THE ACID-SPLITTING REACTION OF ACETOACETIC ACID AND THE ENZYMATIC FORMATION OF ACETIC ACID

FROM ACETOACETIC ACID*

BY ALBERT L. LEHNINGER

(From the Department of Physiological Chemistry, Medical School, University of Wisconsin, Madison)

(Received for publication, December 5, 1941)

There is very little direct chemical evidence on the mechanism of the breakdown of the ketone bodies in the extrahepatic tissues. Among the several theories offered for the catabolism of acetoacetic acid, it has often been suggested (l-3) that it undergoes hydrolysis to 2 molecules of acetic acid prior to further catabolism. However, it has never been demonstrated that this reaction can take place either by enzymatic or non-enzymatic catalysis under any conditions approaching the physiological.

It was the purpose of this investigation to study the conditions, kinetics, and catalysis of the hydrolysis in purely chemical systems in vitro, and then to determine whether any chemical data can be obtained for the existence of an enzyme or enzymes which bring about a formation of acetic acid from acetoacetic acid in excised tissues, tissue extracts, or microorganisms.

EXPERIMENTAL

ConJirmation and Extension of Prezkous Data-Although Wislicenus (4) and von Euler and Olander (5) had previously studied the hydrolysis of acetoacetic acid by strong bases, a complete analysis of the reaction has not been reported. It seemed desirable to confirm and extend certain of their data.

Sodium acetoacetate solutions were prepared throughout this study by the method of Ljunggren (6). In the experiments reported in Table I the classical Messinger-Huppert method (7) was used for the determination of acetoacetic acid, the method of Folin (8) for acetone, and the methods of Virtanen and Pulkki (9) for acetic acid identification and determination.

* This work was supported in part by a grant from the Wisconsin Alumni Research Foundation.

A preliminary report of this work was given at the meeting of the American Society of Biological Chemists at Chicago, April, 1941 (Proc. Am. Sot. Biol. Chem., J. Biol. Chem., 140, p. lxxvi (1941)).

147

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148 ACETIC ACID FROM ACETOACETIC ACID

The data on the course and kinetics of ketone- and acid-splitting at various concentrations of sodium hydroxide generally confirm those of von Euler and olander and need not be discussed further. However, it can be seen from the data (Table I) that acetic acid was the only volatile acid formed in the reaction, that it accounted for all of the hydrolyzed aceto- acetic acid, and that no side reactions occurred. This last observation was unexpected and is important.

E,#‘ect of Possible Catalysts on Rate of Acid-Splitting Reaction--Since no catalyst other than strong bases has been reported for the acid hydrolysis, a series of experiments was then set up as in Table I, with various compounds

TABLE I

Hydrolysis of Sodium Acetoacetate by Sodium Hydroxide at 3Y”

The initial concentration of acetoacetate is approximately 0.069 M in each case. The changes in the reactants are expressed in mM.

Expi+i?=t NaOH Time Initial Acetoacetate concentration acetoacetate hydrolyzed

Acgiit;id Recovery

__- I I N hrs. per cent

0.1 24 17.01 All hydrolyzed to acetone 3 18 16.95 8.92 16.76 99.5 3 24 16.95 10.01 19.90 99.1 5 24 17.05 12.38 24.60 99.2 8 12 17.60 8.72 17.34 99.2

11 48 16.93 16.02 31.80 99.3

Identijication of Acid by Duclaux Constants

Per cent volume distilled

25 per cent 50 per cent 15 per cent 87.5 per cent

Unknown....................... 16.94 36.30 59.20 73.80 Pure acetic acid................ 16.91 36.32 59.16 73.85

substituted for the sodium hydroxide as possible catalysts for the reaction. The compounds and concentrations used (necessarily selected somewhat at random) were 3.5 N NH,, 1 M Na3P04, 1 M Na2HP04, 3 M NH&l, saturated NH&l, saturated NaCl, 0.1 M HgSOI, 1 per cent HgO (freshly precip- itated), 1 N FeSOd, 1 per cent colloidal Fe(OH)s, alumina gel, 1 per cent colloidal Al(OH),, 10 per cent Ca(OH)2 suspension, 10 per cent Cu(OH)z, 1 per cent freshly precipitated AgzO, 1 per cent sodium silicate, silica gel, 1 per cent MnOz suspension, and the common buffer mixtures (bicarbonate, phosphate, glycine, acetate, borate). In no case could any breakdown of acetoacetate into acetate be established.

Another series of experiments was then performed in which the solutions

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A. L. LEHNINGER 149

of acetoacetic acid were buffered at pH 7.4 by 0.05 M phosphate buffer and the possible catalytic effect of a series of compounds known to be important in cellular metabolism was then determined. The concentration of aceto- acetate was uniformly 0.01 M. Acetoacetic acid was determined by the method of Messinger-Huppert but in cases in which interference with the method was expected the results were checked by means of the standard Van Slyke procedure (10). The compounds examined were succinate, malate, fumarate, oxalacetate, citrate, a-ketoglutarate, pyruvate, lactate, dl-alanine, aspartic acid (all in 0.01 M concentration), 0.005 M I-cysteine, 0.1 per cent HzOz, 0.01 M dl-glyceraldehyde, 0.005 M sodium hexose mono- phosphate, 10 mg. per cent cytochrome (crude preparation from beef heart), 0.01 M glucose, 0.01 M acetaldehyde, 0.01 M hydroquinone, 20 units per 100 ml. of insulin (Lilly’s iletin), 1 per cent hemoglobin, and the com- mon physiological anions and cations in 0.01 M concentration. There was no measurable decrease in acetoacetic acid in any case.

Enzymatic Formation of Acetic Acid from Acetoacetic Acid-Since it seemed unlikely from the results of these simple experiments that aceto- acetic acid is hydrolyzed to acetic acid by a non-enzymatic catalysis other than by strong alkalies, the possibility of an enzymatic catalysis of this reaction was then investigated. This search for an enzyme was begun with minced rabbit muscle, and this was the preparation studied in the following series of experiments.

Technique and Methods

The reaction vessels were 1 liter Erlenmeyer flasks. 10 gm. of minced rabbit muscle (Latapie mill) were added to 100 ml. of medium (Krebs- Ringer-phosphate buffer of pH 7.4 ($1) containing the neutralized sub- strate) which had been previously equilibrated at 38”. The appropriate gas was bubbled through the mixture for 10 minutes; the flask was then closed off, and shaken in a thermostat bath at 38” for 2 hours. Initial and final filtrates were prepared for analysis. In all operations involving acetoacetic acid, care was exercised to prevent any possibility of excess alkali causing hydrolysis to acetic acid artificially.

Acetoacetic acid and /?-hydroxybutyric acid were determined on aliquots of copper-lime filtrates by Edson’s micro modification of Van Slyke’s method (12). In some experiments the acetoacetic values were checked by Dewan and Green’s modification (13) of the manometric aniline citrate method, with the standard Warburg apparatus.

The determination of the very small amounts of volatile fatty acid formed in the experiments offered some difficulties. For the analysis of small samples (5 ml.) containing not less than the equivalent of 0.5 ml. of 0.01 N

NaOH the method described by Leloir and Munoz (14) was found to be

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150 ACETIC ACID FROM ACETOACETIC ACID

satisfactory and was used when acetate was the substrate. However, in the determination of smaller amounts of acetate in large volumes of buffer the method is unsatisfactory, since the solutions must first be concentrated by alkaline evaporation before distillation, and hydrochloric acid (from buffer chlorides) was found to be present in the distillates from such concen- trated filtrates, rendering the determinations worthless.

It was found possible to eliminate these difficulties by steam-distilling the concentrated tissue filtrate, by use of the apparatus and reagents described by Friedemann (15) ; 300 ml. of the distillate were collected. This distillate contains hydrochloric acid and the fatty acid to be estimated and is made slightly alkaline and evaporated down to 30 ml. on a water bath and the distillation repeated with the addition of 0.05 gm. of freshly prepared AgzO to the reagents used in the distillation. The distillate is caught in 25 ml. fractions in Erlenmeyer flasks, boiled for 7 to 8 seconds, and titrated with 0.01 N NaOH from a micro burette with phenolphthalein. After the fifth fraction has been collected, the titration value usually has dropped to that of a water-reagent blank distillation, which is performed beforehand and which is subtracted from the titration value of each frac- tion. The total of the corrected titration values represents the total vola- tile fatty acids.

This method was found to be capable of determining as little as 30 y of acetic acid in a 200 ml. volume of buffer with 10 per cent accuracy and larger quantities with much greater accuracy. All fractions were tested after titration with silver nitrate and barium chloride for the presence of chloride and sulfate.

Variation in Recovery of Acetic Acid from Tissues-In some recovery experiments it was found that the added acetate could not be completely recovered. To determine the method of deproteinization which allowed best recovery some experiments were designed to measure recovery of acetate when added to muscle mince and immediately treated to remove proteins. In these experiments acetate was added to 100 ml. of buffer containing 10 gm. of rabbit muscle mince, filtrates prepared as indicated, and analyses of acetic acid performed. See Table II.

Apparently acetate is bound to some extent by tissue proteins and is not removed completely even in the acid Folin-Wu and phosphotungstic acid procedures. The binding of acetic acid by tissue proteins has also been observed by Dr. E. M. MacKay (personal communication) and by Stadie, Zapp, and Lukens (16). This recovery error must be taken into account in any quantitative consideration.

Identification of Residual Endogenous Fatty Acids in Tissue Filtrates- In the experiments reported later under the formation of steam-volatile fatty acids, there was a small Ctratable acidity on the distillation of the

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A. L. LEHNINGER 151

initial filtrates. To determine the nature of these acids the initial value distillates of Experiments 1 to 7 (Table V) were pooled and treated as in the previous section. Duclaux distillation yielded the constants given in Table III.

From the data it can be seen that. acetic acid and possibly a trace of a less steam-volatile acid make up the endogenous volatile fatty acids encoun- tered in the blank controls and the initial value experiments.

Inhibition of Acetate Utilization-Since acetate is rapidly utilized by most tissues (2), some means of preventing this utilization must be employed if

TABLE II Recovery of Added Acetate from Rabbit Muscle Mince

10 gm. of mince in 100 ml. of buffer containing 10.0 micromoles of acetate were used in each case. The recoveries of acetate are reported in micromoles.

Experiment No. Treatment RCXXlVtXy

Copper-lime, pH 8.3 8.4 Same, pH 10.5 7.3 Folin-Wu 8.9 Saturated HgClz 7.6

‘I phosphotungstic acid 7.0 Theoretical recovery 10.0

TABLE III Duclaux Constants o.f Endogenous Volatile Fatty Acids of Rabbit Muscle Mince

Per cent volume distilled

25 per cent 50 per cent 75 per cent 87.5 per cent

Unknown....................... 17.4 37.3 63.1 77.6 Pure acetic acid., 16.6 36.2 59.7 74.6

the formation of acetic acid from acetoacetic acid is to be demonstrated. Some experiments with malonate, arsenite, fluoride, selenite, etc., as inhibitors showed that both acetoacetate and acetate breakdown were inhibited almost equally, making them worthless for this purpose. How- ever, it was found that the utilization of acetate depends on the oxygen tension, and that when the gas phase is air instead of oxygen the utilization of acetate is depressed, while that of acetoacetate remains essentially unchanged from that in pure oxygen (see Table IV). Thus, with air as the gas phase, the most likely conditions for obtaining acetoacetate break- down and retarding acetate breakdown are realized.

Formation of Steam-Volatile Fatty Acids from Acetoacetic Acid-With air

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152 ACETIC ACID FROM ACETOACETIC ACID

as the gas phase, experiments were then performed to determine whether any steam-volatile acids were formed when acetoacetic acid was utilized,

TABLE IV

Effect of Oxygen Tension on Utilization of Acetoacetate and Acetate by Rabbit Muscle Mince

10 gm. of rabbit muscle mince suspended in 100 ml. of buffer containing added acetoacetate or acetate in the concentrations given below were shaken for 2.0 hours at 38” as described under “Technique and methods.” The figures below represent the utilization of the metabolite in terms of micromoles per 10 gm. of mince per 2 hours. In the case of acetoacetate they represent true utilization and do not include conversion to P-hydroxybutyric acid, which was also measured but not included in this table.

Acetoacetate

Acetate

Concentration

micromoles per 100 ml.

1000 500

80 1000

500 80

Gas phase

oxygen Air Nitrogen

120 85 19

230 155

42

TABLE V

-

140

14 90

15

None

None ‘I

5

Formation of Steant-Volatile Fatty Acid from Acetoacetic Acid by Rabbit Muscle Mince

The gas phase was air, and the incubation period was 2 hours at 38”. The data - _ are reported in micromoles.

I Experiment No. Initial aceto-

acetic acid

Acetoacetic acic reduced to Br

hydm.xbdutyn Disappeared

~cetoacetic acid Acid formed Per cent aceto- acetic acid re- covered as acid

1 80.6 12.7 11.3 8.4 37 2 80.6 12.1 21.4 41.0 96 3 81.0 12.9 15.1 11.4 38 4 81.0 13.1 None None 0 5 81.1 14.3 9.4 7.1 38 6 86.0 17.1 None None 0 7 89.0 14.2 17.6 4.4 12 8 74.0 12.0 12.4 8.4 34 9 93.0 9.4 13.1 2.1 8

10 76.4 13.3 None None 0 - -

according to the technique described. The volatile acid figures are calcu- lated as acetic acid. Blank control experiments were run in each case, and the figures in Table V are corrected for the small blank experiment changes.

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A. L. LEHNINGER 153

Parallel experiments in every case on autoclaved tissue and tissue treated with 0.01 N NaCN and 0.005 M HgCI, showed no utilization of acetoacet&te, indicating that the action is probably enzymatic in nature. The presence of 40 units of insulin or 0.01 M glucose or both had no noticeable effect on the transformation.

For the identification of the acid formed, the distillates of the final filtrates from seven experiments were pooled, made slightly alkaline, and evaporated to 50 ml. The acids were then distilled as previously described, giving a total of about 4 ml. of 0.01 M acid. A portion of the sample yielded a positive lanthanum reaction (17), indicating the presence of acetic acid. The other portion was then subjected to a Duclaux distillation, yielding the data given in Table VI.

The Duclaux constants show that the acid formed from acetoacetic acid is mainly acetic acid, with possibly a trace of a less steam-volatile acid, probably formic.

Duclaux Constants o.f Unknown Acid Formed from Acetoacetic Acid by Muscle Mince

Per cent volume distilled

25 per cent 50 per cent 7s per cent 87.5 per cent

Unknown. 17.4 37.2 62.0 77.0 Pure acetic acid................ 16.6 36.2 59.7 74.6 Fraction 2 of unknown redis-

tilled. 17.4 37.1 57.3 76.0

In considering the data in Table V it is quite apparent that the results show an extremely great variation bdth in amount of acetoacetate metab- olized and in amount of acetate formed. Several factors may be enumer- ated as being at least partly responsible. First, the analytical methods are not very accurate or specific (the Edson method is described as being accurate only to within 10 per cent and it is known from his work that pyruvate, if present, interferes considerably; the acetate determination is accurate to only 10 per cent and is also subject to a loss in recovery as described). Secdnd, it is possible that there are variations in the metabolic state of the tissue. Also, the inherent difficulties of the excised tissue technique must be considered.

However, although the data are not quantitatively reproducible from animal to animal, they show qualitatively that acetate is produced only when acetoacetate disappears and that these changes do not appear when the tissue is autoclaved or treated with cyanide or mercuric chloride.

In order to confirm this suggestion attempts were then made to obtain the enzyme free of the cells.

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154 ACETIC ACID FROM ACETOACETIC ACID

Formation of Acetic Acid by Homogenized Tissue Preparations-Muscle mince-buffer suspensions homogenized by the technique of Potter and Elvehjem (18) and kidney mince ground with sand and 0.05 M phosphate buffer at pH 7.4 and then centrifuged to remove insoluble cell material showed a formation of volatile acid as acetoacetate disappeared. This acid was likewise identified as acetic acid. With the kidney preparation the results were readily reproducible although the changes were small. Both preparations retained an active oxygen uptake and metabolized acetic acid.

The formation of a volatile acid by the kidney preparation confirms Quastel and Wheatley’s (19) finding of an increased number of acid groups, as acetoacetate was utilized by kidney slices in bicarbonate buffer.

TABLE VII

Formation of Steam-Volatile Fatty Acid from Acetoacetate by Extracts of Muscle and Kidney

The incubation period was 1 hour; temperature, 38”. The data are reported in micromoles and the fatty acid figures are calculated as acetic acid.

Extract

Muscle.................... ‘I . . . . . ‘I

No substrate.............. Kidney.

‘I . . . . . . . . ‘I

No substrate.. Substrate -I- 0.01 M NaCN. Extract heated to 100”.

Initial Final acetoacetate acetoacetate

80.0 69.9 5.2 81.2 70.2 4.5 76.5 62.0 8.4

1.1 1.8 None 81.0 68.0 7.1 81.0 67.4 7.0 81.0 69.0 6.4

1.0 0.6 0.2 81.0 83.7 0.4 81.0 85.3 0.4

Acgtic;;id Per cqrt

acetoacetate recovered as acetic acid

23 21 29

27 26 27

These data, then, confirm the suggestion of the previous mince experi- ments that an enzymatic formation of acetic acid from acetoacetic acid can occur.

Many extracting procedures and media were used without success in attempts to prepare a more active enzyme concentrate. A cofactor may possibly be necessary, since well washed muscle mince did not give the reaction nor was the wash water (when concentrated in vacua) alone active. The enzyme when obtained in homogenized form was very unstable and became inactive in 1 hour at 38” and 3 hours at O”, rendering further purification difficult. The data are given in Table VII.

Formation of Acetic Acid from Acetoacetic Acid by Escherichia co&-In the search for a biochemical catalyst, a few bacterial cultures were tried.

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A. L. LEHNINGER 155

It was found that pure cultures of Escherichia coli produced acetic acid from acetoacetic acid under anaerobic conditions on a synthetic medium, on peptone, and on an autoclaved kidney extract, the reaction being of much greater extent than in the tissue experiments just described.

The synthetic medium employed was composed of 0.1 per cent K2HP04, 0.1 per cent (NH&S04, 1.0 per cent CaC03, and 0.5 per cent sodium aceto- acetate. The peptone medium was composed of 0.2 per cent peptone, 0.2 per cent (NH&SO+ 25 per cent 0.25 M phosphate buffer (pH 7.0), and 0.5 per cent sodium acetoacetate. The kidney extract was prepared by homogenizing 100 gm. of minced beef kidney in 1 liter of the Krebs-Ringer

TABLE VIII

Formation of Acetic Acid from Acetoacetic Acid by Escherichia coli

The incubation period was 24 hours at 38”. The gas phase was 95 per cent Nz, 5 per cent COZ. The data are given in micromoles.

“lflzt No. -

Synthetic medium + acetoacetic acid “ “

+ “ “ ‘I “ + “ “ ‘I ‘I without acetoacetic acid ‘I “ + 0.01 M NaCN “ “ + 0.001 M HgClz

Kidney extract + acetoacetic acid I< I, + Ii “ ‘I “ without acetoacetic acid

Peptone + acetoacetic acid * ‘I without acetoacetic acid

Initial aceto- rcetate

840 842 856

0 856 856 160 170

0 880

12

L

Final aceto- rcetatc

‘4;;; formed

97 725 90 522

110 543 0 0

833 0 863 0

32 251 34 174

4 4 210 970

22 42

1

*i

-

Per cent aceta-

acetate ecovered ts acetic

acid

49 35 37

98 64

73

buffer, centrifuging, and autoclaving the supernatant. The media were set up with the usual sterile precautions and inoculated with a loopful of a saline suspension of Escherichia coli. Blank control experiments were set up to determine the endogenous acid production. Data are given in Table VIII.

The acid formed was subjected to a Duclaux distillation and the con- stants found were identical with those of pure acetic acid.

Control experiments with an equivalent amount of acetone as substrate showed only a very slight formation of acetic acid, showing that acetone was probably not an intermediate in the reaction.

These results on the anaerobic metabolism of acetoacetic acid by Escheri- chia coli supplement those of Deotto (20) who found that aerobically

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156 ACETIC ACID FROM ACETOACETIC ACID

acetate was not an intermediate in the metabolism of acetoacetic acid by this organism.

The data on the kidney extract cultures show that, when the organism has another source of carbon, acetate is not attacked, and quantitative recovery of 2 molecules of acetic acid from 1 of acetoacetate was possible. In these experiments large amounts of acetoacetate are utilized and the more reliable macromethods can be used. These experiments conclusively show that an acid hydrolysis of acetoacetic acid is a possible cellular process.

DISCUSSION

Although the data here presented on the muscle mince experiments are admittedly not quantitatively significant, the qualitative observation that acetic acid may be formed from acetoacetic acid is well established by the experiments on the homogenized preparations and the bacterial cultures. These data show that this reaction can take place in certain cellular proc- esses and provide the first experimental basis for postulating such a reaction for the breakdown of acetoacetate.

Although it has often been suggested that acetoacetic acid is hydrolyzed to 2 molecules of acetic acid in the extrahepatic tissues, there has been no direct chemical evidence for such an assumption. Rumpf (21) found that diabetics excreted as much as 5 gm. of acetic acid in the urine per day. This may have originated by such a reaction or may have been an end- product of fatty acid oxidation in the liver, although S tadie et al. (16) were unable to show any formation of acetic acid by excised liver slices from depancreatized cats.

The results here reported show a formation of acetic acid from aceto- acetic acid which is small and irregular but definite. The mechanism of this formation is not clear from the experiments but three possibilities exist: (1) an actual hydrolysis into 2 molecules of acetic acid; (2) a phosphorolysis into acetic acid and acetylphosphoric acid; and (3) formation of acetic acid by dismutation of pyruvic acid, which may be formed from acetoacetic acid, according to the suggestions of Hoff-Jorgensen (22). In view of the evidence here presented these theories of acetoacetic acid catabolism should be retained until more chemical evidence is obtained on this long standing problem.

Very little is known about the metabolism of acetic acid. From the work of MacKay et al. (23) it is known that the phlorhizinized dog converts acetic acid into ketone bodies, but this reaction is believed to take place only in the liver and not in the extrahepatic tissues. It is more probable that acetic acid is utilized in extrahepatic tissues by some other mechanism.

All attempts to reverse this reaction in muscle mince met with failure. Although the results obtained with tissues and bacterial cultures indicate

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-49. L. LEHNINGER 157

the presence of an enzyme capable of forming acetic acid from acetoacetic acid, nothing can be said at present about the physiological importance of this transformation, except that it must be included in any consideration of the catabolism of acetoacetic acid until further evidence is obtained.

The author wishes to express his appreciation for the advice and interest of Professor E. J. Witzemann.

SUMMARY

1. Acetic acid was the only product obtained when acetoacetic acid was hydrolyzed by strong bases. No other catalyst could be found for the reaction.

2. Rabbit muscle mince formed small quantities of acetic acid from acetoacetic acid, when incubated at 38” for 2 hours.

3. Pure cultures of Escherichia coli formed large quantities of acetic acid anaerobically from acetoacetic acid.

4. The catalytic system involved in the muscle mince and the bacteria was thermolabile and possessed other classical properties of an enzyme.

BIBLIOGRAPHY

1. Loeb, A., Biochem. Z., 47, 118 (1912). 2. Edson, N. L., and Leloir, L. F., Biochem. J., 30, 2319 (1936). 3. Mathews, A. P., Physiological chemistry, Baltimore, 6th edition, 221 (1939). 4. Wislicenus, J., Ann. Chem., 190, 257 (1877). 5. von Euler, H., and iilander, A., 2. anorg. U. allg. Chem., 147,295 (1925). 6. Ljunggren, G., Ber. them. Ges., 66, 2469 (1923). 7. Neuberg, C., Der Harn, Berlin, 303 (1911). 8. Folin, O., J. Biol. Chem., 3, 177 (1907). 9. Virtanen, A. I., and Pulkki, L., J. Am. Chem. Sot., 60, 3138 (1928).

10. Van Slyke, D. D., J. Biol. Chem., 32, 455 (1917). 11. Krebs, H. A., 2. physiol. Chem., 217, 191 (1933). 12. Edson, N. L., Biochem. J., 29, 2082 (1935). 13. Dewan, J. G., and Green, D. E., Biochem. J., 31, 1074 (1937). 14. Leloir, L. F., and Munoz, J. M., Biochem. J., 33, 734 (1939). 15. Friedemann, T. E., J. BioZ. Chem., 123, 161 (1938). 16. Stadie, W. C., Zapp, J. A., and Lukens, F. D. W., J. Biol. Chem., 137,75 (1941). 17. Kruger, D., and Tschirch, E., Ber. them. Ges., 62, 2776 (1929); 63, 826 (1930). 18. Potter, V. R., and Elvehjem, C. A., J. BioZ. Chem., 114, 495 (1936). 19. Quastel, J. H., and Wheatley, A. H. M., Biochem. J., 29, 2773 (1935). 20. Deotto, It., Enzymologia, 8, 289 (1940). 21. Rumpf, L., BerZ. klin. Woch., 32, 669 (1895). 22. Hoff-Jorgensen, E., Z. physiol. Chem., 266, 56 (1940). 23. MacKay, E. M., Barnes, R. H., Carne, H. O., and Wick, A. N., J. BioZ. Chem.,

136, 157 (1940).

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Albert L. LehningerACID FROM ACETOACETIC ACID

ENZYMATIC FORMATION OF ACETICACETOACETIC ACID AND THE

THE ACID-SPLITTING REACTION OF

1942, 143:147-157.J. Biol. Chem. 

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