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STUDIES

OF ACETOACETATE FORMATION CARBON*

I. EXPERIMENTS

WITH PYRUVATE, WASHED LIVER BY

(From

the

DANA

Department

ACETATE, AND HOMOGENATES

I. CRANDALL

of Physiological of Pennsylvania,

WITH

AND

SAMUEL

Chemistry, School Philadelphia)

(Received for publication,

FATTY

LABELED ACIDS

IN

GURIN of Medicine,

University

June 1, 1949)

EXPERIMENTAL

Organic Syntheses-Acetate labeled with Cl3 in the carboxyl position was prepared from NaCY3N by the Walden (4) reaction. Pyruvate containing Cl3 or Cl4in the a!and @positions was in someinstances synthesized by the method previously reported by Sakami, Evans, and Gurin (5) ; it was also prepared from doubly labeled acetate by way of acetyl bromide, pyruvonitrile, and pyruvamide (6). Carboxyl-labeled octanoic and hexanoic acids were prepared from NaCY3N and the appropriate alkyl bro*Aided

by a grant from the American Cancer Society administered by the Comof the National Research Council. Some of the material in this paper has been published (Cold Spring Harbor Symposium on Quant. Biol., 13, 118 (1948)). a29

mittee on Growth

Downloaded from http://www.jbc.org/ by guest on December 11, 2017

Lehninger (1) has described the preparation of a liver homogenate which converts pyruvate or fatty acids into acetoacetate in good yield and which consumeslittle or no oxygen and does not produce acetoacetate endogenously. The parallel behavior of octanoate and pyruvate in this preparation with regard to acetoacetate formation and the suppression of this process by the addition of members of the tricarboxylic acid cycle (1) emphasize the question of whether pyruvate and fatty acids give rise to identical intermediates in the process of forming acetoacetate or tricarboxylic acids. In the experiments reported here an attempt has been made by use of substrates labeled with Cl3 or CY to determine whether or not pyruvate and octanoate give rise to a common intermediate in the process of forming acetoacetate and whether, if different, these intermediates can interact to produce acetoacetate of dual origin. It was also felt worth while to repeat, in this system, the experiments of Weinhouse et al. (2) and Buchanan et al. (3) on the conversion of carboxyllabeled octanoate to acetoacetate and also to study the distribution of isotope in the acetoacetate formed from isotopically labeled pyruvate.

830

ACETOACETATE

FORMATION.

I

mides. After hydrolysis of the resulting nitriles, the acids were distilled in vucuo. For the preparation of octanoic acid labeled in the p position (see the accompanying diagram), carboxyl-labeled hexanoic acid was * * CHa(CHJCOOGH6 + CHa(CH&:HzOH + CHs(CH&aCHsBr -+ * CH~(CHZ)&H&H(COOCZH~)~ ---) CHs(CHJr:H&H&OOH


converted to the ethyl ester and reduced with LiAlHd to hexyl alcohol. The alcohol was converted to hexyl bromide and condensed with ethyl malonate (7). After saponification and decarboxylation of the resulting product, b-labeled octanoic acid was separated and purified by distillation. Other Compounds--Adenosine triphosphate (ATP) was prepared by the method of Kerr (8) as modified by Polis and Meyerhof (9). It was also obtained commercially as the tetrasodium salt from the Rohm and Haas Company. Crystalline sodium pyruvate was prepared from redistilled pyruvic acid which was dissolved in a minimal quantity of water, neutralized with sodium carbonate, evaporated in vucuo until crystallization began, and treated with small amounts of ethyl alcohol to induce further crystallization. This product was then recrystallized from water and ethyl alcohol. N-Octanoic acid was obtained from the Eastman Kodak Company. Malonic acid was purified via the calcium salt (10). Preparation and Incubation of Homogenates-Washed homogenates of rat liver were prepared in a cold room (3”) according to the procedure described by Lehninger (11) with the slight modifications mentioned below. Since large quantities of homogenate were needed, a 100 ml. glass Potter-Elvehjem homogenizer was used. In order to obtain active preparations with this unusually large homogenizer it was necessary to run it, previously chilled, at 90 R.P.M. and not to attempt a complete excursion of the plunger without intermittent chilling of both plunger and cylinder. After one excursion of the plunger, the crude homogenate thus obtained was filtered through gauze, distributed among a sufficient number of 15 ml. conical centrifuge tubes, chilled again, and centrifuged for 7 minutes at 4000 R.P.M. in an angle head centrifuge. Although in Lehninger’s original procedure the crude homogenate was centrifuged and resuspended four times, we found that the last two washings could be omitted without changing the character of the homogenate. The homogenates were incubated in a medium essentially that of Lehninger (11). The incubation mixture consisted of one-third by volume of the washed homogenate and cont.ained phosphate buffer, pH 7.7,0.0085 M, MgSOa 0.0053 M, and ATP 0.0021 M. Malonate was included in the medium at a final concentration of 0.01 M except in the experiments

D.

I.

CRANDALL

AND

S.

GURIN

831

1 We are indebted analyses.

to Dr. Sidney Weinhouse

and the Sun Oil Company for these


involving octanoic and hexanoic acids. The concentrations of substrates are listed in Tables I to V. The substrates were added to the medium prior to the addition of cold homogenate in an attempt to preserve the activity of the enzyme systems. In order to obtain sufficient amounts of acetoacetate for chemical degradation and isotopic analysis, 75 ml. of combined homogenate and medium were incubated in each experiment. The gas phase was air. It was found that rapid shaking of the incubation flask inactivated the fatty acid oxidase system so that, in order to minimize the amount of shaking necessary for oxygen diffusion, the 75 ml. of fluid were equally divided among four 125 ml. Erlenmeyer flasks which contained alkali insets and which were tightly stoppered. The flasks were then shaken gently in a water bath at 25” until acetoacetate formation reached a limit. In order to follow the course of the reaction, small aliquots of the reaction mixture were incubated simultaneously in Warburg manometer vessels. The relative shaking rates of the manometer vessels and the Erlenmeyer flasks had been previously adjusted so that the oxygen uptake of the manometer vessels gave indication of the extent of oxidation taking place in the large flasks. At the end of the incubation, the contents of the flasks were combined and small aliquots withdrawn for the determination of pyruvate or acetoacetate. Acetoacetate was determined manometrically by the method of Edson (12) and pyruvate by the method of Friedemann and Haugen (13). The bulk of the homogenate was then divided into two portions. From approximately two-thirds of it COZ, collected as BaC03, was obtained from the carboxyl group of acetoacetate by a procedure previously described, involving treatment with aniline citrate (3). The mercury-acetone complex derived from the acetoacetate was obtained from a copper-lime filtrate prepared from the remaining third of the homogenate (3). The BaC03 was analyzed for Cl3 by a mass spectrometer’ or for Cl4 in a Geiger counter (14). The mercury-acetone complex was oxidized to COZ and water, and this CO2 was analyzed for Cl3 or U4. Since it has been previously shown that all of the isotope in the acetoacetate formed from carboxyl-labeled octanoate is located in the carbonyl and carboxyl carbons of acetoacetate (2), we calculated the concentration of isotope in the carbonyl carbon by multiplying the value obtained for acetone by 3 for all acetoacetate derived from monolabeled fatty acids. A few degradations of acetoacetate were carried out by the heat decarboxylation of copper-lime filtrates prepared from entire homogenates. Here, liberation of carboxyl COZ and the formation of the mercury-acetone

832

ACETOACETATE

FORMATION.

I

complex were accomplished simultaneously by heating the COz-free filtrate to 100” in the presence of DenigBs’ reagent. Results

Acetoacetate Formatic

In f

-

TABLE I iom Pyruvate Containing

1Ynitial concen tration of PYmT;g; in

Pyruvate utilized

t ExplYiP

Final total acetoacetate

-M

1 2A 2B 3A 3B t Cl3 used.

0.0120 0.0222 0.00 0.0112 0.00

C’s or Cl4 in LY-and &Carbons Atom per cent excess C’s or counts carbon per min. for

PM

PM

244 545

137 176 0 238

735

-

In this experiment

cc- and j3carbons of added pynlvate

Final EH3~OEHl-

1.341 27$

56 711

4606

per mg.

acetoacetate

--EOOH 1.37t 321 4555

O I the respiratory

CO* contained

0.07 atom per cent

03. 1 Wused.

carbons equal to the concentration of isotope in the acetyl carbons of the added pyruvate. Furthermore, the carbon dioxide formed in the reaction should contain no isotope, since the carboxyl group of the added pyruvate contained none. The experiments (Table I) show that the concentrations of isotope in the acetone and carboxyl carbons of the acetoacetate formed in the homogenate were essentially equal, indicating that all 4 carbons of the acetoacetate contained an equal concentration of isotope. In Experiment 1, metabolic carbon dioxide obtained from the incubation mixture contained little more than a trace of isotope. Since in this preparation COZ does not arise endogenously (1 l), this COZ probably arose from the unlabeled carbon of pyruvate. These results are in agreement with the conclusion that the cw,p-carbons of pyruvate contribute equally to both halves of acetoacetic


Conversion of cr,p-Labeled Pyruvate to Acetoacetute-The formation of acetoacetate from pyruvate by liver and other tissues is a well established metabolic reaction (15-17). It has been generally assumed that an acetoacetate molecule arises from the acetyl portions of 2 pyruvate molecules whose carboxyl carbons have been eliminated as COZ. If only the acetyl carbons of pyruvate are utilized in this reaction, incubation of ar,p-labeled pyruvate in this system would be expected to give rise to acetoacetate containing a concentration of isotope in each of its

D.

I.

CRANDALL

AND

S.

GURIN

833

* A personal communication from Dr. John M. Buchanan.


acid, while the carboxyl carbon is lost as COZ. Further evidence for this conclusion is provided by the formation of unlabeled acetoacetate when carboxyl-labeled pyruvate was incubated with rat liver slices.2 In Experiments 2A and 3A a comparison of the concentrations of isotope in the acetyl carbons of the added pyruvate with that of the carbons of acetoacetate shows that an almost 2-fold dilution of isotope occurred during this conversion. The nature of this dilution is unexplained. That these particular homogenates did not produce or contain acetoacetate in the absence of added substrate is shown in control Experiments 2B and 3B. Except in the case of Experiment 1, the disappearance of pyruvate was more than adequate to account for the acetoacetate formed. In contrast to this, a relatively slight dilution of isotope was observed in the acetoacetate formed from isotopically labeled fatty acids (Table IV). Conversion of Carboxyl-Labeled Acetate to Acetoacetate-Preliminary analytical studies in this laboratory showed that although, in agreement with Lehninger (ll), acetate alone will not form acetoacetate in this homogenate, when supplemented with pyruvate it gave slight but reproducible increases in the yield of acetoacetate. An attempt to determine the extent of incorporation of acetate carbon into the two halves of acetoacetate was made by incubating carboxyl-labeled acetate and unlabeled pyruvate together in the homogenate. The results (Table II) show that in the presence of unlabeled pyruvate a small but significant incorporation of acetate carbon into acetoacetate occurred, and that within the limits of experimental error the isotope was equally distributed between the carbonyl and carboxyl positions of the acetoacetate. On the average, 5 per cent of the total acetoacetate formed arose from labeled acetate. The amount of incorporation of isotope was independent of the concentration of acetate in the medium in the range studied (0.020 to 0.001 M). A possible interpretation of these results is that amounts of acetoacetate, too small to be detected by the analytical methods used, are actually formed from acetate, thus accounting for the small concentration of isotope found in the acetoacetate of Experiments 1, 2,3A, and 4A (Table II). This possibility was eliminated, however, by the control Experiments 3B and 4B in which portions of the same homogenates used in Experiments 3A and 4A, respectively, were incubated with carboxyl-labeled acetate alone. An amount of unlabeled acetoacetate comparable to that formed from pyruvate in Experiments 3A and 4A was mixed with the homogenate either before or after the incubation, and at the end of the incubation it was decomposed into acetone and CO2 for isotopic analysis. Here the traces of isotope found were relatively insignificant, indicating that the

834

ACETOACETATE

FORMATION.

I

conversion of acetate to acetoacetate depended entirely on pyruvate in these experiments. Similar experiments were carried out with carboxyl-labeled acetate and unlabeled octanoate as the active cosubstrate. In these experiments (Table III) it was not possible to include malonate in the medium, since washed homogenates of the liver of rats of the Wistar strain do not oxidize octanoate in the presence of malonate. These homogenates were highly active in the absence of malonate, but, unlike Lehninger’s preparaTABLE

Initial concentmtion~no~fsu~ate Expelimeat No.

1

2 3A 3B 4A 4B

Carboxyllabeled acetatef ~---___-M

0.020 0.006 0.006 0.006 0.001 0.001

II

of Isotope in Acetoacetate Derived from Carboxyl-Labeled Presence of Non-Labeled Pyruvate

Acetoacetate formed

Acetoacetate added

Total scetoacetate

(A) II

PM

264 195 185.5 (1.1) 276 (0.8)

PM

cx

0 0

264 195 186 213 276 164

21:g 16;,,

“istrp;o rcetoacetatl

-

Pymvate

0.015 0.015 0.015 0 0.008 0

Atom per cent excess C’a in total acetoacetate t0 (W

EOOH (0

Acetate in

EO

Acetate converted to acetoacetate3 (calculated)

OOH

lrdl

0.21 0.36 0.39 0.03 0.30 0.03

0.23 0.39 0.35 0.03 0.30 0.03

0.91 0.92 1.11 1.00

19.5 24.4 22.9 2.1 27.6 1.6

t Acetoacetate derived from acetate= micromoles total acetoacetate (atom y. excess Cl3 in carboxyl + carbonyl carbons) atom $!Jcexcess 08 in carboxyl carbon of acetate Column A X Columns B + C = atom y. excess Cl3 in carboxyl carbon of acetate t: The carboxyl carbon contained 6.0 atom per cent excess 03. $ Added after incubation period prior to degradation of acetoacetate into acetone and CO,. 11Added at beginning of incubation period.

tions, exhibited a slight residual oxygen uptake and were capable of converting acetate to acetoacetate to an extremely small but variable extent. No acetoacetate was formed in the absence of added substrates. It was also observed that frequently concentrations of acetate above 0.001 M partially inhibited the oxidation of octanoate. Experiments 1, 2, and 3 (Table III) show that in the presence of nonlabeled octanoate a 2- to 6-fold increase in the conversion of labeled acetate to acetoacetate occurred. Only 6 to 10 per cent of the total acetoacetate formed arose from labeled acetate. The distribution of Pa between the


Incorporation

D. I.

CR4NDALL

AND

835

S. QURIN

carbonyl and carboxyl positions of the aeetoacetate was significantly uneven and when Experiments 1A and 2A were compared with their respective controls, Experiments 1B and 2B, which were run on portions of the same homogenate in each case, it was possible to calculate, as shown in Table III, the distribution of isotope between the carbonyl and carboxy1 positions of the increment of isotopic acetoacetate whose formation depended upon the simultaneous oxidation of unlabeled octanoate. The Acetate in

-

EXperimerit No.

Initial concentrations of substrates in medium I

Acetoacetate formed

?z?%l Octaacetatetmate

Total acetorcetate

acetate

(A)

EO (B)

EOOH (Cl

PM

PM 0

I.rx 49

0.51 0.01

IA 1B

0.002 0.002

0.001 49.1 0 (0.4)

227

227

0.27 0.01

2A 2B

0.004 0.004

0.001163 0 (8)

0 215

163 223

0.51 0.21

0.66 0.20

3A 3B

0.001 0.001

0.001 136 0 (2)

0 165

136 167

Lost “

0.57 0.07

L

t Calculation acetoacetate =

-

of micromoles

Distribution of$ce~~~ acetoacetate

C:HadO0)

--CI&~OOH 09

CI&-CHr&3OH

_

-.

lb

Acetate converted to acetoacetate~ (calculated) --

added nitially

--

II

Atom per cent excess Cl3 in total acetoacetate

-

of acetate converted

lr-+f

NM

2.2 0.4 1.85 13.9 7.8 G

4.2 0.4 E$ 18.0 7.4 lo.sg 12.9 2.0

-

to CH&O-

0.478

0.588

or -CH&OOH

Column A X Column B or C = atom ‘% excess 03 in carboxyl carbon of acetate (6.0) $ Carboxyl carbon contained 6.0 atom per cent excess 08. g These figures concern the increment of acetate carbon whose incorporation acetoacetate is due to the presence of non-labeled octanoate.

of

micromoles

into

60:600H ratios3 for these increments show that approximately twice as much acetate carbon was incorporated in the carboxyl half of the acetoacetate as in the carbonyl half. This indicates that at least 50 per cent of this labeled acetoacetate must have been purely carboxyl-labeled and therefore must have arisen as a result of the acetylation of carboxyl-labeled acetate (or an active derivative) by non-labeled 2-carbon compounds formed by octanoate. s To be used henceforth to designate the ratio of the concentration of labeled carbon in the carbonyl position to that in the carboxyl position of acetoacetate.


TABLE III of Isotope in Acetoacetate Derived from Carboxyl-Labeled Presence of Non-Labeled Octanoate

Incorporation

836

ACETOACETATE

FORMATION.

I


The incubation of carboxyl-labeled acetate with liver slices has been reported by Weinhouse, Medes, and Floyd (18) to result in the formation of asymmetrically labeled acetoacetate with a EO:EOOH ratio of 0.81, suggesting that in the liver slice the endogenous oxidation of fatty acids exerted a similar influence on the conversion of acetate to acetoacetate. Conversion of Labeled Fatty Acids to Acetoacetate-In the experiments of Weinhouse, Medes, and Floyd (2) the incubation of carboxyl-labeled octanoate with rat liver slices resulted in the formation of labeled acetoacetate, with an equal distribution of the isotope between the carbonyl and carboxyl carbons. These results, considered together with the evidence reported by Buchanan, Sakami, and Gurin (3) that carboxyl-labeled acetoacetate is not converted to symmetrically labeled acetoacetate by rat liver slices, are in accord with the theory proposed by Ma&Kay et al. (19) that fatty acids are completely converted into a single species of 2-carbon units which undergo random condensation to form acetoacetate. In an attempt to repeat the experiments of Weinhouse et al. (a), Buchanan et al. (3), using identical conditions, ibta$red labeled acetoacetate from carboxyl-labeled octanoate with a CO:COOH ratio of 0.65. These authors considered this asymmetrically labeled acetoacetate to have arisen partially by multiple alternate oxidation. In view of the experiments reported in this paper another explanation seems more likely. When carboxyl-labeled octanoate was incubated alone in the washed liver homogenate, the distribution of the isotope in the resulting acetoacetate was highly unequal, a large excess of isotope appearing in the carboxyl position (Table IV, Experiments 1, 2, 3A, 4A, 5A, 5B, and 6). The average ?!O:;OOH ratio was 0.66. Similar results were obtained with carboxyl-labeled hexanoate (Experiments 10 and 11). In each of these experiments the dilution of isotope accompanying the conversion of the labeled fatty acid to acetoacetate was calculated by comparing the average concentration of Cl3 in the carbons of acetoacetate with the average concentration of Cl3 in the carbons of the added monolabeled fatty acid. It was found that the acetoacetate contained, on the average, 85 per cent of the over-all Cl3 concentration of the original fatty acid; the highest value observed was 94 per cent. The figures indicate either slight dilution by endogenous acetoacetate formation or perhaps slight contamination of the mercury-acetone fraction, which will be discussed below. Whatever the cause, the dilution is relatively small in comparison with the dilution of isotope observed in the acetoacetate formed from isotopically labeled pyruvate (Table I) in which only 61 per cent of the expected concentration of isotope was found in the acetoacetate.

D.

I.

CRANDALL

AND

S.

837

GURIN

If the appearance of more Cl3 in the carboxyl than in the carbonyl position of the acetoacetate was due to the occurrence of some multiple alternate oxidation of the carboxyl-labeled octanoate, then ,&labeled octanoate should give rise to acetoacetate with an equal preponderance of TABLE

Acetoacetate Formation boxy&Labeled

Fatty

IV

in Washed Homogenate of Acids Alone and in Presence

Rat Liver

-

itom per ten excessCY in

Substrates

carboxyl carbon

Initial :oncentrstior of substrate in medium

-

I Xstribution

Atom per cent excess Cl3 in final acetoacetate I 2

Car-

with lruvate

C’S in final acetoacetate

of

_-

Final Lcetoacetatr


Experi. merit NO.

Incubated of Non-Labeled

EO EO

EOOH

EOOH .-

bf

1

2 3At 4At 5AS 5BS.

68 7 8 3Bt 4Bt 9§ 10 II

Octanoate I‘ I‘ I‘ ‘I “ “ Octanoate + pyruvate Octanoate + pyruvate Octanoate + pyruvate Octanoate + pyruva,te Octanoate + pyruvate Hexanoate “

-

7.68 7.68 23.8 23.8 23.8 23.8 23.8 7.68 None 7.68 None 23.8 None 23.8 None 23.8 None 23.8 23.8

0.0010 0.0010 0.0010 0.0010 0.0010 0.0010 0.0010 0.0010 0.0040 0.0010 0.0040 0.0010 0.0080 0.0010 0.0080 0.0010 0.0040 0.0015 0.0010

PM

117 144 161 156 75 199 253 250

1.32 1.47 3.72 4.17 3.45 3.84 4.51 0.84

6.64 6.07 6.05 5.92 6.54 0.84

0.66 0.82 0.56 0.69 0.57 0.65 0.69 1.00

258

0.87

1.01

0.86

112

1.62

1.99

0.81

223

1.86

2.28

0.82

222

2.30

2.82

0.82

134 172

4.95 4.65

8.48 8.36

0.58 0.56

2.01 1.79

A and B run on the portions of the same homogenate. $ Homogenate 3 times more concentrated in Experiment B than in Experiment 5 Heat, degradation of acetoacetate; all others by aniline citrate.

t Experiments

A.

CY in the carbonyl position (i.e., a 6O:EooH ratio of 1.34). Accordingly, P-labeled octanoate was incubated in the washed homogenate (Table V, Experiments 1 and 2) and the resulting acetoacetate was found to have an average ;0:6OOH ratio of 0.50, thus eliminating the possibility of multiple alternate oxidation as a significant factor in these experiments. Although this ratio is slightly lower than the average value

838

ACETOACETATE

FORMATION.

I

obtained in the experiments with carboxyl-labeled octanoate, this difference can be partially attributed to the rather low yields of acetoacetate obtained in the experiments with p-labeled octanoate. Errors accompanying low yields of acetoacetate are discussed below. The fatty acid oxidase activity of the different homogenates used in these experiments varied several fold and the possibility that variations in

TABLE

V

Acetoacetate Formation in WashedHomogenate of Rat Liver from &Labeled Alone and in Presence of Non-Labeled Pyruvate

Octanoate

-

Experiment No.

Substrates

Atom per cent con;$?r$tion excess of C’” of substrate in ,%carbon in medium

Atom per cent cxcfss C** in final acetoacetate

Distribution of Cl8 in final acetoacetate

Final acetoacetate ‘CO EO

EOOH EOOH

-L

II

Octanoate ‘( Octanoate + pyruvate Octanoate + pyruvate

4.8 4.8 4.8 None 4.8 None

--

PM

0.0010

110

0.0010

103 118

0.63 0.63 0.33

0.39

0.46 0.53 0.85

192

0.48

0.55

0.87

0.0005 0.0040 0.0005 0.0020

1.36

1.19

?!O:;OOH ratios for acetoacetate formed by the diluted and concentrated homogenate were 0.57 and 0.65 respectively, suggesting that variations in the rate of fatty acid oxidation did not affect the distribution of isotope significantly. In order to test the possibility that pyruvate and octanoate might interact to produce a composite species of acetoacetate, experjment.s were carried out in which either carboxyl-labeled octanoate (Table IV, Experiments 7, 8, 3B, 4B, and 9) or P-labeled octanoate (Table V, Experiments It was found 3 and 4) was incubated together with unlabeled pyruvate. in preliminary studies of such incubations that pyruvate and octanoate at concentrations of 0.004 and 0.001 M respectively (which are equivalent for acetoacetate production) were oxidized at maximal and nearly equal The incubarates when incubated in portions of the same homogenate. tion of both substrates together in another portion of the same homoge-


the ?O:cOOH ratio of acetoacetate might be related to the speed of fatty acid oxidation was tested in Experiments 5A and 5B (Table IV). Here two portions of the same homogenate with tissue concentrations adjusted to give a 3-fold difference in the rates of oxygen consumption in the presence of 0.001 M carboxyl-labeled octanoate were compared. The

D.

I.

CRANDALL

AND

6.

GURIN

839

DISCUSSION

The data presented here (Table I) and previous related evidence (14, 20) leave little doubt that acetoacetate is derived from the acetyl groups of pyruvate. Whether these acetyl groups are converted into 2-carbon 4 A gift from Dr. Sidney Weinhouse.


nate resulted in an increased rate of oxygen consumption which was essentially equal to the sum of the rates obtained when the two substrates were incubated separately. Under these conditions it was assumed that both pyruvate and octanoate were being converted to acetoacetate simultaneously and that their intermediates had the opportunity to mingle and possibly interact. In all of the isotope experiments of this type, a significantly higher ??O:*COOH ratio was observed than in experiments in which carboxyl* * or p-labeled octanoate was incubated alone. The average CO:COOH ratio was 0.86 as compared with 0.63 for the acetoacetate formed from carboxyl- or B-labeled octanoate incubated alone. In Experiments 3 and 4 (Table IV) this comparison was made on different portions of the same homogenate. The possibility that the discrepancy between the uniform distribution of CY in the acetoacetate formed from carboxyl-labeled octanoate in the found experiments of Weinhouse et al. (2) and the uneven distribution consistently in this laboratory could be a technical artifact was critically examined during the later stages of this study. Carbon dioxide and acetone were prepared from synthetic acetoacetate4 containing equal concentrations of isotope in the carbonyl and carboxyl positions by both of the degradation procedures used in the biological experiments (described above). Furthermore, some of these degradations were carried out in copper-lime filtrates made from homogenates which had been incubated with and without non-labeled pyruvate or octanoate. It was found that in general a small but detectable contamination of the ,mercury-acetone occurred. The magnitude of this contamination appeared to be unaffected by the presence of copper-lime filtrates of homogenates during the degradation but rather to vary inversely with the total amount of acetone recovered (e.g. 7 per cent for the isolation of 162 PM and 16 per cent for 73 PM of acetone). The effect of these errors upon the results obtained in the biological experiments reported here was estimated and found not to change them sufficiently to warrant the calculation of new values. Furthermore, they are insufficient to account for the difference between the previous results of Weinhouse et al. (2) and those obtained here on the distribution of CYS in the acetoacetate formed from carboxyl-labeled octanoate in liver tissue.

849

ACETOACETATE

FORMATION.

I

to form asymmetrically labeled acetoacetate with an average :O:(?OOH ratio of 0.53 (Table III) is an indication that octanoate differs from pyruvate in the production of an intermediate which preferentially acetylates acetate (or “active” acetate). Furthermore, the (!?O:;OOH ratio of this acetoacetate agrees within the limits of experimental error with the average values found for this ratio in the acetoacetate formed from carboxyl-labeled (Table IV) and p-labeled (Table V) octanoate, suggesting that acetate and the first 4 carbons of octanoate give rise to an identical 2-carbon precursor of acetoacetate. The conversion, in this homogenate, of carboxyl- and /Mabeled octanoate into acetoacetate with ?!O:??OOH ratios of 0.65 and 0.50, respectively, conclusively rules out multiple alternate oxidation as an explanation for this uneven distribution of isotope. A modification of the P-oxidation-condensation hypothesis (19), however, which will explain these results is that two species of 2-carbon fragments arise from a fatty acid, with the result that condensation between these fragments to form acetoacetate is not completely random. According to this mechanism,


compounds in the process of forming acetoacetate is not known. Attempts to establish 5- or 6-carbon compounds as intermediates have, to date, been unsuccessful. Acetopyruvate, reported by Krebs (16) to form acetoacetate readily in liver slices, was found to be inactive in the washed liver homogenate (1). Parapyruvic acid, reported by Annau (21) to form acetoacetate in liver homogenate, was found in this laboratory to be inactive in the washed liver homogenate, as were itaconic, citraconic, and mesaconic acids. These fragmentary negative results, however, do not rule out the possibility that some 5- or 6-carbon intermediate exists. Suggestive evidence that pyruvate gives rise to a a-carbon precursor of acetoacetate is provided by the effect of pyruvate in inducing carboxyllabeled acetate to form symmetrically labeled acetoacetate (Table II) in the washed liver homogenate. It is reasonable to suppose that the stimulatory effect of pyruvate involves the conversion of acetate into an active 2-carbon intermediate. From the symmetrical distribution of isotope in the mixed acetoacetate formed from the isotopically labeled “active” acetate and the non-isotopic intermediates formed from pyruvate, it would appear that these intermediates are capable of undergoing random condensation with each other and are, therefore, identical. This interpretation may be in conflict with the evidence of Bloch and Rittenberg (22), confirmed by Anker in rats of the Wistar strain (20), that pyruvate, unlike acetate, will not readily acetylate aromatic amines in tivo, a matter which will be discussed more fully in Paper II (23). The interaction of carboxyl-labeled acetate with non-labeled octanoate

D. I.

CRANDALL

AND

Y. GURIN

841

fragment and therefore lead to a reduction of the (?O:?JOOH ratio of the acetoacetate derived from it. Consequently one would expect a small difference between ;O:?JOOH ratios in experiments with carboxyllabeled octanoate and hexanoate, whereas much larger differences in the values would be obtained by comparing carboxyl-labeled butyrate and octanoate. Our figures show an average value of 0.65 for the ;O:?JOOH ratio of acetoacetate derived from carboxyl-labeled octanoate as compared with 0.57 for acetoacetate derived from carboxyl-labeled hexanoate. This small difference, although in accord with theoretical expectations, is also within the limits of experimental error and therefore not significant. A comparison of carboxyl-labeled butyrate and carboxyl-labeled octanoate may be drawn from the experiments of Weinhouse et al. in which these substances were incubated with liver slices (2, 24). In these experiments, unlike those reported here, symmetrically labeled acetoacetate was formed from carboxyl-labeled octanoate, and under the same conditions carboxyllabeled butyrate 0.58.

gave rise to acetoacetate

with a (?O:cOOH

ratio of

To account for the observation that the ?JO:(?OOH ratio for acetoacetate produced by a mixture of either carboxyl- or ,&labeled octanoate and unlabeled pyruvate was significantly greater than the ratio found in aceto-


asymmetrically labeled acetoacetate would result from the condensation of 2-carbon units derived from monolabeled fatty acids. This would imply that the a-carbon fragment derived from the carboxyl and a-carbons of octanoate (carboxyl fragment) and the fragment derived from the & and y-carbons (p-fragment) are preferentially acetylated by a-carbon fragments arising from the remaining carbons of the fatty acid chain. In terms of this hypothesis, the /3- and carboxyl fragments of octanoate are identical, since both /3- and carboxyl-labeled octanoate give rise to acetoacetate, with essentially the same distribution of isotope. The fragment derived from the 6- and t-carbons (6 fragment) would be expected to be identical with the p fragment, since both arise from a structurally similar portion of the fatty acid chain. By elimination, the fragment derived from the r- and w-carbons (terminal fragment) of octanoate should differ from the other three, and t-labeled octanoate should therefore give rise to predominantly carbonyl-labeled acetoacetate. Experiments showing the conversion of c-labeled octanoate into acetoacetate containing the distribution of isotope required by this hypothesis will be presented in Paper II (23). According to this mechanism, shortening the chain of a carboxyllabeled fatty acid should increase the relative importance of the terminal

342

ACETOACElTATE

FORMATION.

I

We wish to thank Dr. Adelaide M. Delluva for analytical aid and to express our appreciation to Dr. D. Wright Wilson for his interest and advice. SUMMARY

The incubation of a,&labeled pyruvate in washed homogenates of rat liver resulted in the formation of unlabeled carbon dioxide and of acetoacetate containing equal concentrations of isotope in the acetone and carboxyl carbons, in agreement with the conclusion that the a- and Pbut not the carboxyl carbons of pyruvate are used in the biosynthesis of acetoacetate. Pyruvate and octanoate stimulated the conversion of acetate into acetoacetate in the washed homogenate. Octanoate, however, differed from pyruvate in the production of asymmetrically labeled acetoacetate from carboxyl-labeled acetate. Carboxyl- and p-labeled octanoate both gave rise to acetoacetate containing more Cl3 in the carboxyl than in the carbonyl position, thus eliminating multiple alternate oxidation as a significant processin this pathway. In both instances, simultaneous incubation of the monolabeled fatty acid with non-labeled pyruvate partially equalized the distribution of the isotope between the two positions. In the presence of octanoate, pyruvate and acetate showed a similar tendency to introduce their carbons preferentially into the carboxyl portion of acetoacetate and, in this respect, were similar to the 2-carbon fragments which arose from the first 4 carbons of octanoate. A hypothesis that fatty acids give rise to more than one type of a-carbon


acetate produced when either monolabeled fatty acid was incubated alone (Tables IV and V), it is assumed that pyruvate must have contributed more carbon to the carboxyl than to the carbonyl portion of acetoacetate. In this respect its action is similar to that of acetate and the carboxyl and p fragments of octanoate. The formation of asymmetrically labeled acetoacetate from carboxyllabeled octanoate in the experiments reported here and in previous experiments carried out with liver slices in this laboratory (3) does not in our opinion conflict with the experiments of Weinhouse et al. (2) in which symmetrically labeled acetoacetate was obtained from carboxyl-labeled octanoate in liver slices. It is quite possible that in the latter experiments the magnitude of endogenous pyruvate and long chain fatty acid catabolism was sufficiently large nearly to equalize the distribution of isotope in the final acetoacetate, in accord with the phenomena observed here.

D.

I.

CRANDALL

AND

8.

GURIN

843

intermediate in the process of forming acetoacetate is proposed as an explanation for the uneven distribution of isotope in the acetoacetate formed from both carboxyl- and p-labeled octanoate. BIBLIOGRAPHY

20. 21. 22. 23. 24.

Lehninger, A. L., J. Biol. Chem., 161, 291 (1946). Weinhouse, S., Medes, G., and Floyd, N. F., J. Biol. Chem., 166, 143 (1944). Buchanan, J. M., Sakami, W., and Gurin, S., J. Biol. Chem., 169,411 (1947). Walden, P., Ber. them. Ges., 40, 3214 (1907). Sakami, W., Evans, W. E., and Cur-in, S., J. Am. Chem. Sot., 69,lllO (1947). Anker, H. S., J. Biol. Chem., 176, 1333 (1948). DOX, A. W., J. Am. Chem. SOL, 46, 1708 (1924). Kerr, S. E., J. Biol. Chem., 139, 121 (1941). Polis, B. D., and Meyerhof, O., J. Biol. Chem., 169, 389 (1947). Blatt, A. H., Org. Syntheses, ~011. 2 (1943). Lehninger, A. L., J. Biol. Chem., 161. 437 (1945). Edson, N. L., Biochem. J., 29,2082 (1935). Friedemann, T. E., and Haugen, G. E., J. BioZ. Chem., 147,415 (1943). Gurin, S., Delluva, A. M., and Wilson, D. W., J. BioZ. Chem., 171, 101 (1947). Embden, G., and Oppenheimer, M., Biochem. Z., 66,335 (1913). Krebs, H. A., and Johnson, W. A., Biochem. J., 31,772 (1937). Evans, E. A., Biochem. J., 34, 829 (1940). Weinhouse, S., Medes, G., and Floyd, N. F., J. BioZ. Chem., 166, 411 (1945). MacKay, E. M., Barnes, R. H., Came, H. O., and Wick, A. N., J. BioZ. Chem., 136, 157 (1940). Anker, H. S., J. BioZ. Chem., 176. 1337 (1948). Annau, E., 2. physiol. Chem., 224, 141 (1934). Bloch, K., and Rittenberg, D., J. BioZ. Chem., 169,45 (1945). Crandall, D. I., Brady, R. O., and Gurin, S., J. BioZ. Chem., 161, 845 (1949). Medes, G., Weinhouse, S., and Floyd, N. F., J. BioZ. Chem., 167,35 (1945).


1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

STUDIES OF ACETOACETATE FORMATION WITH LABELED CARBON: I. EXPERIMENTS WITH PYRUVATE, ACETATE, AND FATTY ACIDS IN WASHED LIVER HOMOGENATES Dana I. Crandall and Samuel Gurin J. Biol. Chem. 1949, 181:829-843.

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