the metabolism of pvruvate in the tricarboxvlic acid cvcle

8/12/2019 The Metabolism of Pvruvate in the Tricarboxvlic Acid Cvcle

http://slidepdf.com/reader/full/the-metabolism-of-pvruvate-in-the-tricarboxvlic-acid-cvcle 1/4

The Metabolism of Pvruvate in the Tricarboxvlic Acid Cvcle*J J J

AARON D. FREEDMANt AND SAMUEL GRAFF

prom the Department of Biochemistry, College of Physicians and Surgeons, Columbia University, New York

(Received for publication, April 4, 1958)

Pyruvate is a branch point (1) in the catabolic sequence of

glucose since it has several different metabolic pathways avail-

able. 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 en-

tering 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 pyruva te entering the TCA cycle

by these routes has been estimated in this study by injecting

nL-alanine-Z-Cl4 into rats and determining the relat ive radio-

act ivi ty 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 Murphy-

Sturm lymphosarcoma subcutaneously 7 days before the experi-

ment.

nL-Alanine-2-C14 (Isotope Specialties Co.), with a specifi c ac-

tiv ity of 1 mc. per mmole was injected intraperitoneally in

a dose of 0.1 mc. per kilo of rat.

Each animal afte r 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 fulfil lment of the requirements for the

degree of Doctor of Philosophy of the Faculty of Pure Science of

Columbia Universi ty, New York. This work was done in part

during the tenure of a Fellowship of the National Cancer Institu te,

National Institutes of Health, United States Public Health Serv-

ice.

1 The abbreviations used are: TCA, tricarboxyl ic acid; AKG,

a-ketoglutaric acid; PEP, phosphoenolpyruvic acid; OAA, ox-

alacetic acid; CoA, coenzyme A.

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 disti lled water until the

eluate was ninhydrin negat ive, glutamic and aspartic acids were

eluted separately by 0.5 N acetic acid. Glutamic acid hydro-

chloride 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 crysta ls

were dissolved in a minimal volume of water and precipitated by

HCl gas to constant act ivi ty. 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 con-

tinued 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 act ivi ty. The results in Tables II and

III are expressed as percentage of the total radioactivi ty calcu-

lated 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-C’4 CoA.

2. Column B by conversion of dicarboxylic acids of the

TCA cyc le labeled in the central carbons to OAA or PEP with

subsequent conversion to acetyl CoA which then will be radio-

active 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.

292



August 1958 A. D. Freedman and 1.9.Gra 293

TABLE I

Theoretical isotope distribution in TCA intermediates after intro-

duction of radioactive compounds with arbitrary activity oj 10

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 prev iously 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 withtissues is rapid, apparently the OAA formed in the course of the

TCA cycle has surprising stabil ity over the 1 hour o f time used.

Carbon 5 of the glutamate isolated from the livers o f 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-Vis oxidatively decarboxylated to acetyl-l-U4 Cob, both carbons 1

and 5 become radioactive but the act ivi ty 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 act ivity , carbon 1 activ ity 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 act ivi ty among

carbons 1, 2, and 3 may, in part, result from the presence of

pools of intermediates, but is probably chiefly a result o f averag-

ing 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 revolu-tion o f the TCA cycle at the time when the AKG was trans-

aminated to glutamate.

Livers of Fed Animals-The primary eff ect 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 degrada-

tion 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

Relative activity of individua l carbon atoms of

glutamic acid of liver

Carbon No.I

Percentage activity =activity of individual carbon

activity of total combustion X 5x 100

A C DIsotopic compound

entering TCA Cycle

ketyl-1-W CoA OAA-2-P OAA-4-P

10

5

5

2

5

10

5

5

10

5

5

3

5

10

5

5

10

5

5

2.<

3.:

8.1

8.:

3.1

1

I-

10

10

2

-

2

-

5

5

0

5

5

0

2.

7.

7.

2.

Number of cycles

Citric acid

Carbons

1 COOH

6 -COOH

AKG

Carbons

1 COOH

I2 7”3 CHz

4 HZ

I5 COOH

OAACarbons

l rooH

2 ;:O

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 con-

version 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 mecha-

nism labeling carbons 2 and 3. It would be labeled also by

oxidative decarboxylation of pyruvate to an extent not greater

than one-half that o f carbon 5.

It should be possible, therefore, to determine the pathway

chosen by pyruva te for entrance into the TCA cycle from therelative radioactivity of the carbons of glutamic acid.

Livers of Fasted Animals-The radioactivity of carbon 4 of

--/

Rat L Rat S Rat ST Rat F

% % YO %

6.2 7.7 14.2 15.3

27.5 25.3 18.1

90.8* 56.3 54.2 40.7

0.8 0. 1.1

3.0 3.1 3.9 20.3

95.4 97.6 95.5

Rat FT

YO

20.5

12.3

19.2

2.0

40.4

94.4um

* By difference.



294 Metabolism of Pyruvate TCA Cycle Vol. 233, No. 2

derived from the degradation of glutamic acid of the liver o f 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 effect s 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 administereddose 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 o f Rat FT was

obtained from a tumor-bearing animal, a host eff ect 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 acti vity 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 isocit ritase reaction. The hesose monophos-

phate shunt route is, of course, possible but is rather unlikely

to aff ect 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, hasformed totally labeled acetyl CoA. It appears likely that OAA

has less stabili ty 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 radioact ivity 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 IIIRelative activity of individual cc&o n atoms

o.f glutamic acid o.f tumor

Carbon No.

Percentage activity =activity of individual carbon

activity of total combustion X 5

IRat ST Rat FT

per cent per cent

19.8 23.9

14.1 8.6

33.9 18.8

9.0 7.7

21.0 40.2

97.8 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 act ivi ty of carbon 4 from that of carbon 5 we

still find excess activi ty 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 iswell labeled in the tumors of well fed animals is analogous to the

findings in liver, and is readily interpreted to signify that pyru-

vate 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 affa irs 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 ofthe TCA cycle may be supplied by transamination of preformed

amino acids. This would decrease the relat ive 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

shi ft 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, fa tty acids

may fai l to provide sufficient acetyl CoA, and the animal, there-

fore, decarboxylates pyruvate to supplement its needs. Sincethe tumor has this obligatory energy requirement, it fails ful ly

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 shi ft 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 availabi lity 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 otherhand, 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 effect s 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 dicar-

bosylic 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 Murphy-

Sturm 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 osalaceticacid to a two carbon fragment.

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).

REFERENCES

10. CUTTINELLI, C., EHRENVARD, G., REIO, I,., SALUST E, E., AND

STJERNHO LM, R., Acta Chem. Stand., 5, 353 (1951).

4. MOSBACH, E. H., PHARES, E. F., AND CARSON, S. F., Arch.11. CUTT INELLI, C., HOGSTROM, G., REIO, I,., SALUST E, E., AND

Biochem. Biophys., 33, 179 (1951).STJERNHOLM, R., A&iv. Kemi, 3, 315 (1951).

5. KOEPPE, R. E., AND HILL, R. J., J. Biol. Chem., 216, 81312. CARSON, S. F., In W. D. MCELROY AND H. B. GLASS (Editors),

(1955).Phosphorus metabolism, Vol. T, Johns Hopkins Press, Balti-

6. GRAFF, J., AND RITTENBERG, D., Anal. Chem., 24, 878 (1952).more, 1951, p. 276.

7. BLACK, A. L., AND KLEIBER, M., Biochem. et Biophys. Acta,13. BLACK, A. L., AND KLEIBER, M., Biochem. et Biophys. Acta,

1’7, 346 (1955). 23, 59 (1957).

8. WANG, C. H., CHRISTENSEN B., AND CHALDELIN, V. H., J.14. HILL, R. J., HOBBS, D. C., AND KOEPPE, R. E., J. Biol. Chem.,

Biol. Chem., 201, 683 (1953). 230, 169 (1958).

9. HOGSTROM, G., Acta Chem. Stand., 7, 45 (1953). 15. BUSCH, H., Cancer Research, 15, 365 (1955).

the metabolism of pvruvate in the tricarboxvlic acid cvcle

Documents