INCORPORATION OF THE CARBONS OF ACETONE, FORMATE, AND CARBONATE INTO ACETOACETATE” G. W. E. PLAUTt AND HENRY A. LARDY (From the Department of Biochemistry, College of Agriculture, University of Wiswnsin,, Madison) (Received for publication, June 3, 19.50) During the past 15 years it has been demonstrated repeatedly that acetone appears to be metabolized in the animal body. Several workers (l-4) have reported an appreciable rise in the P-hydroxybutyrate and acetoacetate content of various tissues and in the urine of animals which were given acetone. It was not certain from these studies whether acetone was actually converted to P-hydroxybutyrate and acetoacetate or whether it merely stimulated the production of these substances from other sources. The demonstration by Polonovski and Valdiguie (5) that acetate was pro- duced when acetone was incubat,ed in adrenal extract provided more concrete evidence for such a metabolic conversion. The studies of Price and Rittenberg (6) leave no doubt that acetone is actively metabolized in the rat. They administered acetone labeled with Cl4 in the methyl groups to rats and observed that an appreciable amount of the radioactivity appeared in the respiratory CO2 and in urinary acetyl compounds. As a result of these studies, the mechanism of the conversion of acetone to a 2-carbon fragment became of interest. Two pathways for this trans- formation were considered: (a) acetone could be split to CZ and Cl frag- ments, and (5) acetone might be condensed with a Cl compound to form acetoacetate, which in turn could split into two Cz fragments. With re- gard to the latter mechanism, an enzyme has been demonstrated in Clos- tridium acetobutylicum (7) which catalyzes the decarboxylation of aceto- acetic acid. A similar catalyst has been claimed to occur in dog blood (8, 9). Evidence will be presented in this study which demonstrates that the carbons of acetone, formate, and carbon dioxide can be incorporated into acetoacet,ic acid by rat liver preparations. Methods and Results Experiments with Slices-10 gm. of rat liver slices (0.5 mm. thick), pre- pared with the apparatus of Stadie and Riggs (lo), were suspended in * Published with the approval of the Director of the Wisconsin Agricultural Experiment Station. Supported in part by a grant from the Nutrition Foundation, Inc., and the United States Public Health Service (RG 313). t Predoctoral Fellow, National Gancer Institute. 706 by guest on March 17, 2020 http://www.jbc.org/ Downloaded from
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INCORPORATION OF THE CARBONS OF ACETONE, FORMATE, AND CARBONATE INTO ACETOACETATE”
G. W. E. PLAUTt AND HENRY A. LARDY
(From the Department of Biochemistry, College of Agriculture, University of Wiswnsin,, Madison)
(Received for publication, June 3, 19.50)
During the past 15 years it has been demonstrated repeatedly that acetone appears to be metabolized in the animal body. Several workers (l-4) have reported an appreciable rise in the P-hydroxybutyrate and acetoacetate content of various tissues and in the urine of animals which were given acetone. It was not certain from these studies whether acetone was actually converted to P-hydroxybutyrate and acetoacetate or whether it merely stimulated the production of these substances from other sources. The demonstration by Polonovski and Valdiguie (5) that acetate was pro- duced when acetone was incubat,ed in adrenal extract provided more concrete evidence for such a metabolic conversion. The studies of Price and Rittenberg (6) leave no doubt that acetone is actively metabolized in the rat. They administered acetone labeled with Cl4 in the methyl groups to rats and observed that an appreciable amount of the radioactivity appeared in the respiratory CO2 and in urinary acetyl compounds.
As a result of these studies, the mechanism of the conversion of acetone to a 2-carbon fragment became of interest. Two pathways for this trans- formation were considered: (a) acetone could be split to CZ and Cl frag- ments, and (5) acetone might be condensed with a Cl compound to form acetoacetate, which in turn could split into two Cz fragments. With re- gard to the latter mechanism, an enzyme has been demonstrated in Clos- tridium acetobutylicum (7) which catalyzes the decarboxylation of aceto- acetic acid. A similar catalyst has been claimed to occur in dog blood (8, 9).
Evidence will be presented in this study which demonstrates that the carbons of acetone, formate, and carbon dioxide can be incorporated into acetoacet,ic acid by rat liver preparations.
Methods and Results
Experiments with Slices-10 gm. of rat liver slices (0.5 mm. thick), pre- pared with the apparatus of Stadie and Riggs (lo), were suspended in
* Published with the approval of the Director of the Wisconsin Agricultural Experiment Station. Supported in part by a grant from the Nutrition Foundation, Inc., and the United States Public Health Service (RG 313).
t Predoctoral Fellow, National Gancer Institute. 706
10 ml. of a medium of the following composition: 40.3 mM of K2HP04, 8.85 mu of KHZOI, 76.6 mM of KCl, 0.38 mM of MgS04, and 10 mM of acetone. The final pH of the medium was adjusted to 7.4. NaHCY403 and HC’400K were added to this mixture as indicsted.
The reactions were carried out in 125 ml; Warburg vessels which were shaken in a 37” water bath for 2 hours. At the end of the incubation period the reaction was stopped by the addition of 5 ml. of 20 per cent copper sulfate’ solution. 0.5 mM of sodium acetoacetate (11) was then ,added as a “carrier.” The slices were removed by filtration through cheese-cloth. Freshly prepared calcium hydroxide’ suspension was added to the mixture to pH 8 to 8.5. The precipitate was centrifuged and the residue was washed by suspension in 20 ml. of water, followed by centrifuging. The supernatant solutions were combined and acidified to pH 2 to 3 with 1 ml. of 2 N HCl and diluted to 150 ml. with water. Free acetone and dissolved carbon dioxide were removed by distillation of the solution in vucuo to a volume of 10 ml. This was followed by replenishment of the volume to 150 ml. with water and another concentration in ZKWAO to 10 ml. Solid carbon dioxide was added to displace residual radioactive carbon dioxide. Oxalacetate and oxalosuccinate, which might still be present, were de- carboxylated by incubation of this mixture for 2 hours at room temperature in the presence of aluminum sulfate2 (12) while a rapid stream of COZ- free nitrogen passed through the solution. Dry ice was then added to displace any radioact.ive CO2 which may have arisen as a result of the alu- minum sulfate treatment. The excess carbon dioxide was removed with nitrogen. Aniline citrate was added to decarboxylate acetoacetate (12). The resulting carbon dioxide was swept out with nitrogen and absorbed in saturated barium hydroxide.
The acetone resulting from the decarboxylation of acetoacetic acid was separated from the residual solution by distillation. The acetone in the distillate was precipitated as the 2,4-dinitrophenylhydraxone in acid solu- tion (13). The radioactivities of the solid samples were determined in a Q-gas counter. Reported values have been corrected for background and self-absorption (14) with the self-absorption curve obtained for BaCOs. The limit of accuracy of the counts has been calculated as the standard error in all cases. All organic compounds were recrystallized to constant radioactivity; their melting points checked with those of the corresponding pure authentic non-isotopic compounds.
The experiments with slices reveal that the carbon of formate and bi-
1 In studies with homogenates perchloric acid was employed for deproteinization because the use of copper-lime tended to give erratic results.
f In a few experiments aniline citrate (12) at 0’ was used for this purpose instead of aluminum sulfate with similar results.
carbonate is incorporated predominantly into the carboxyl group of aceto- acetate (Table I). The insertion of the carbon of formate into the carboxyl of acetoacetate was enhanced when liver slices from fasted rats were used. This might be expected from the studies in ti&o of Annau (15), who observed an increased net production of acetoacetic acid in the livers of fasted rats as compared to that in fed rats.
Homogenate Experiments-Since the slice technique is cumbersome, it was of interest to find a homogenate system in which the CB to C1 conden- sation could be carried out. The reaction mixture of Potter and Reck- nagel,3 which in addition to the buffer salts contained malonate and 0.0007 M adenosinetriphosphate, was used for the subsequent homogenate studies.
TABLE I Incorporation of Cl4 from NaHCldOa and HWOOK into Acetoacetate in
Rat Liver Slices
Ex%Y’t Addition, counts per min. Radioactivity
Carbon 1 1 Carbons 2,3. and 4
9.9 J&M HC’400K, 1.34 X lo6 9.9“ ‘( 1.34 x 10’ 9.9“ ‘( 1.34 x 106 100 (‘ NaHCY03, 1.06 X lo* 100 “ “ 1.06 x lo6
c.p.m. per mdb 1,280 f 30
23,800 f 386 9,350 f 92 2,280 f 44
12,400 f 100
e.p.m. per m.w
170 f 79 370 f 58 590 f 89 400 f 79
In Experiment 1 the rat was fed and in Experiments 2 to 5 the animals were fasted 36 to 48 hours prior to the experiment. Incubated at 37” in air for 2 hours.
The amounts of radioactive indicators and the enzyme concentrations employed are noted in Tables I to VI. The amount of acetoacetate carrier added and the subsequent method of degradation were the same as those described for the experiments with slices.
As in the case of the slices, the homogenates incorporated a significant amount of the carbon of formate and of carbonate into the carboxyl group of acetoacetate. Even when NaHC1403 of very high specific activity was employed and a particularly large incorporation of Cl4 into the carboxyl group of acetoacetate resulted, the activity of the acetone portion of the molecule was very small (Table II). It is uncertain whether formate is in- corporated into the carboxyl grou’p of acetoacetic acid as such or whether it has to be converted to carbon dioxide prior to being thus utilized. Under the conditions of our experiments enough carbon dioxide is formed from
J We wish to thank Dr. Van R. Potter and Dr. R. 0. Recknagel for providing this information before publication.
9.6 ml. of 20 per cent homogenate in isotonic KC1 were added to all the flasks except in Experiment 1 in which 4.8 ml. of 10 per cent homogenate were used. Rat liver was used in all the experiments except in Experiments 10 and 11. Final vol- ume 17 ml. Incubated at 37” in air for 2 hours except in Experiments 9 to 12.
* 70 per cent of the radioactivity of the added formate was recovered in the respiratory COz after stopping the reaction with HC104. The respiratory CO* had a specific activity of 1.57 X lo7 c.p.m. per mM.
t Homogenate heated in a boiling water bath for 15 minutes. $ Incubated for 15 minutes. 8 Heart homogenate. 20 PM of acetoacetate present during the 15 minute in-
cubation period. 11 Kidney homogenate. 20 PM of acetoacetate present during the 15 minute
incubation period. 7 This value represents the actual specific activity of the carboxyl group of
acetoacetat,e and was derived as follows: after the addition of HC104 at the end of the 15 minute incubation period, the resulting mixture was divided into two portions. Acetoacetate carrier was added to an aliquot and the specific activity of the carboxyl group was determined as usual (4900 c.p.m. per mM). The aceto- acetate concentration in the filtrates with and without added carrier acetoacetate was determined (18) and was found to be 50.5 X 10-s rnM per ml. and 0.19 X 10-J mM per ml. respectively. Then the specific. activity of -COOH (plus carrier) X (acetoacetate (carrier present))/(acetoacetate (carrier absent)) = 4900 X ((50.5 X 10-s)/(0.19 X lo+)) = 1.3 X lo6 c.p.m. per mM = actual specific activity of -COON.
formate to account for the insertion of the carbon of formate into aceto- acetate via carbonate (Experiment 3, Table II). Fixation of carbon dioxide
into the carboyxl group of atietoacetate was observed with kidney.homo- genates but it is not. certain whether this reaction occurs to any significant extent in heart. muscle preparations (Table II).
Incorporutian of &etone--Carbonyl-labeled acetone was prepared by pyrolysis of carbmyl-labeled barium acetate (14). The degradation meth- ods employed in these experiments were identical with those described except that during the course of the removal of the residual, free isotopic acetone in vacua large quantities of non-isotopic acetone (2.7 moles) were added. This treatment was adequate to prevent significant contamination of acetone obtained from the acetoacetate degradation by the labeled com- pound initially added.
TABLE III Incorporation of Cl4 from Carbon&Labeled Acetone into Acetoacetate
in Rat Liver Homogenates
Experiment No.
1 2
Malonate
Y
0.0028 0.0071
Carbon 1
C.).rn. jer nzY
2110 f 37 790 f 17
Radioactivity
Carbons 2, 3, and 4 Respiratory COP
C.#.rn. )W n&M c.p.m. per m24
9,800 * 98 1480 f 26 10,000 zt 84 875 f 33
33 PM of carbonyl-labeled acetone, 1.11 X 106 c.p.m., and 9.6 ml. of 20 per cent rat liver homogenate per flask. Final volume 17 ml. Incubated at 37’ in air for 2 hours.
* The specific activity of the respiratory COz cannot be compared directly with that in the acetoacetate since no “carrier” was present during the incubation period.
The results of this study (Table III) indicate that the carbons of added acetone are incorporated into the acetone portion of acetoacetate. In the case in which only 0.0028 M malonate was used to inhibit the tricarboxylic acid cycle, .‘a considerable portion of the carbonyl carbon of the added .acetone was found in the carboxyl groups of the formed acetoacetate. When the malonate concentration was increased to 0.0071 M, the incor- poration into the carboxyl groups was decreased while the radioactivity of the acetone portion remained constant. A small amount of the activity was also fo,und in the respiratory COZ. This conversion of acetone to car- bon dioxide may be due to incomplete blocking of the Krebs cycle by malonate or to the exchange of the carboxyl of formed acetoacetate with the surrounding medium.
While the incorporation of the carbonyl carbon of acetone into aceto- acetate was demonstrated (Table III), the insertion of CY4-carbonate into the carboyxl group was not enhanced by the addition of acetone to the homogenate or washed residue enzyme systems (Table IV). It was con-
sidered that ‘the ladk of effect of acetone here might be caused by the endogenous production of an adequate amount of acetoacetate by these systems. A requirement for acetone could, however, not be shown even in the presence of atoxyl (Table IV), which Jowett and Quastel (16) found to decrease endogenous acetoacetate formation. Furthermore, the addi- tion of acetoacetate to incubating liver homogenates did not lead to an increased fixation of CO:, into the carboxyl group. However, the incorpora- tion of carbon dioxide into the carboxyl group of acetoacetate was enhanced by the addition of pyruvate (which yields acetoacetate under’ these experimental conditions ((17); see also foot-note 3) to the incubation
TABLE TV Eflect of Acetone on Zncorporalion oj Cl4 jrom iVaHPOa into Carboxyl
Group oj Acdoacetate
Experi- ment No.
- _
-
Homogenate “
Washed residue I‘ “ “ ‘L “ ‘I “ “ 4‘ “ “ “
- .
-
Addition
1 pM acetone No acetone 1 #bf acetone 1 “ “ + 1 PM or-ketoglutarate 1 “ or-ketoglutarate 1 “ acetone No acetone 1 PM acetone, 0.01 M atoxyii No acetone, 0.01 M atoxyi
15,303 f 125 29,700 * 211 33,500 i 261 11,500 i 151 9,200 i 143
5.4 PM of NaHCY40r at 8.17 X 106 c.p.m. were added to all t.he flasks. Incubated at 37” in air for 2 hours; 0.0071 M malonate present.
* From rat liver tissue. t Sodium arsanilate.
mixture (Experiment 8, Table VI). It is not clear whether this effect is due to the production of a nascent form of acetoacetate from pyruvate or to the generation of energy utilizable in the condensation reaction. The chemical methods employed may not differentiate between acetoacetate and possible derivatives of this compound which may be present. The actual endogenous acetoacetic acid formation was determined4 by Leh- ninger’s unpublished modification of the method of Greenberg and Lester (18) in one experiment in which a washed liver homogenate was used as the enzyme. On the basis of this assay and the specific activity of added NaHC403, it was calculated that on a molar basis 0.83 per cent of the
4 We are indebted to Miss Gladys Feldott who conducted the dietary experiments and performed the acetoacetate determinations.
harboxyl groups and of the acetoacetate present had been derived from bicarbonate (Experiment 12, Table II).
Experiments with P-Hydroxybulyraie- To eliminate the clement of un- certainty regarding the specificity of the direct degradation of aceto- acetate in the deproteinized medium it seemed desirable to use a more specific method of degradation. Since /%hydroxybutyric acid and accto- acetic acid are interconvertible in the liver (19), a procedure, based on the isolation of &hydroxybutyric acid and its subsequent degrttdation, was developed. At the end of the incubation period, 180 mg. of carrier sodium nn-fl-hydroxybutyrate were added to the reaction mixture. After de- proteinization with copper-lime, the filtrate was acidified with 1 ml. of 7 N sulfuric acid. Volatile fatty acids were removed by steam distillation. The residual solution was neutralized to pH 5 to G with sodium hydroside. 1 gm. of sodium bisulfite per 15 ml. of solution was added to combine with
keto acids. 7 gm. of MgS04*7H,O and 0.45 ml. of 7 N HS04 were added and the solution was continuously extracted with ether for 96 hours. The extract was evaporated to dryness in vacua. The /3-hydroxybutyric acid in the residue was converted to crotonic acid by refluxing with 50 per cent H&O4 according to the method of Darmstadter (20). The crotonic acid in the distillate was extracted into ether. The ether layer (400 to 500 ml.) was washed two to three times with 5 ml. of water and dried with anhydrous Na304. The dried ether extract was evaporated to 10 to 15 ml. and an ethereal solution containing 0.8 gm. of p-dimethylaminophenylcarbo- diimide was added. The solution was refluxed for several hours and left to stand at room temperature for a few days until the precipitation of the ureide of crotonic acid was complete (21, 22). The ureide (m.p. 151-152’) was recrystallized to constant radioactivity. It was hydrolyzed with 85 per cent H3P04 according to Zetzsche and Riittger (21). The acid solu- tion was diluted with 2 volumes of water and extracted with ether; the sol- vent was washed with a few small volumes of water and the crotonic acid
was extracted from the solvent with 15 ml. of 0.1 N. NaOH. The, aqueous solution was chilled to 0” in an ice bath and the crotonate was oxidized to dihydroxybutyrate (23) by the dropwise addition of dilute KMnOa to the first, tinge of pink. The excess KMn04 was destroyed immediately with a small quantity of sodium hydrosulfite and MnOz was removed by centrif- ugation. The supernatant was acidified with 0.3 ml. of glacial acetic acid and evaporated in vacua to 10 ml. The solution was adjusted to pH 4 to 5 with 0.1 N NaOH. The 2,3-dihydroxybutyric acid was degraded to COZ, formic acid, and acetaldehyde by the addition of 5 ml. of 0.1 M
periodic acid, and the products were collected separately. The carbon dioxide was precipitated as BaC03, acetaldehyde was converted to the 2,4- dinitrophenylhydrazone, and formic acid was decomposed to carbon dioxide
TABLE V
Incorporation of HPOOH and NaHPOJ into &Hydroxybutyrate by Rat Liver
Radioactivity Experi-
ment No. Addition, counts per min. Ureide &irbon of Carbon 1 Carbon 2
crotonic acid Et?: ~~
c.p.m. per mv C.).rn. per mar c.fi;i@r c,yg&fer
1 19.8 PM HCY’OOK 2.68 X lo6 2 5.4 “ NaHWOn: 8.17 X lo6
1210 i 291210 * 20 0 0 1400 f 361225 f 17 0 0
In Experiment 1, slices incubated aerobically for 1 hour; 180 mg. of sodium DL- ,%hydroxybutyrate then added and incubated under Nz for 45 minutes.
In Experiment 2 with homogenate, 0.0071 M malonate present. Homogenate incubated in air for 30 minutes. Then 75 PM of a-ketoglutarate were added and incubated under N2 for 30 minutes. Final volume 17 ml. The oxidation of a-ke- toglutarate is coupled with the reduction of acetoacetate (36).
with mercuric chloride (24). Pilot experiments with non-isotopic crotonic acid revealed that a 90 per cent, recovery of the split-products (Equation 3) could be obtained.
The data obtained with this procedure revealed that the carbons of formate and carbon dioxide were incorporated exclusively into the carboxyl group of P-hydroxybutyric acid (Table V). It should be pointed out that no insertion of the carbon of formate or carbon dioxide into @-hydroxy- butyrate could be demonstrated unless the additional anaerobic reductive steps described in Table V were used. This may indicate that very little or no P-hydroxybutyrate is formed under our aerobic experimental condi- tions.
E$ect of Biotic-Biotin has been implicated in a number of carbon dioxide fixation reactions (25-29). A study was made to ascertain whether a similar situation prevailed in the acetoacetate reaction.
Weanling Sprague-Dawley rats were grown for 10 weeks on a purified diet? containing raw egg white without added biotin (27).4 An equal number of rats was fed a similar diet but with added biotin and without raw egg white, and their food intake was restricted to that of a biotin- deficient animal. The animals fed the egg white diet developed typical symptoms of biotin avitaminosis.
TABLE VI Effect of Biotin Deficiency on Incorporation of 04 from NaHC1403 into Carboxyt
5* 6
7
8A SBt.
Group of Acetoacetate
I Radioactivity - Control ration fed
Control anims1
c.@?z. per n&M
+ dietary biotin, pair-fed 44,200 f 398 “ ‘I “
; <‘ ‘L I‘ 30,200 f 151 20,600 f 144
+ “ “ “ 48,000 i 215 Stock ration ad libitum 50,300 zk 302 + dietary biotin, pair-fed 22,400 f 163 Biotin-deficient + injected biotin,? 52,500 i 210
9.6 ml. of 20 per cent rat liver homogenate (variations of tissue dry weights be- tween different flasks did not exceed f5 per cent); final volume 17 ml., 0.0071 M malonate present. Homogenates incubated in air at 37” for 120 minutes, except Experiments 5 and 8. 5.4 PM of NaHCP03 containing 8.17 X 106 c.p.m. added ex- cept in Experiment 8 in which 2.7 PM of NaHWOa containing 4.08 X lo6 c.p.m. were used.
* 20 PM of acetoacetate present during the 15 minute incubation period. t 50 y of biotin injected intraperitoneally daily for 4 days. $60 NM of sodium pyruvate present during the 15 minute incubation period.
A smaller incorporation of Cl4 from carbonate into the carboxyl group of acetoacetate was obtained in every case when liver homogenates from biotin-deficient rats were used in comparison with enzyme preparations from either deficient animals injected with biotin or from control rats (Table VI). These differences were particularly apparent when the in- cubation time was reduced from 120 to 15 minutes (Experiments 5 and 8, Table VI).
6 The vitamin levels quoted previously (27, 28) were for 10 gm. of ration rather than 100 gm., as stated.
It appears from this data that the state of biotin nutriture influences this new carbon dioxide fixation reaction too.
DISCUSSION
The equilibrium of the reaction
Acetoacetic acid e acetone + CO*
is so far in the direction of decarboxylation that it would be impossible to explain the incorporation of Cl4 into acetoacetate found here merely by an equilibrium exchange of the reactants and products.” Therefore, it is likely that an energy-coupling process is a part of the mechanism of this reaction.
The failure of Swendseid et al. (30) to demonstrate any incorporation of Cl302 into ketone bodies in the whole rat may have been due to the ex- cessive dilution of the isotope in the body, the rapid loss of the carton in the respiratory carbon dioxide, and the competition of more rapid carbon dioxide fixing reactions.
The C3 + C1 condensation leading to acetoacetate could provide a path- way by which carbon dioxide could be inserted into the carboxyl group of “acetate.” This mechanism may account for Schubert and Armstrong’s (31) observation that the carboxyl groups and presumably the other odd carbons of fatty acids were tagged as a result of the administration of CL4- carbonate to rats. It has already been pointed out by others (32,33) that such a labeling of the fatty acids could not occur by the metabolism of COZ via the tricarboxylic acid cycle.
The finding by Coon and Gurin (34) that the y-carbon of leucine appears in the carbonyl group of acetoacetate could be explained by the reaction studied here, particularly since their evidence points to the formation of an acetone-like fraction from the isopropyl group of leucine.’ In this con- nection it has been observed by Valdiguie and Seguelas (35) that acetone was produced when cholesterol and coprosterol were incubated in liver es-
B The equilibrium constant was calculated from the free energy of formation of the reactants and products at 37.6’. The values of the free energies of formation are for 1 molal activity, except for water;. in the latter case they are for pure water. These values are from unpublished work of Professor Henry Borsook, California Institute of Technology. We wish to thank Professor Borsook for providing these data.
AF = -16,300 calories; K = ((acetone) (HC03)/(acetoacet,ate-) (HZO)) = 3 X 10”. 7 In a personal communication Dr. M. J. Coon and Dr. S. Gurin have informed us
that they too have observed the incorporation of the carbon of carbon dioxide into the carboxyl group of acctoacetate.
tracts. These workers are of the opinion that this acetone is derived from the isopropyl portion of the sterol side chain.
SUMMARY
The carbons of formate and carbonate were incorporated into the car- boxy1 group of acetoacetate by rat liver slices and homogenates. This finding was confirmed by the isolation and degradation of ,&hydroxybutyric acid, the reduction product of acetoacetate. Additions of pyruvate to the reaction mixture increased the fixation of carbon dioxide into acetoacetate, but acetone and ar-ketoglutarate did not enhance this incorporation of COZ.
Formate was rapidly oxidized to carbon ,dioxide by rat liver prepara- tions and possibly was incorporated into acetoacetate only after this con- version.
Carbon dioxide was also fixed into the carboxyl group of acetoacetate by kidney homogenates and perhaps in the presence of heart enzyme prepara- tions.
In rat liver homogenates the radioactivity of carbonyl-labeled acetone was incorporated predominantly into the acetone portion of acetoacetate. When low concentrations of malonate were used to inhibit the tricarboxylic acid cycle, a considerable portion of the carbonyl carbon of added acetone was also found in the carboxyl group of acetoacetate. A small portion of the Cl4 from added acetone was also detected in the respiratory carbon dioxide.
The fixation of carbon dioxide into acetoacetate by liver homogenates from biotin-deficient rats was much less than by homogenates from rats receiving biotin.
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