The

Metabolism

of Pvruvate
J

in the
AND

Tricarboxvlic
SAMUEL GRAFF

Acid

Cvcle*
J

AARON

D. FREEDMANt

prom the Department

of Biochemistry,

College of Physicians for publication,

and Surgeons, Columbia April 4, 1958)

University, New York

(Received

Pyruvate is a branch point (1) in the catabolic sequence of glucose since it has several different metabolic pathways available. It is significant, however, that pyruvate can enter one of these routes, the tricarboxylic acid (TCAI) cycle, in two different ways by condensation with COZ to form a dicarboxylic acid, or by oxidative decarboxylation to acetyl CoA. Pyruvate entering the TCA cycle as a dicarboxylic acid yields a net increase in the mass of cycle intermediates, and permits their use in synthesis. Pyruvate entering as acetyl CoA provides no net increase in intermediates and permits use of the TCA cycle for energy purposes only. The relative proportion of pyruvate entering the TCA cycle by these routes has been estimated in this study by injecting nL-alanine-Z-Cl4 into rats and determining the relative radioactivity of the individual carbon atoms of L-glutamic acid isolated from liver and tumor. It was found that the nutritional state of the animal markedly directed the pathway chosen in the livers and in subcutaneous Murphy-Sturm lymphosarcomas of fed and fasted animals.
EXPERIMENTAL

Adult Sprague-Dawley rats were used. Animals L, S, and ST were fasted 40 hours before injection. Animal F was fasted 40 hours, and then given 2.5 gm. of glucose by stomach tube 30 minutes before injection. Animal FT was fed ad Zibitum and given 2.5 gm. of glucose by stomach tube 30 minutes before injection. Rats ST and FT were implanted with MurphySturm lymphosarcoma subcutaneously 7 days before the experiment. nL-Alanine-2-C14 (Isotope Specialties Co.), with a specific activity of 1 mc. per mmole was injected intraperitoneally in a dose of 0.1 mc. per kilo of rat. Each animal after injection was kept in a glass jar through which air was slowly passed. At the end of 1 hour, the rat was killed by cervical dislocation, livers and tumors quickly removed, homogenized in 1 N HCl for 30 seconds in a Waring Blendor, and appropriately diluted with HCl to make a final volume which * This investigation was supported in part by a research grant (C-3141) from the National Institutes of Health, United States Public Health Service. t Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy of the Faculty of Pure Science of Columbia University, New York. This work was done in part during the tenure of a Fellowship of the National Cancer Institute, National Institutes of Health, United States Public Health Service. 1 The abbreviations used are: TCA, tricarboxylic acid; AKG, a-ketoglutaric acid; PEP, phosphoenolpyruvic acid; OAA, oxalacetic acid; CoA, coenzyme A. 292

was 20 times the original volume of tissue and was 6 N in HCl. Tissues were hydrolyzed by refluxing for 18 hours, humin was precipitated by phosphotungstic acid (2), and the solution was filtered and concentrated to 30 ml. in vacua. The concentrate was washed with five 20 ml. portions of amyl alcohol, the residual amyl alcohol removed from the concentrate by washing with ethyl ether, the aqueous solution evaporated in vacua to a brownish glass, and placed in a vacuum dessicator over sodium hydroxide overnight. The residue was dissolved in 100 ml. of distilled water, stirred with a small amount of charcoal for 0.5 hour, and filtered, yielding a clear colorless solution. This was placed on a column 25 x 2 cm. made up of Dowex-1 resin in the acetate cycle (3) cross-linked 10 times. After slowly loading the column and washing it with distilled water until the eluate was ninhydrin negative, glutamic and aspartic acids were eluted separately by 0.5 N acetic acid. Glutamic acid hydrochloride was isolated by passing HCl gas through the effluent after addition of an appropriate amount of nonradioactive glutamic acid and concentration to a small volume. The crystals were dissolved in a minimal volume of water and precipitated by HCl gas to constant activity. The glutamic acid was degraded by the procedure of Mosbach et al. (4), as modified by Koeppe and Hill (5). For total activity, a sample of glutamic acid was converted to CO2 by dry combustion (6), and collected as barium carbonate. All barium carbonate samples were washed, dried, and plated at infinite thickness on Teflon planchets having a sample area of 1 sq. cm. and counted in a Tracerlab gas flow counter using a Berkley decimal scaler. Counting was continued until an accuracy of within 3 per cent was obtained in all samples except carbon 4 of the liver glutamate of S, ST, F, and FT which had very low activity. The results in Tables II and III are expressed as percentage of the total radioactivity calculated from total dry combustion of glutamic acid.
RESULTS AND DISCUSSION

In Table I are seen the labeling patterns expected in TCA intermediates after introduction of isotope in the following fashions : 1. Column A by oxidative decarboxylation of pyruvate-2-Cl4 to acetyl-l-C4 CoA. 2. Column B by conversion of dicarboxylic acids of the TCA cycle labeled in the central carbons to OAA or PEP with subsequent conversion to acetyl CoA which then will be radioactive in Position 2. 3. Column C by conversion of pyruvate-2-V plus CO2 to a dicarboxylic acid radioactive in Position 2. 4. Column D by conversion of pyruvate plus Cl402 to a dicarboxylic acid labeled in Position 4.

August

1958
TABLE

A. D. Freedman and 1.9. Gra$

I
TCA intermediates after introwith arbitrary activity oj 10
C
CoA 2 3 2 D

293

Theoretical isotope distribution duction of radioactive compounds

A
Isotopic entering compound TCA Cycle ketyl-1-W Number of cycles

in

OAA-2-P

OAA-4-P
1 I-

Citric acid Carbons 1 COOH

5 5 5 0

10

10

10 5 2.<

6 -COOH AKG Carbons 1 COOH

I 2 7 3 CHz
4 5 &HZ

5 5 5 0

10

COOH

10

10

10

OAA Carbons 5 l 2 rooH ;:O 5 5 5 5 5 2. 7. 7. 2. 3.: 8.1 8.: 3.1

-

These theoretical patterns correspond quite well to those experimentally found in glutamic acid after administration of acetyl-1-W (5, 7-ll), acetyl-2-P (9-11, 13), NaHW03 (5, la), and pyruvate-2-C14 (14). In accordance with Table I, one would expect the following: 1. Carbon 5 labeling in glutamate would occur by the conversion of pyruvate-2-Cl4 to acetyl-l-Cl4 CoA. 2. Carbon 4 labeling in glutamate would occur by the conversion of dicarboxylic acids to acetyl CoA. 3. Carbons 3 and 2 of glutamate would result from conversion of pyruvate to dicarboxylic acids via CO2 fixation. 4. Carbon 1 of glutamate would be labeled by the mechanism labeling carbons 2 and 3. It would be labeled also by oxidative decarboxylation of pyruvate to an extent not greater than one-half that of carbon 5. It should be possible, therefore, to determine the pathway chosen by pyruvate for entrance into the TCA cycle from the relative radioactivity of the carbons of glutamic acid. Livers of Fasted Animals-The radioactivity of carbon 4 of

the L-glutamic acid of the livers of fasted rats (L, S, and ST, Table II) is insignificant, indicating that there is negligable conversion of the OAB synthesized de no~o to acetyl CoA. Since it has been previously indicated that carbon 4 would be most heavily labeled by this conversion, labeling of the other carbons of glutamate by methyl-labeled acetyl is eliminated. Alt.hough enzymatic decarboxylation of OAA incubated with tissues is rapid, apparently the OAA formed in the course of the TCA cycle has surprising stability over the 1 hour of time used. Carbon 5 of the glutamate isolated from the livers of fasted rats (ST and L, Table II) contains about 3 per cent. of the total label in glutamate. Since carbon 4 labeling is vanishingly small, this carbon 5 radioactivity is taken as a measure of the conversion of pyruvate to active acetate, and this latter must also be small. Carbons 2 and 3 are a measure of the conversion of pyruvate to a dicarboxylic acid, and in the glutamate from livers of fasted rats (L, S, and ST, Table II), over 80 per cent of the total radioactivity resides in these carbons. Carbon 1 becomes radioactive by both the mechanisms that label carbons 5 and 3. It has previously been noted that when pyruvate-2-V is oxidatively decarboxylated to acetyl-l-U4 Cob, both carbons 1 and 5 become radioactive but the activity of carbon 1 will not exceed one-half that of carbon 5. In the glutamate from the livers of fasted rats, since carbon 5 accounts for from 3 to 4 per cent of the total activity, carbon 1 activity by the acetyl CoA formation mechanism will not exceed 2 per cent. The bulk of the radioactivity in carbon 1, therefore, results from dicarboxylic acid synthesis. The marked difference in specific activity among carbons 1, 2, and 3 may, in part, result from the presence of pools of intermediates, but is probably chiefly a result of averaging the radioactivity of molecules which have been active in the TCA cycle for varying lengths of time. It suggests that a considerable portion of the AKG had not completed one revolution of the TCA cycle at the time when the AKG was transaminated to glutamate. Livers of Fed Animals-The primary effect of feeding glucose (F and FT, Table II) is the marked increase in the proportion of radioactivity found in carbon 5 of glutamate, and the somewhat smaller increase found in carbon 1. The data in Table II under F are derived from the degradation of liver glutamate of an animal fasted for 40 hours, and then given 2.5 gm. of glucose by stomach tube, 30 minutes before injection of alanine-2-Cr4. The data in Table II under FT are
TABLE

II
carbon of liver atoms of

Relative

activity

of individual glutamic acid

activity =

Carbon

No.

Percentage

activity of individual carbon x activity of total combustion X 5 Rat ST Rat % F

100

Rat %

Rat %

-/

Rat F T

6.2
90.8*

3.0 Sum * By difference.

7.7 27.5 56.3 0.8 3.1 95.4

YO 14.2 25.3 54.2 0. 3.9 97.6

15.3
18.1

YO 20.5 12.3
19.2

40.7
1.1

20.3 95.5

2.0 40.4 94.4

294

Metabolism

of Pyruvate

TCA

Cycle

Vol. 233, No. 2

derived from the degradation of glutamic acid of the liver of a tumor-bearing rat which had been fed ad Zibitum and then given 2.5 gm. of glucose 30 minutes before the injection of alanine-2-C14. Two effects may account for the difference of labeling of carbon 5 of glutamate. Animal FT may be presumed to have had adequate levels of liver glycogen and so the administered dose of glucose was far in excess of needs. In Rat F, starvation had depleted liver glycogen, and carbohydrate, even in large doses, was not in excess but was undoubtedly utilized in part for glycogenesis. In addition, since the liver of Rat FT was obtained from a tumor-bearing animal, a host effect may have played an additional role. The importance of the conjectural host tumor interrelationship cannot be evaluated here since the comparable data are not available. It is noteworthy that the considerable labeling of carbons 2 and 3 in both F and FT testifies to the continuing need for dicarboxylic acid synthesis in the liver. Tumor-The most striking finding in the tumor study is the significant activity of carbon 4 in both the fasted and fed states (Table III). Labeling in carbon 4 can be accounted for in the following ways: (a) Two successive decarboxylations of OAA containing isotope in Position 3, or (6) the hexose monophosphate shunt, or (c) the isocitritase reaction. The hesose monophosphate shunt route is, of course, possible but is rather unlikely to affect the results of the present esperiment since extensive dilution by all the sugars present in the cell would be expected, and the short time interval chosen for study would further minimize this rather long circuiting. The isocitritase reaction has thus far not been observed in animal tissue. The most reasonable explanation of the labeling in carbon 4 of AKG, therefore, is that 098-2, 3.Cl4 produced by the TCA cycle has been decarboxylated to pyruvate-2, 3-U4 which in turn, has formed totally labeled acetyl CoA. It appears likely that OAA has less stability in the tumor than in the liver, and is more readily decarboxylated there. The relatively heavy carbon 5 label in tumors is in part related to the label in Position 4. It was previously shown that there is equal labeling of all OAA carbons by the second turn of the TCA cycle. Two decarbosylations of this OAA would lead to acetyl Coh with the same level of radioactivity in each carbon, and condensation of this acetyl CoA with the OAA from which it was produced would lead to AKG and glutamate equally labeled in carbons 4 and 5. vnder these conditions,
TABLE Relative activity III atoms

of individual cc&on o.f glutamic acid o.f tumor

activity =

Percentage Carbon No.

I--

activity of individual carbon activity of total combustion X 5 Rat F T per cent

Rat ST per cent

19.8 14.1 33.9 9.0 21.0 97.8

23.9 8.6 18.8 7.7 40.2 99.2

AKG and glutamate would be produced in which the labeling in carbon 4 is equal to that portion of the labeling of carbon 5 of glutamate due to the double decarboxylation of OAA. Thus if we deduct the activity of carbon 4 from that of carbon 5 we still find excess activity of carbon 5 in the glutamate of tumors, indicating a comparatively great utilization of pyruvate by decarboxylation to acetyl CoA. That carbon 5 of glutamate is well labeled in the tumors of well fed animals is analogous to the findings in liver, and is readily interpreted to signify that pyruvate in excess of that needed for dicarboxylic acid formation is being supplied by degradation of the fed glucose and, therefore, pyruvate is being decarboxylated to acetyl CoA. It is seen, however, that in t.he tumor glutamate of a fasting animal, carbon 5, even after correction for carbon 4 radioactivity, is moderately well labeled, implying utilization of considerable sugar for acetyl formation. Various interpretations of this state of affairs suggest themselves. If, as suggested by Busch (15)) tumors tend to utilize blood amino acids and proteins rather than TCA intermediates for their required glutamic and aspartic acids, it may be suggested that dicarboxylic intermediates of the TCA cycle may be supplied by transamination of preformed amino acids. This would decrease the relative labeling of carbon 2 and 3 of AKG, and thus, correspondingly increase the relative labeling of carbon 5. One could, on the other hand, suppose that tumors are not so subject to the regulatory processes which alter metabolic pathways in liver when the animal is fasted, and that they continue to consume glucose for acetyl Cob formation even when the animal is fasted. Tumors show a qualitative shift of labeling pattern of carbon 3 versus carbon 5 of glutamate similar to that of liver. If we assume that tumors, with their large energy requirements, are oxidizing maximally, fatty acids may fail to provide sufficient acetyl CoA, and the animal, therefore, decarboxylates pyruvate to supplement its needs. Since the tumor has this obligatory energy requirement, it fails fully to respond to the process taking place in the liver which decreases pyruvate decarboxylation, and the tumor continues to use blood glucose for acetyl CoA formation. At branch points, where a substrate has the opportunity to follow more than one pathway, the results, in terms of body economy, of a shift in the route selected may be far reaching and yet the extent of utilization of this substrate may be unaltered. Pyruvate entering the TCA cycle as a dicarboxylic acid increases the availability of glutamate and aspartate for protein synthesis, and also increases the concentration of TCA cycle intermediates so that more acetyl CoA, whether from fat or carbohydrate, can be utilized, and more energy can be generated. On the other hand, with no change in the amount of pyruvate utilized, if the substrate is diverted from dicarboxylic acid synthesis to acetyl CoA formation, nonessential amino acids will be available for protein synthesis in reduced amounts, the concentration of TCA cycle intermediates will fall, the rate of condensation of acetyl CoA may decrease because of the nonavailability of OAA, and osidatively generated energy production may decline. The factors then, in determining the pathway followed by pyruvate are of great consequence, and their effects can be inferred from the labeling patterns of glutamate.
CONCLUSIONS

1. In the fasted animal, carbohydrate is used by the liver tricarboxylic acid (TCA) cycle primarily as a source of dicarbosylic acids rather than a source of acetyl coenzyme A.

August

1958

A. D. Freedman and S. Gra$

295

2. In the fasted animal, the principal energy source for the liver is probably fat. Although the TCA cycle is labeled by a three carbon precursor, the label has entered as a dicarboxylic acid. 3. In a rat given glucose, an appreciable amount of pyruvate entering the TCA cycle does so by decarboxylation to a two carbon fragment. 4. In livers of fasted and fed rats only a minor proportion

of oxalacetic acid is decarbosylated to a two carbon fragment over a 1 hour period. 5. In contrast to the findings in the liver, in the MurphySturm sarcoma of the rat there is a significant production of a two carbon fragment from pyruvate, even in the fasted condition. 6. In the Murphy-Sturm sarcoma of the rat, in both fasted and fed animals, there is significant decarbosylation of osalacetic acid to a two carbon fragment.

REFERENCES 1. KREBS, H., Endeavor,, 16, 125 (1957). 2. CANNON, P. R., J. Biol. Chem., 162, 406 (1943). 3. BUSCH, H., Cancer Research, 13, 789 (1953). 4. MOSBACH, E. H., PHARES, E. F., AND CARSON, S. Biochem. Biophys., 33, 179 (1951). 5. KOEPPE, R. E., AND HILL, R. J., J. Biol. Chem.,
10. CUTTINELLI, C., EHRENVARD, G., REIO, I,., SALUSTE, E., AND STJERNHOLM, R., Acta Chem. Stand., 5, 353 (1951). 11. CUTTINELLI, C., HOGSTROM, G., REIO, I,., SALUSTE, E., AND STJERNHOLM, R., A&iv. Kemi, 3, 315 (1951). 12. CARSON, S. F., In W. D. MCELROY AND H. B. GLASS (Editors), Phosphorus metabolism, Vol. T, Johns Hopkins Press, Balti-

F., Arch.

216, 813 (1955). 6. GRAFF, J., AND RITTENBERG, D., Anal. Chem., 24, 878 (1952). 7. BLACK, A. L., AND KLEIBER, M., Biochem. et Biophys. Acta, 17, 346 (1955). 8. WANG, C. H., CHRISTENSEN! B., AND CHALDELIN, V. H., J. Biol. Chem., 201, 683 (1953). 9. HOGSTROM, G., Acta Chem. Stand., 7, 45 (1953).

more, 1951, p. 276. 13. BLACK, A. L., AND KLEIBER, M., Biochem. et Biophys. 23, 59 (1957). 14. HILL, R. J., HOBBS, D. C., AND KOEPPE, R. E., J. Biol. 230, 169 (1958). 15. BUSCH, H., Cancer Research, 15, 365 (1955).

Acta, Chem.,