THE CHEMISTRY OF GLUCONEOGENESIS.’

III. THE FATE OF ISOBUTYIUC, ISOVALERIANIC AND ISOCAPROIC ACIDS IN THE DIABETIC ORGANISM, WITH CONSIDERA-

TION OF THE INTERMEDIARY METABOLISM OF LEUCINE AND VALINE.

BY A. I. RINGER, E. M. ‘FRANKEL AND L. JONAS.

(From the Department of Physiological Chemistry of the University of Penn- sylvania, Philadelphia, Pa.)

(Received for publication, April 30, 1913.)

For the proper understanding of the processes involved in the metabolism of the different foodstuffs it is essential to know the fate of the individual products of metabolism in the body. For the past few years this question has been the center of attack from different singles. The difficulties associated with these researches appeared insurmountable at times, but with the latest developments in the application of organic chemistry to biological processes more and more light is being thrown on the subject of the intermediary metabolism of foodstuffs.

There are a number of methods that have been devised of late for the prosecution of these researches, and while it must be ad- mitted that every one of them may be associated with a certain amount of error, they have nevertheleis all helped to bring to light certain definite reactions. The entire subject has been recently reviewed2 and much space need not be given here to this phase of the problem.

In these researches animals were made diabetic by daily injec- tions of 1 gram of phlorhizin ground up in a mortar with 10 cc. of olive oil.. The urine was collected by catheter and at the end of each period of twelve hours the bladder was washed three times with warm distilled water.

1 Aided by a grant from the Rockefeller Institute for llledical Research. 2 Dakin: Oxidations and Reductions in the Animal Body, Longmans,

Green and Company, London, 1912.

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526 Chemistry of Gluconeogenesis

The urine was analyzed for its optical activity, nitrogen, glucose, ammonia, acetone, aceto-acetic acid and /?-hydroxybutyric acid. In these determinations we were assisted by Mr. C. A. Horn- berger.

The original purpose of these researches was to find the chemical configuration that a substance required in order to become con- verted into glucose in the diabetic organism. It soon became evi- dent that the study of this problem was so closely interwoven with questions of the intermediary metabolism of foodstuffs and sub- stances chemically related to them, that it was deemed necessary to extend the scope of this investigation to a broader field.

The method employed in these researches permits-of the study of the following problems:

I. The fate of different substances in the animal body with refer- ence to their ability to form glucose.

II. The fate of different substances with reference to their ability to give rise to P-hydroxybutyric acid, aceto-acetic acid and ace- tone.

III. The chemical changes that different substances undergo in the process of catabolism in the animal body before their conversion into glucose or acetone bodies.

IV. The influence of different substances on the metabolic pro- cesses of the diabetic organism-especially antiketogenesis.

V. The role of different substances in the pathology of diabetes. In the first two papers of this series it was shown that the normal

fatty acids in the processes of catabolism in the diabetic animal undergo very definite reactions, by becoming oxidized in the P-position, giving rise to an acid with two carbons less. This con- clusion was reached after having established the fact that propionic acid (when fed to diabetic dogs) is completely converted into glu- cose, and that of the higher fatty acids only those give rise to glu- cose which stand in relationship to propionic acid by having two (or a multiple thereof) carbons more. Thus n-valerianic and n-hep- tylic acids, containing five and seven carbons respectively, give rise to glucose, whereas n-butyric and n-caproic acids, containing four and six carbons respectively, do not give rise to glucose, but to an increase in the acetone bodies. These findings add increased support to Knoop’s hypothesis of p-oxidation.

In this communication are recorded the results of experiments

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Ringer, Frankel and Jonas 527

with isobutyric, isovalerianic and isocaproic acids fed to diabetic dogs.

The methods employed were essentially the same as those of the previous experiments, with the exception that the phlorhizin was administered according to the Coolen method. It yields very satis- factory results, and with greater comfort to both operator and ani- mal. The P-hydroxybutyric acid was determined by Shaffer’s method.

The iso compounds enumerated above stand in very close rela- tionship to several important amino-acids,

CHr CH3 CH3 CHs CH, CH3 CH3 CH, CH, CH3

x x x x x

COOH CH2 CH-NH2 CH2 CH2

I I I COOH COOH CH-NH2 CH2

I I COOH COOH

Isobutyric Isovalerianic l Valine. Leucine. Isocaproic acid. acid. acid.

and the results of our experiments seem to throw light on the inter- mediary metabolism of these amino-acids.

The effect of isobutyric acid.

CH3

CH3 >

CH-COOH

Baer and Blum3 gave 20 grams of isobutyric acid to a diabetic patient with the object of ascertaining whether it would cause increased elimination of the acetone bodies. They did not get any increase in the /3-hydroxybutyric acid or acetone. Embden, Salomon and Schmidt4 perfused the liver with blood containing isobutyric acid and found no increase in the acetone formation.

In this series of experiments isobutyric acid was fed with a double object in view. First, both Baer and Blum and Embden

s Baer and Blum: Arch. f. exp. Path. u. Pharm., Iv, p. 89,1906. 4 Embden, Salomon and Schmidt: Hofmeister’s Beitriige, viii, p. 129.

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528 Chemistry of Gluconeogenesis

suggested the possibility of demethylation in the catabolism of the isobutyric acid molecule, with the introduction of either an H or an OH in the a-carbon:

CH3 CH3 CH3 ; CHJ CH3 : CH3

x I I

------+ (332 Or CHOH I I

COOH COOH CHOH

If this were true, isobutyric acid would give rise to propionic acid or lactic acid, either of which should give rise to extra glucose. Second, it was of great importance to study the possible effects of isobutyric acid on acidosis.

In experiment XI, period VI, the animal was given subcutane- ously 10 grams of isobutyric acid as sodium salt. The glucose elimination rose from 11.5 to 14.03 grams. The nitrogen elimina- tion sank from 3.6 in the fore period to 3.29 and 2.55 in periods VI and VII, respectively, to rise again to 3,.61 in period VIII. The amount of extra glucose eliminated was 5.52 grams (calculated by the method suggested by Lusk and Ringer).5 The most remarkable effect of the isobutyric acid was on the acetone and P-hydroxy- butyric acid elimination. In the fore period the acetone elimina- tion was 0.223 gram; it dropped down to 0.059 and rose again to almost the original level in period VII. The /3-hydroxybutyric acid, which stood at 1.1 grams for two successive periods, dropped to 0.238 gram and returned to 1.325 grams as the effect of the isobutyric acid wore off. In period XII of the same experiment a similar dose of isobutyric acid was administered to the same dog. The effects were essentially the same, except that the amount of extra glucose was less than in the first case.

In periods IX and XII of experiment XII the animal received 10 grams of isobutyric acid. The yield of extra glucose was 7.6 grams in period IX and 4.3 grams in period XII.

In period VI of experiment XII the animal received 20 grams of isobutyric acid, which resulted in the’elimination 0:’ 9.15 grams of extra glucose. In period XII of the same experiment the animal was given subcutaneously 10 grams of isobutyl alcohol, which re- sulted in the elimination of 10.3 grams of extra glucose.

6 Ringer and Lusk: Zeitschr. f. physiol. Chem., Ixvi, p. 106, 1910.

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Ringer, FrankeI and Jonas 529

All these experiments prove beyond question that isobutyric acid gives rise to considerable quantities of extra glucose. In most of the cases there was a very marked diminution in the nitrogen output, immediately following the isobutyric acid administration. This phenomenon is very marked in experiments XI, XII and XIV. It is, however, absent in experiment XIII, periods VI and VII.

Another point of great interest is the effect of isobutyric acid on the elimination of acetone bodies. In experiment XI the effect was very marked, while in the others it was hardly noticeable. These individual differences point very strongly to the existence of certain factors in the metabolism of the diabetic organism which determine the action of isobutyric acid and similar substances. Since these experiments were performed, we have administered iso- butyric acid to several patients, some with very severe and others with moderately severe acidosis. It was found that in severe cases of diabetes most of the isobutyric acid is converted into glucose, no effect whatsoever being exerted on the acidosis. In the milder cases it is similarly converted into glucose, but very distinct anti- ketogenetic effects are noted. The influence of isobutyric acid on the nitrogen metabolism in animals and on acidosis in human diabetes will form the subject of a separate communication in the near future.

The e$ect of isovalerianic acid.

CH3

CHS > CH-CH2-COOH

That isovalerianic acid gives rise to large quantities of p-hydroxy- butyric acid, aceto-acetic acid and acetone has been proven beyond question by Baer and Blum6 in the diabetic patient and by Embdenl who found an increase in the aceto-acetic acid formation on perfusing an excised liver with blood containing 2 grams of isovaIerianic acid.

In this research it was our object to see whether isovalerianic acid has any influence on the glucose elimination. In experiment XI period IX, in experiment XII period VI and in experiment XIV

6 Lot. cit. 7 Lot. cit.

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530 Chemistry of Gluconeogenesis

period XI, IO grams of isovalerianic acid as sodium salt were admin- istered subcutaneously. In none of these experiments was there any increase in the glucose. The elimination of the acetone bodies was increased to a remarkable extent, thus confirming the findings of Baer and Blum and of Embden.

The effect of isocaproic acid (isobutyl acetic acid).

CH3)CH-CHa-C~I-COOH CH3

Embdea perfused a dog’s liver with blood containing isocaproic acid and obtained no increase in the aceto-acetic acid formation. We performed our experiments with this substance to see what influence it has on acidosis and whether it will give rise to extra glucose. Reasoning a priori, one would expect this substance to give rise to glucose. The process of demethylation having been established in iso compounds in the case of isobutyric and isovaleri- anic acids, there is every reason to suppose that such a process takes place in the isocaproic acid molecule. Valerianic acid would be formed first, which after undergoing &oxidation, would give rise to propionic acid and to glucose.

CH3 CH3 CH3 j CH3 CH3

kc

.._....

-+ CHz -+ CH2 -- CsH120r

I

O-7 O-F

COOH

a-CH2 a- CH2 CHs

I I I COOH COOH COOH

In period III of experiment XIII, 11.6 grams of isocaproic acid were giyen subcutaneously as sodium salt. The glucose elimina- tion rose from 24.6 grams in the fore period to 25.19 grams, in spite of the drop in the nitrogen. The D: N ratio, which was 3.38 in the fore period and 3.36 in the after period, rose to 3.65. The extra glucose eliminated was 1.95 grams. In experiments XIV periods XVIII and XIX and in experiment XV period IX, 10 grams of isocaproic acid as sodium salt were given per OS. The results in both cases corroborate the results in the first experiment in showing

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Ringer, Frankel and Jonas 531

that extra glucose is formed from isocaproic acid. It is true that the amount of extra glucose does not come up to the theoretical value, but, as will be seen from other experiments, theoretical yields are not always obtained.

Intermediary metabolism of fatty acids containing an isopropyl radical.

From the work of Baer and Blum, Embden and our own, itis ap- parent that all compounds containing an isopropyl radical undergo demethylation. The question of interest in this connection is, in what way does the methyl radical dissociate from the rest of the molecule? Baer and Blum have advanced the suggestion that the CH, may leave the molecule and an OH radical take its place. They based this suggestion upon the fact that after feeding 20 grams of isobutyric acid they obtained 0.5 gram of zinc lactate from one-half of the twenty-four hours’ urine, and also upon the fact that isovalerianic acid gave rise to P-hydroxybutyric acid. We are inclined to believe that the methyl radical is removed by a process of hydrolysis, the OH going to the methyl radical, forming CHSOH, which is oxidized in the body, while the H radical goes to replace the CH,, forming an acid of the normal series. Thus isobutyric acid gives rise to propionic acid which in turn may give rise to lactic acid. Isovalerianic acid gives rise to butyric acid which in turn produces p-hydroxybutyric acid. This also explains why Baer and Blum obtained ,&hydroxybutyric acid from a-methylbutyric acid and failed to get it from cr-hydroxybutyric acid.

Intermediary metabolism of amino-acids containing an isopropyl radical.

Embden and Marx* in their perfusion experiments found that a-amino-valerianic acid gave rise to aceto-acetic acid, while neither a-amino-butyric nor a-amino-caproic acids showed any evidence of

fi Embden and Marx: Hofmeister’s Beitriige, xi, p. 318, 1908.

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532 Chemistry of Gluconeogenesis

yielding aceto-acetic acid. They drew the conclusion that cr-amino- acids suffer oxidation in the ar-position giving rise to acids with one carbon atom less, while the carboxyl radical is split off, in all proba- bility in the form of COz.

In the protein molecule we find two amino-acids which contain the isopropyl radical-leucine and valine. Halsey9 fed leucine to phlorhizinized dogs, and, of six feedings, two showed considerable amounts of extra glucose, and four gave entirely negative results. Recently Dakin published one experiment in which he fed 15 grams of i-leucine and obtained 4 grams of extra glucose. Neither Halsey nor Dakin are inclined to attribute glucogenetic properties to leucine.

When we consider leucine in the light of our experiments with unsubstituted fatty acids with branched chains, and having in mind Embden’s results, in which he showed that leucine is a strong aceto-acetic acid builder, there remains only one possible conclusion with regard to the catabolism of leucine. As far as our knowledge goes, and as will be shown in a following communication, we are fully justified in assuming that all carbons in the a-position having an amino radical lose the amino radical and become oxidized possibly to the carboxyl state, giving rise to compounds (acids) with one carbon less.

For the present we do not include glycocoll or alanine in this category. Experiments are in progress which will throw light on the role of the carboxyl in the formation of glucose from these sub- stances.

Leucine in its catabolism can be assumed to pass through the following intermediary stages :

CH, CH3 CH, CH3 CH3 C& CH3 CH,

kc v I

I - CH

I --+ CH2 + CHOH z CO __f CO

I I I I CH2 CH2 CH2 CH2 CH2 CHe

I CH-NH2 COOH COOH COOH COOH I

dOOH

9 Halsey: Amer. Journ. of Physiol., x, p. 229, 1903.

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Ringer, Frankel and Jonas 533

Ordinarily leucine cannot be classed as a glucose-yielding sub- stance, but under certain circumstances it may give rise to glucose. There are certain flora of intestinal bacteria that seem to possess the power of effecting a-oxidation. In one of our experiments butyric acid, which, when given subcutaneously never yields even a trace of glucose, when fed per OS gave rise to as much as 3 grams of glucose. This reaction cannot be explained in any other way, and it seems very possible that the same factors obscured Halsey’s two experiments.

When we found that isobutyric acid gave rise to glucose and that isobutyl alcohol could yield an amount of glucose that corres- ponds to the conversion of three of its carbons into glucose, we were led to the belief that valine in its catabolism was broken down to isobutyric acid, and that it would be found to be one of the amino- acids that give rise to glucose.

CH3 CH3 CHs \/ I

2 CH - 2CH:, CG&‘&

I I - CHzOH CHzOH

148 : 120 : 180 10 : 8.1 * ’ - 12.1 (found 10.3)

In his paper on the “Intermediary Metabolism of Amino-acids,” Dakin’O showed that valine yields practically no glucose. This of course makes our theory more difficult of interpretation, but we do not believe it robs the theory of its strength, for the antiketo- genetic properties of valine are very similar to those of isobutyric acid, and as will be shown in the future, this property is possessed only by compounds capable of forming glucose. (The reverse, ‘however, is not true, i.e., not all compounds that are capable of forming glucose possess antiketogenetic properties.)

SUMMARY.

Experiments were performed on phlorhizinized dogs. I. It was found that isobutyric acid and isobutyl alcohol give

rise t,o glucose, probably by undergoing demethylation and by giving rise to normal fatty acids (propionic acid).

lo Dakin: This Journal, xiv, p. 321, 1913.

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534 Chemistry of Gluconeogenesis

II. Isovalerianic acid does not give rise to glucose, but to large quantities of ace&acetic acid, acetone and j3-hydroxybutyric acid.

III. Isocaproic acid was found to give rise to glucose, probably having, by a process of demethylation, formed normal valerianic acid, which became oxidized to propionic acid.

IV. Isobutyric acid, in certain cases, possesses very marked anti- ketogenetic properties.

V. It is suggested that isovalerianic acid is one of the intermedi- ary stages in the catabolism of leucine.

VI. It is also suggested that isobutyric acid may be an inter- mediary body in the catabolism of valine.

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A. I. Ringer, E. M. Frankel and L. JonasLEUCINE AND VALINE

INTERMEDIARY METABOLISM OFCONSIDERATION OF THE

DIABETIC ORGANISM, WITHAND ISOCAPROIC ACIDS IN THE

OF ISOBUTYRIC, ISOVALERIANICGLUCONEOGENESIS: III. THE FATE

THE CHEMISTRY OF

1913, 14:525-538.J. Biol. Chem. 

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