acid with an ammon- iacal solution of a cupric salt. It is moderately soluble in alcohol and gives a bright red color on addition of caustic soda.

ANALYSIS: 0.1225 gram gave 0.0228 gram N = 18.6 per cent N. C9H904N3 requires 18.8 per cent N.

The substance obtained from lactic acid was identical with the product obtained from pyruvic acid as described by Hyde’ and also prepared for comparison by us. The melting point given by Hyde is 219”-220”.

A number of experiments were made in which additions of other substances were made to the lactic acid mixture in the hope of accelerating its decomposition into methyl glyoxal and water. The following were tried: spongy platinum, aluminum oxide, chrom- ium oxide, mercuric iodide, uranium acetate, glycine, sulphuric acid and calcium lactate. None of them proved effective.

It should be noted that while the yield of crystalline substances from the lactic acid is small, the greater part of the acid remains unchanged and may be treated over and over again with fresh nitrophenylhydrazine.

7 Berichte, xxxii, p. 1815.


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3. The formation of methyl glyoxal and ammonia from alanine.

The formation of methyl glyoxal from alanine is readily demonstrated by allowing an aqueous solution of the amino-acid with a little nitrophenylhydrazine and a few drops of an acid to stand at room temperature, or better in the incubator at 39”. After a few hours, the separation of a red precipitate commences and its quantity gradually increases from day to day. From time to time it is advisable to filter off the precipitate and to add more nitrophenylhydrazine. If no acid be added to the mixture, a precipitate is still obtained but it contains little or none of the methyl glyoxal derivative. Comparative experiments in which equivalent amounts ‘of sulphuric and acetic acids were used, failed to show any marked difference.

In one experiment, a filtered solution containing alanine (25 grams), nitrophenylhydrazine (1.5 grams) and acetic acid (5 cc.) and water (500 cc.) was digested at 39”. After three hours, the separation of a precipitate was noticeable and the amount gradually increased during the following four days, when almost all the nitrophenylhydrazine had disappeared. The precipitate was filtered off and additional nitrophenylhydrazine (1 gram) dissolved in the filtrate. A second precipitation soon commenced and the whole process was eventually repeated four times.

The combined precipitates which weighed rather less than a gram, were purified by washing successively with hot 10 per cent sodium carbonate solution, water and alcohol. The residue was then crystallized from a mixture of nitrobenzenk and toluene and was obtained in the form of red needles melting at 302’ identical with methyl glyoxal dinitrophenylhydrazone prepared from other sources. It gave the color reaction with caustic soda in typical fashion. On mixing the substance from alanine with a preparation from methyl glyoxal, the melting point of the mixture was unchanged.

ANALYSIS: 0.1367 gram gave 0.0333 gram N = 24.4 per cent N. ClsH~404Na requires 24.6 per cent N.

In addition to the above typical experiment, we have made a number of others in which the reaction and concentration of the acid was varied but without obtaining materially different results.


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The yield of the methyl glyoxal derivative is small but it must be remembered that most of the alanine may be recovered unchanged, so that it is likely that the yield is relatively large com- pared with the amount of amino-acid decomposed.

The formation of methyl glyoxal from alanine necessitates the simultaneous liberation of ammonia and we have made a number of experiments which indicate that small amounts of ammonia are liberated from amino-acids with much greater ease than has been commonly supposed. We find, for example, that if a weak solution (2 per cent) of ordinary sodium phosphate is boiled in a distilling flask attached to a condenser until the distillate is perfectly free from ammonia when tested with Nessler’s reagent and then a gram or so of an amino-acid, such as alanine, is added, the second distillate will be found to contain very definite traces of ammonia. On continuing the distillation, the amount of ammonia slowly diminishes but does not disappear entirely, and on allowing the previously boiled mixture to stand in the distilling flask for a short time (e.g., 1 hour), a fresh formation of ammonia is apparent. Similar results were obtained on substituting sodium borate, prepared from boiled ammonia-free caustic soda and ignited boric acid, for the phosphate.

On adding a little freshly distilled ammonia-free acetic acid to a dilute alanine solution which has been well boiled with a little caustic soda to remove any ammonia present as an impurity and then digesting the mixture in the distillation apparatus at about 50” for an hour or two, we find that on making alkaline with caustic soda and redistilling, there is no difficulty in detecting ammonia in the distillate. Digestion of amino-acids with weak caustic soda solution (&J also appears to lead to ammonia formation.

It need hardly be added that in all of the above experiments appropriate blank tests were constantly carried out and every effort made to guard against accidental contamination. We pro- pose to study the reaction quantitatively.

4. The formation of methyl glyoxal from glucose.

Methyl glyoxal was shown by Pechmann to be somewhat vol- atile with steam and we made use of this property for its isolation from the complex mixture of substances resulting from the action of salts upon glucose.


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Glucose (50 grams) and sodium phosphate crystals (25 grams) were dissolved in water (500 cc.) and the mixture was distilled until about 300 cc. of distillate were obtained. An addition of 300 cc. of 5 per cent phosphate was then made and the distillation repeated until finally about 3 liters of distillate were obtained. The distillate gave a marked iodoform reaction and on treatment with p-nitrophenylhydrazine dissolved in acetic acid gave a red flocculent precipitate. The precipitate was collected and crystallized from either pyridine or better from nitrobenzene in deep crimson needles melting at 300”. The melting point was unchanged on mixing with pure methyl glyoxal dinitrophenylhydrazone.

ANALYSIS : 0.0882 gram dried at 150” gave 0.0214 gram N = 24.3 per cent N. C&HI~O,N~ requires 24.6 per cent N.

The,yield of precipitate was small, about 0.5 gram, but no doubt only a very small proportion of the glyoxal formed was obtained in the distillate. Reference may be made here to the interesting experiments of Henderson8 upon the loss of optical activity of glucose solutions on digestion with phosphates. The reaction un- doubtedly deserves careful study. It is possible that the methyl glyoxal derivative we obtained is derived from acetol, but while this is doubtful it is not a matter of great importance for the pur- pose of the present experiments.

5. The formation of other &etonic aldehydes from a-hydroxy-acids and warn&o-acids.

We have been able to observe the formation, from a number of hydroxy- and amino-acids, of insoluble nitrophenylhydrazones giving reactions indicative of their being derived from oc-ketonic aldehydes; but in many cases we must defer an accurate description of the properties of the substances until we have had opportunity to study them more closely. The experiments were conducted in similar fashion to those already described with lactic acid and alanine, so that repetition will be unnecessary.

Glycollic acid. Glycollic acid (20 grams), p-nitrophenylhydrazine (2 grams ) and water (200 cc.) were heated together, cooled, filtered and then digested at 39”. After a day a fine red granular

* This JournuZ, x, p. 3, 1911.


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H. D. Dakin and H. W. Dudley =37

precipitate separated out which gave all the reactions of glyoxal dinitrophenylhydrazone. The quantity of precipitate increased steadily, but after a few days bright yellow crystals began to separate, which dissolved in caustic soda to give a bright red color. They proved to be the nitrophenylhydrazone of glyoxylic acid. The precipitate which first separated crystallized from nitrobenzene and toluene in small glistening deep red crystals melting with evolution of gas at 302”. On mixing the substance with glyoxal dinitrophenylhydrazone prepared from glyoxal, the melting point was ‘unchanged. A trace of the substance on warming with caustic soda solution and a few drops of alcohol, gives a transitory greenish-blue color, passing to a deep blue and slowly changing to violet and finally brown red. The yield of pure substance was insufficient for analysis.

For comparison, glyoxal dinitrophenylhydrazone was prepared from glyoxal precisely as in the case of the methyl glyoxal derivative. The yield is practically theoretical. The substance has also been obtained by Wohl and Neuberg from glycollic aldehyde.g

The precipitate appearing during the later stages of the glycol- lit acid digestion was filtered off and washed with water and then dissolved in much boiling alcohol. On filtering, a small red precipitate consisting chiefly of glyoxal dinitrophenylhydrazone separated out. On concentrating the filtrate, yellow crystals of the nitrophenylhydrazone of glyoxylic acid were obtained. This substance has an indefinite melting point, beginning to decompose at a temperature somewhat above 200”, and on repeated crystallization, passes over into a less soluble modification, which is very sparingly soluble even in boiling nitrobenzene. The hydrazone dissolved in caustic soda to give a bright red color and was identical with the substance previously described by one of us, which was prepared directly from glyoxylic acid.*O

Glyceric acid. The conversion of glyceric acid in 5 or 10 per cent solution on digestion with nitrophenylhydrazine (1 per cent) into the nitrophenylosazone of glyceric aldehyde is effected with remarkable ease. The osazone separates out in abundance after one to two hours and its quantity steadily increases as long as unchanged nitrophenylhydrazine is present.

9 Ber. d. deutsch. them. Gesellsch., xxxiii, p. 3107, 1900. lo This JoumuZ, iv, p. 235, 1908.


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The precipitate was filtered off and recrystallized from nitrobenzene and toluene. The osazone crystallizes in long thin scar- let needles melting at about 315’ with evolution of much gas. It is sparingly soluble in alcohol, ether or amyl alcohol.

ANALYSIS: 0.1292 gram gave 0.0301 gram N = 23.3 per cent N. CIF,HI~O~N~ requires 23.8 per cent N.

A trace of the substance boiled with caustic soda and a little alcohol gives successively greenish-blue, deep blue, violet red and brown-red colors.

An attempt to prepare the above osazone from glyceric aldehyde, for purposes of comparison, gave a disappointing yield.

Mandel~c acid. Experiments with mandelic acid similar to those with glycollic acid, gave a complicated mixture of substances. On washing the precipitate successively with hot sodium carbonate solution, water and alcohol, and then recrys- tallizing the residue from nitrobenzene, a small quantity of substance was obtained which resembled phenyl glyoxal dinitrophenylhydrazone in every respect. The amount of pure substance was insufficient for analysis.

The sodium carbonate washings on acidification gave a small quantity of phenyl glyoxylic acid nitrophenylhydrazone, crystal- lizing in hair-like needles melting at 163”-165”. Both of the above-mentioned hydrazones were prepared by independent meth- ods for purposes of comparison.

Phenyl glyoxal dinitrophenylhydrazone was prepared from phenyl glyoxal (1 mol.) and nitrophenylhydrazine (2.2 mols.) dissolved in acetic acid (33 per cent). The red precipitate was washed with alcohol and recrystallized from nitrobenzene and toluene. It crystallizes in bright red needles melting at 302”-304”.

.~NALYSIS: 0.1551 gram gave 0.3370 gram CO2 and 0.0559 gram H20. Calculated for

Found: CzoHaN&: C.. . _. _. _. _. _. 59.3 59.4 H....................................,....,. 4.1 4.0

A trace of the substance warmed with caustic soda and alcohol gives successively carmine red, purple, clear light red fading finally to a light brown color.


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Phenyl glyoxylic acid nitrophenylhydrazone was prepared by adding nitrophenylhydrazine dissolved in a slight excess of 5 per cent sulphuric acid to phenyl glyoxylic acid.‘l The hydrazone is precipitated as a yellow substance which crystallizes from weak alcohol, in which it is readily soluble, in hair-like needles melting at 163”-165”. It dissolves in caustic soda to give a bright red color.

ANALYSIS: 0.1056 gram substance gave 0.01568 gram N = 14.8 per cent N. CLHIINSO~ requires 14.7 per cent N.

Glycine. The experiments with this substance were similar in every respect to those with alanine. There was no difficulty in detecting the formation of glyoxal dinitrophenylhydrazone, but the yield of precipitate was distinctly smaller than in the case of alanine.

Aspartic acid. 0.n digesting a 1 per cent solution of aspartic acid with nitrophenylhydrazine at 39”, there is an abundant formation of a dinitrophenylhydrazone..- The substance crystallizes from nitrobenzene and toluene in the form of small thick prisms, and on warming with caustic soda gives successively a greenish- blue, clear deep blue, followed by a more persistent violet-blue, finally turning reddish brown. The substance is apparently the dinitrophenylhydrazonc of the ol-ketonic aldehyde corresponding to aspartic acid (COOH.CH,.CO.CHO). It will be studied further.

We have also obtained dinitrophenylhydrazones from other amino-acids including valine, leucine, phenylalanine, proline. These substances are sparingly soluble compounds with high melting points and give characteristic color reactions with caustic soda. We prefer to reserve their detailed description until we have had an opportunity of studying them more closely.

11 A most convenient method for the preparation of phenylglyoxylic acid is as follows : Mandelic acid (10 grams) is neutralized with caustic potash and diluted to 500 cc. with ice and water. Potassium permanganate in 4 per cent solution (200 cc.) is added drop by drop to the cooled potassium mandelic sol&ion, using a mechanical stirrer. Half an hour after all the permanganate has been added sulphur dioxide is passed in to dissolve the oxides of manganese. Sulphuric acid is then added in excess and the phenylglyoxylic acid extracted with ether. The yield is 90 per cent of the calculated one. (Cf. Evans: Zoc. cit.)


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6. The fate of methyl glyoxal and phenyl glyoxal on perfusion through the liver. The formation of phenyl glyoxylic acid.

The perfusions of dogs’ livers with methyl and phenyl glyoxals were carried out as in the case of similar experiments reported from this laboratory,12 with the exception that sodium phosphate was added to the perfusion mixture in order to provide for the prompt neutralization of any acid that might be formed.

Methyl glyoxal. The dog (14 kgm.) was starved for two days before operation. Th e 1 iver was perfused for half an hour with a mixture containing blood, 500 cc. ; phosphate, 500 cc. 5 per cent; methyl glyoxal, 3 grams; salt solution, 250 cc. After perfusion, the mixture was heated to coagulate protein and the liver was also cut up and boiled with water and the filtrates combined. The filtrates were evaporated almost to dryness, acidified with phosphoric acid and then taken up with gypsum. The dry powder was extracted with ether in the usual way. The ether extract was taken up in water and was found to be strongly laevo-rotatory. It was boiled with zinc carbonate and gave two crops of dextro- rotatory zinc lactate (4 grams). The rotations and analyses showed that both d- and l-lactic acids were present, the latter being in excess.

Crop I. (2.1 grams): 0.3302 gram dried at 120” lost 0.0585 gram H,O = 17.7 per cent.

ROTATION: 0.2667 gram air dried salt in 10 cc.; 1 = 2 dm.; 01 = 0.1”. [a], = + 2.28”

Crop II. (1.9 grams): 0.2025 gram dried at 120” lost 0.0340 gram Hz0 = 16.8 per cent.

ROTATION: 0.1685 gram of dry salt in 10 cc.; 1 = 2 dm.; 01 = 0.20”. [cY]D = + 5.93”

.0.2702 gram of the mixed salts gave 0.0904 gram ZnO = 33.5 per cent ZnO. CsH606 requires 33.4 per cent ZnO.

A second perfusion was made in which no methyl glyoxal was added. The dog (7 kgm.) had not been starved and the liver contained much glycogen. 500 cc. of 5 per cent phosphate solution were added to the blood, which after a perfusion lasting half an hour was analyzed as before. 1.7 grams of zinc lactate were obtained, all of which was derived from dextro lactic acid.

i2 This Journal, ix, p. 146, 1911.


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H. D. Dakin and H. W. Dudley 141

ANALYSIS: 0.2516 gram dried at 120” lost 0.0335 gram Hz0 = 13.2 per cent. 0.2150 gram gave 0.0714 gram ZnO = 33.2 per cent.

ROTATION: 0.2838 gram in 10 cc.; 1 = 2 dm.; CY = - 0.42’. [a], = - 8.53”.

Phenyl glyoxal. Two experiments were made with phenyl glyoxal which were essentially similar to the methyl glyoxal experiment . In one experiment,, 3 grams of phenyl glyoxal and 200 cc. of 5 per cent phosphate were added to the blood saline mixture, and perfusion carried on for one and three quarters hours. In the second experiment 4 grams of phenyl glyoxal and 400 cc. of phosphate were added and perfusion lasted one hour.

The aqueous filtrates from blood and liver were concentrated and, after acidifying with phosphoric acid, extracted with ether in a continuous extractor. The ethereal solution in each case was shaken twice with 10 cc. of saturated sodium bisulphite solution to separate any phenyl glyoxylic acid. The main ether extract was then evaporated to dryness and the crystalline residue of mandelic acid recrystallized from boiling toluene. The yield of mandelic acid varied from l-l.6 grams. It was practically all the laevo-rotatory variety and melted at 131”.

ROTATION: 0.5776 gram in 20 cc.; 1 = 2 dm.;a = - 8.52”. [alo = - 148”.

The sodium bisulphite extracts were strongly acidified wit,h sulphuric acid and extracted with ether. The ether residue, in addition to phenyl glyoxylic acid, contained much mandelic acid which apparently may be extracted from ether solutions by sodium bisulphite to a rather surprising extent.

TLe residue gave the benzene, thiophene, sulphuric acid test for phenyl glyoxylic acid in typical fashion.13 The acid was further characterized as the nitrophenylhydrazone. The residue was dissolved in water, filtered and a clear solution of nitrophenylhydrazine in dilute sulphuric acid added. A bright yellow precipitate of phenyl glyoxylic acid p-nitrophenylhydrazone at once separated and was purified by recrystallization from dilute alcohol. In one experiment 0.2 gram was obtained, in the second 0.1 gram. The

I3 Some samples of technical benzene do not contain enough thiophene to give the reaction. It is therefore advisable to add thiophene separately.


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142 Metabolism of Carbohydrates and Proteins*

substance melted at 163”165” and was identical with the product prepared directly from phenyl glyoxylic acid (section 5).

ANALYSIS: 0.1100 gram gave 0.0161 gram = 14.6 per cent N. C14HllN304 requires 14.7 per cent N.

It may be noted here that on simple digestion of muscle tissue extracts with phenyl glyoxal, we have been able to detect readily the formation of phenyl glyoxylic acid.

‘7. The fate of methyl glyoxal and of l-lactic acid in the glycosuric organism.

For these experiments, we made use of dogs rendered glycosuric by daily injections of phlorhizin (1 gram) suspended in olive oil. The conditions of the experiments were similar to those of recently published experiments.14

Methyl glyoxal. A preliminary experiment was made in which 1.5 grams of methyl glyoxal in aqueous solution were given sub- cutaneously to a rabbit (1.5 kgm.) without effect, showing that it was relatively non-toxic. The methyl glyoxal used for the following experiment was freshly prepared by hydrolyzing the acetal according to Meisenheimer’s method. It, was given by stomach t,ubc and produced no particular symptoms. The nitrophenylhydrazine test showed that no ‘unchanged methyl glyoxal was excreted in the urine. The urine was collected in six-hour periods.

NTTBOGEN ACETOACETIC ) DLUCOsE -I~- -c,:~N-~--~---- ~~~~ / ACID 1 ~~ SUBBTANCE GWEN

----I- 3.67 14.40 j 3.92 ~ 0.012 i 3.16 19.37 6.13 ~ 0.018 9 gms. methyl glyoxal 4.43 17.15 I 3.88 : 0.066

1 3.81

The rise in G : N ’ ratio on giving the tiethy glyoxal is very marked. Using 3.87 as the average ratio, it is calculated that 9 grams of methyl glyoxal gave a little over 7 grams of “extra glucose.”

In a second experiment in which the methyl glyoxal was given by subcutaneous injection, the G: N ratio rose from 3.7 to 7.66.

I4 This Journal, xiv, p. 321, 1913.


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l-lactic acid. The acid was prepared by resolving inactive lactic acid with morphine according to Irvine’s15 excellent method. The crystalline morphine l-lactate was decomposed by ammonia and the alkaloid filtered off. The ammonium lactate was con- verted into the calcium salt by prolonged boiling with lime. The pure crystallized calcium Z-lactate was 6nally decomposed by heating with an equivalent weight of sodium sulphate and the calcium sulphate removed by filtration. The sodium lactate was given by stomach tube and evoked no symptoms.

G:N

3.72 I 13.10

3.81 3.80 6.33

3.52 3.55

0.155 0.043

0.013

12 gms. Z-lactic acid as sodium salt.

Adopting 3.62 as the average G:N rat,io, it is found that 12 grams of lactic acid furnished slightly over 9 grams of glucose.

I5 Transactions of the Chem. Sot., lxxxix, p. 935, 1906.


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H. D. Dakin and H. W. Dudley-KETONIC ALDEHYDES. PART IIαAND

-HYDROXY-ACIDSα-AMINO-ACIDS, αTHE INTERCONVERSION OF

1913, 15:127-143.J. Biol. Chem.

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