CONVERSION OF LACTATE TO LIVER GLYCOGEN IN THE INTACT RAT, STUDIED WITH ISOTOPIC LACTATE*

BY VICTOR LORBER, NATHAN LIFSON, HARLAND G. WOOD, WARWICK SAKAMI, AND WALTON W. SHREEVEt

WITHTHE TECHNICAL ASSISTANCE OF JULIEN BORDEAUX AND MARGARET COOK

(From the Department of Biochemistry, School of Medicine, Western Reserve University, Cleveland, and the Department of Physiology, University of

Minnesota Medical School, Minneapolis)

(Received for publication October 15, 1949)

The distribution of isotope in liver glycogen deposited following the feeding of a labeled compound may be used as an indicator in viva of inter- mediary reactions linking the fed compound and glycogen. When acetate or butyrate labeled with isotopic carbon is administered together with non-isotopic glucose to a fasted rat, the resulting liver glycogen contains isotope (3, 4). If the distribution of the isotope in the glycogen is de- termined by hydrolysis and degradation of the resulting glucose, a number of characteristic patterns are observed (4). These distribution patterns are consistent with the reactions of the tricarboxylic acid cycle and of glycolysis as the main pat.hs for the transfer of carbon from these non- carbohydrate sources to glycogen.

In the present experiments the metabolism of lactic acid has been studied by this technique. Three types of labeled lactate,’ CH3. C3HOH.- COONa, C13H3. C13HOH * COONa, and C14H3 * C13HOH. COONa have been fed to fasted rats, and the resulting liver glycogen hydrolyzed to glucose and degraded. The distribution of isotope has been found to be in har- mony with predictions based on the schemes of glycolysis and the tricar- boxylic acid cycle. The results, when interpreted in terms of these schemes, indicate that the major fraction of the administered lactate car-

* This work was supported in part by a grant from the American Cancer Society on the recommendation of the Committee on Growth of the National Research Council, and by a grant to the Department of Biochemistry from the Prentiss Foun- dation. Preliminary accounts of some parts of this study have appeared (1, 2). The radiocarbon used in this work was obtained on allocation from the United States Atomic Energy Commission.

t Fellow of the American Cancer Society. r Racemic mixtures of the D and L forms were fed in each case. Calculations

involving the amount of isotope administered have been baaed on the isotope content of the total lactate fed, although it might have been more appropriate to consider only the isotope contained in the natural form. The possible metabolic role of the unnatural isomer in determining the results of the present experiments has otherwise been left out of consideration.

517

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518 LACTATE CONVERSION TO GLYCOGEN

bon which was converted to glycogen traversed reactions of the tricar- boxylic acid cycle prior to this conversion.

Methods

The lactate labeled with Cl3 in the cy and /3 positions was synthesized according to the method of Cramer and Kistiakowsky (5) with some modi- fications. The C13-a-labeled lactate was prepared from CHS. CY300H by esterification with butyl alcohol, and hydrogenation of the ester to the alcohols with copper-barium-chromium oxide catalyst, yielding CHI*C’~- HzOH and CH3. CH2. CH2. CHtOH. The mixture of alcohols was con- verted to the corresponding iodides and fractionated. The ethyl iodide was converted by the Grignard reaction to propionic acid, CH,. CY3H, * COOH, which was isolated as the sodium salt. The final steps may be represented as follows :

CHa.Cl3H2.COONa P + B; CH~.CY3HBr~COOH Na&03 CH~.Cr3HOH.COONa >

The C14H3. C13HOH. COONa was prepared in a generally similar manner. C14H3C1300H was made from C14H31 and KC?3N. Conversion of the ethyl iodide to propionate was accomplished by nitrile formation and hydrolysis. The propionate was boiled with benzoyl chloride and the resulting propionyl chloride was removed by distillation. Bromination and hydrolysis yielded the isotopic lactate. Purity of the different acetic and propionic acids was checked by partition coefficient (6), which agreed with theory within experimental error. In all cases, the lactic acid was removed from the hydrolysis mixture by continuous ether extraction. Total acid, by titration, agreed with lactate determined calorimetrically (7).

The general plan of the experiments and the methods employed were similar to those described previously (4, 8). Male white rats, after a 24 hour fast, were given the isotopic lactate as the sodium salt by stomach tube, together with non-isotopic glucose to enhance glycogen deposition.2 In several experiments, the respiratory CO2 was collected. 3 hours after giving the lactate, the livers were removed under amytal anesthesia, and the glycogen isolated and hydrolyzed to glucose. When necessary, the glucose from more than one liver was pooled.

The glucose was converted to lactate by fermentation with Lactobacillus

2 In some experiments preliminary to the present work, CY%,&labeled lactate was administered in larger doses and without glucose. Not only were the glycogen yields much smaller, but the level of isotope in the glycogen was not much greater than that found in the present experiments. Comparable results were obtained by Vennesland et al. (9) who also administered a,&labeled lactate without glucose. If its use is not contraindicated by other considerations, the administration of glucose thus permits economy of isotopic compound and makes for conveniently large glycogen yields with little decrease in isotope concentration in the glycogen.

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LORBER, LIFSON, WOOD, SAKAMI, AND SHREEVE 519

casei, and the lactic acid was carried through a stepwise degradation to yield the carboxyl and a- and @-carbons of the lactate as separate fractions (4, 8). These fractions correspond to positions 3 and 4,2 and 5, and 1 and 6 of the original glucose, respectively. In the experiments with C1*H3.C13- HOH . COONa (Experiments 6 to 9) the fraction consisting of carbons 1 and 6, obtained as iodoform, was purified by sublimation before conversion to COz in an effort to minimize dilution with adventitious, non-isotopic carbon, known to occur at this step (4). In addition, in Experiments 6 to 9 part of the glucose was converted to the methyl glucoside and de- graded by an alternative procedure (4, 6). This degradation yields three separate fractions containing carbon 3, carbons 1, 2, 4, and 5, and carbon 6 of the glucose, respectively. The carbon 3 fraction is contaminated with small amounts of carbon from other positions. The various fractions

TABLE I Degradation of Swthetic Isotopic Lactate*

Isotope concentrationt in degra-

2gzf d&ion fractions

Type of lactate

%%zl a-Carbon &Carbon ---

CHz-CPHOH-COONa. . . . . . . . . . . . . . . . C’” 0.00 0.58 0.00 C’“Hs-C’aHOH.COONa. . . .

“ . . . . . . . . . . . . 0.01 1.14 1.16

CPHH,.C’aHOH-COONa. . . . . _. . . . . . . . . “ 0.00 1.34 0.00 “ 0’ 0.00 25 2390

* The isotopic compounds were diluted with non-isotopic lactate before degrada- tion.

t In this and subsequent tables, the Cl8 values are given in atom per cent excess, and the CP values in counts per minute per mg. of carbon.

from both types of degradation, after conversion to COZ, were analyzed for their Cl3 content in the mass spectrometer, as was the respiratory COZ. In Experiments 6 to 9 duplicate aliquots of all samples were precipitated as BaC08, and radioactivity determined with an end window Geiger-Miiller tube. The method of sample preparation was very similar to that of Henriques et al. (10). The radioactivity measurement on the duplicate samples of the glycogen fractions agreed within f2 per cent. Duplicates on the respiratory COZ samples, which contained much less material, agreed within *5 per cent.

As a check on the synthesis and degradation procedures, the three types of synthetic lactate, after appropriate dilution with non-isotopic lactic acid, were degraded in the same manner as the biological material. The results, presented in Table I, show that the methods of synthesis and

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520 LACTATE CONVERSION TO GLYCOGEN

degradation are mutually confirmatory. A trace of Cl4 appears in the a-carbon fraction from the CF4H3. C13HOH. COONa.

RESULTS AND DISCUSSION

The general experimental data are summarized in Table II. In Table III are presented the results of the glycogen degradations. It

will be noted that for all types of lactate the isotope occurs predominantly in positions 1, 2, 5, and 6 of the glucose, and to a much lesser extent in carbons 3 and 4. In previous experiments on CO2 fixation with labeled bicarbonate, positions 3 and 4 contained only about one-sixth the concen- tration of isotope found in the respiratory CO, (S), whereas in all the present studies for which data on respiratory CO2 are available (Table IV) the

TABLE II

General Experimental Data on Rats

Experi- ment NO.

-

Body weight

after fast

sm. 185 190 210 200 180 225 190 205 205

Type of lactate administered

CH3.C’3HOH.COONa ‘< “

C~3Ha~CPHOH+COONa “

CYH,~C1~HOH~COONa “ I‘ “

Amoqunt lactate adminis-

tered er 100 gm

body weight

Am:Yt glucose

adminis- tered

er 100 gm body

weight

n&M m&f

0.85 3.00 1.57 1.47 1.42 1.33 1.60 1.40 1.78 1.56 1.96 2.17 1.89 2.10 1.85 2.06 1.81 2.01

Liver Liver

weight glycogen

(as glucose)

gn. mu

6.73 1.18 5.49 6.53

1.15

6.65 0.96 5.73 0.83 7.30 1.33 5.00 0.80 5.50 6.00

0 0.87

relative concentration runs appreciably higher, being about equal to the respiratory COZ in Experiment 5. Although the two types of experiment are not strictly comparable, since the labeled CO2 is being produced meta- bolically in the present studies, it seems very unlikely that the level of isotope appearing in carbons 3 and 4 in the lactate experiments can be accounted for by CO2 fixation alone.

In the experiments with lactate containing Cl3 only in the LY position (Experiments 1 to 3 and 6 to 9), carbons 2,5 of the glucose are seen to contain about one-third more Cl3 than carbons 1,6. When the P-carbon is labeled with Cl4 (Experiments 6 to 9), the relative distribution of isotope between these positions is reversed, although the disparity between carbons 1,6 and 2,5 is not as great as that noted for the Cl3 of the a-carbon.

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LORBER, LIFSON, WOOD, SAKAMI, AND SHREEVE 521

TABLE III Distribution of Isotope* in Liver Glycogen after Administration of Labeled Lactate to

Rats

$ 8 Type of lactate administered s

% P .I % i%

c -

1 2-3

4 5

6t

6-9

CHI. CYHOH. COONa “

CPH3~CPHOH~COONa I‘

C1*H~~C13HOH~COONa

“

C’ “ “ “ “

C’ Cl C’

4.64 0.010.100.0i 4.64 0.050.270.U 5.06 0.150.600.5S 5.06 0.180.940.85 6.50 0.070.420.3(

11,600 204 679 826 6.50 0.100.430.3(

11,600 201 681 742

- 3

l.O!

22: J.300.2< 577 756

5.91 4.23 8.1 10.8 7.36 8.93 4.861 5.54$

* See foot-note to Table I. t 1 mM of glucose was degraded. The remaining 0.33 rnM was combined with

that from Experiments 7 to 9, and the pooled sample divided equally and de- graded by both methods.

- Isotope concen- tration in degra- dation fractions

of methyl glucoside

Carbon atoms of glucose

‘4 “j 6 --

$ These calucations are based on the total isotope administered and the total recovered in liver glycogen in Experiments 6 to 9.

EX- peri-

merit NO.

2 5 7

8

TABLE IV Data on Respiratory COz of Rats

Co~no~llt

Type of lactate administered

1st 2nd 3rd hr. hr. hr.

---

CHs. WHOH*COONa 7.825.836.44 Cl~Ha~CPHOH~COONa 7.196.646.8: C~4H3~CPHOH~COONa 6.99 7.23 6.7f

‘I 6.695.826.4f

Isotope COnCentratiOn’ o?id%$

TY of

in respiratory COa istered isotope

iso- recovered tape in res-

1st hr. 2nd hr. 3rd hr. pir&t,ory 2

-----

03 0.10 0.15 0.08 15.7 ‘I

0.13 0.17 0.21 10.8 “

0.08 0.24 0.28 17.9 04 143 351 476 16.2 Cl3 0.05 0.13 0.20 9.64 C’4 92 243 318 j 9.29

* See foot-note to Table I.

The results obtained by the two degradation procedures in Experiments 6 to 9 are seen to be in good agreement. The values for carbon 6 from the degradation of the methyl glucoside agree, within experimental error, with the values for carbons 1,6 from the glucose degradation. The expected

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522 LACTATE CONVERSION TO GLYCOGEN

concentrations for the fraction containing positions 1,2,4, and 5, calculated from the results for the three fractions of the glucose degradation, are 0.31 atom per cent excess and 576 c.p.m. per mg. of carbon for Cl3 and Cl4 respectively, in agreement with experimental values of 0.30 and 577. Agreement between carbons 3,4 and 3 is satisfactory, in view of the low Cl3 values and the fact that the carbon 3 fraction contains a small amount of material from other positions.

With the C&Y ,fl-labeled lactate, the isotope values for carbons 2,5 and 1,6 in Experiment 4 are equal, within the error of the measurement (&O.Ol atom per cent). In Experiment 5, positions 2,5 appear to contain about 5 per cent more Cl3 than positions 1,6. This is due to a procedural arti- fact. As pointed out previously (4) the degradation procedure as usually employed causes a certain amount of dilution of fraction 1,6 with non- isotopic COZ. This dilution produces a depression of the isotope values for this fraction, which is most marked when the samples are small and when the isotope values are high. In Experiments 2 to 3, at the relatively low Cl3 value of 0.18 atom per cent excess, and large sample size of 0.84 mM, the dilution effect is masked. In Experiment 5, at a higher CY value and smaller sample size (0.89 atom per cent excess and 0.45 mM, respec- tively), the dilution effect becomes apparent. These dilution effects have been observed by degradation of synthetic lactate, equally labeled with Cl3 in the LY and p positions (4).

The data obtained by the simultaneous use of Cl3 and Cl4 in Experiments 6 to 9 permit fairly accurate comparison of the relative contribution of the (Y- and P-carbons of the lactate to liver glycogen and respiratory COZ. Since the Cl3 and Cl4 are administered together in the same compound, and determined in aliquots of the same degradation fractions, the significance of any differences noted will not be influenced by variations between ani- mals or chemical procedures, but will depend solely on the errors involved in the isotopic measurements. The values for the per cent of adminis- tered isotope recovered in liver glycogen in these experiments, presented in the last column of Table III, indicate that, although the transformation of (Y- and P-carbons to glycogen is of the same order of magnitude, a larger contribution has been made by the P-carbon. The observed differences lie beyond the errors of the measurements. (In similar experiments with CY4H,. CYaH2* COONa in which, unlike the present experiments, Cl3 and Cl4 were found to be equally distributed between carbons 2,5 and 1,6 of the glucose, the per cent recovery in the glycogen was identical for both isotopes (ll).) In the last column of Table IV (Experiments 7 and 8) it will be noted that the per cent of administered isotope recovered in the respiratory CO2 is approximately the same for both isotopes, indicating

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LORBER, LIFSON, WOOD, SAKAMI, AND SHREEVE 523

that the OL- and P-carbons of lactate have contributed about equally to the respiratory COZ. It might be expected that the introduction of a relatively larger amount of one isotope in the glycogen would result in the appearance of a relatively smaller amount of this isotope in the respiratory COZ. The differences between the per cent of Cl3 and Cl4 recovered in the respiratory COZ, while compatible with this expectation both in direc- tion and magnitude, lie within the error of the isotopic measurements on the respiratory COZ samples and cannot be considered as significant.

On the assumption that glycogen is formed by reversal of the glycolytic reactions as indicated in Diagram 1 (Reactions 2 and l), the main results of the present study indicate that isotope administered in either the a- or P-carbon of lactate is largely, though not completely, randomized between these positions in pyruvate, and that, when isotope is introduced in equal concentration in both the (II- and P-carbons of lactate simultaneously, the concentration remains equal in the resulting pyruvate.

The reactions of glycolysis and the tricarboxylic acid cycle, presented in abridged fo m in Diagram 1,3 readily account for the main results. On the assumption that lactate enters into metabolism via pyruvate, reversal of the glycolytic reactions will lead to the following types of isotopic glucose,

C--&C--G-&--C or L-C-C-C-C-:, depending on whether the isotope is introduced in the c+ or P-carbon of the lactate (the asterisk in- dicates the location of the isotope). CO2 fixation and the reversible reductive reactions of the tricarboxylic acid cycle (Reactions 3, 10, 9, and 8) followed by Reaction 2, will produce a glucose with isotope equally

*=* *=*

distributed in carbons 1, 2, 5, and 6 (C-C-C-C-C-C), regardless of whether the isotope is introduced in the a! or /3 position of the lactate.

The contribution to the isotopic “pyruvate pool” by decarboxylation of oxalacetate formed via the oxidative reactions of the cycle must also be considered. The oxidative reactions of the tricarboxylic acid cycle (Reactions 5 to 10) will involve initially an isotopic oxalacetate (Reaction 3) as well as an isotopic 2-carbon compound (Reaction 4). In the case of CY4H3. CY3HOH. COONa, COOH * Ci4Hz. C30. COOH and CY4H3. POX will be the initial reactants. Assuming that the initial condensation reaction occurs on the carbonyl group of oxalacetate, as usually depicted, the newly

8 Stern and Ochoa (12) have recently demonstrated citrate accumulation in an aged pigeon liver preparation, using acetate and oxalacetate together in substrate amounts. The preparation was very low in aconitase activity, pointing to citrate as the initial condensation product in the enzyme system involved. Because of this and other recent developments (13, 14), which emphasize the tentative nature of current schemes of the tricarboxylic acid cycle, the individual tricarboxylic acids have been omitted from Diagram 1.

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LORBER, LIFSON, WOOD, SAKAMI, AND SHREEVE 525

formed oxalacetate generated via the cycle will contain both isotopes in all positions, since, according to the scheme of Diagram 1,

C’*Hs . POX 04. CPOOH

+ -4 I

-

COOH.C14H~.C130.COOH COOH~C’“~CP~COOH

C”H2. CPOOH I -

COOH.CY40~C1~H,

CY4Hz. CPOOH I

Reactions 8-10 7 C’400H~C’3H~~CL40~C1300H

C”OOH-C”Hz LC’300HG4Hz~C’~0~C’400H

Any of this oxalacetate going to glycogen via Reactions 3 to 1 will introduce both isotopes into all positions of the hexose chain. Cl3 will be equally distributed among carbons 1, 2, 5, and 6, as will CY4. However, the relative abundance of the two isotopes in these positions will depend upon the isotope dilution occurring in the “acetate” and “oxalacetate pools,” respectively. For example, heavier labeling in the “acetate pool” should yield a hexose relatively richer in Cl4 than in CP. In addition, under this latter condition, the disparity in isotope concentration between posit,ions 1,6 and 2,5 of the final glycogen, produced by direct conver- sion of C14H3. CY3HOH. COONa via glycolysis, should be less pronounced in the case of C4, as was actually found.

It will be noted that the oxidative reactions of the tricarboxylic acid cycle provide a path (in addition to CO2 fixation) for the transfer of iso- tope from the a- and P-carbons of pyruvate to positions 3,4 of glucose. It may be wondered why the relative concentration of isotope in carbons 3,4 of the glucose is not higher than that actually found in the present study. It should be pointed out that in the presence of a respiratory COz low in isotope, as in the present experiments, the CO2 fixation reaction may operate to dilute the isotope in carbons 3,4 of the glucose. The con- trary would be expected when the respiratory COz is relatively high in isotope, and actually it has been found in similar experiments with CY3- H3.Ca00Na (4), in which the isotope level in the respiratory CO2 was several times that obtaining in the present study, that the Cl3 concentra- tion in carbons 3,4 exceeded that in the other positions of the glucose.

Study of the schemes presented in Diagram 1 will also reveal that t,here is no mechanism for altering complete randomness of distribution of isotope between the CY- and P-carbons of pyruvate, once it has been es- tablished. As noted (Table III) in the experiments with C3H3.C3HOH.- COONa, the resulting liver glycogen contains Cl3 equally distributed

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526 LACTATE CONVERSION TO GLYCOGEN

between the 1,6 and 2,5 fractions, in accordance with expectations. The reactions of Diagram 1 require, furthermore, that isotope administered in the carboxyl position of lactate should occur exclusively in positions 3 and 4 of the resulting liver glycogen. In experiments with CH,-CHOH.- U300Na, undertaken in another connection, it has been noted that isotope was present in detectable amounts only in carbons 3 and 4, as expected.

Some idea of the relative amount of isotope that traverses the reactions of the tricarboxylic acid cycle prior to conversion to glycogen via Re- actions 2 and 1 may be gained from a consideration of the distribution of isotope in the glycogen in experiments in which a-labeled lactate has been fed. (Similar considerations would apply to the experiments in which P-labeled lactate was administered.) Glucose formed via Reaction 2, before randomization of the isotope has occurred, will exhibit the following

isotope distribution pattern, C--&C-C-&--C, in the case of a-labeled lactate. As pointed out earlier, CO2 fixation and the reductive reactions of the tricarboxylic acid cycle (Reactions 3, 10, 9, and S), followed by Reaction 2, will distribute isotope equally between carbons 1,6 and 2,5 of

*=* *=* the resulting glucose (C-C-C-C-C-C). The CO2 fixation reaction and the oxidative reactions of the cycle will introduce isotope into carbons 3,4 of the glucose, but will not disturb the equality of distribution between fractions 1,6 and 2,5. The final glycogen in experiments with a-labeled lactate may therefore be considered as having been constituted from es-

sentially two types of isotopic glucose,4 C-&-C-C-&C, and b-h-

&--&-~~. All of the isotope in the second type of glucose, according to the proposed schemes, must have been involved in one way or another in Reactions 3 to 10.

Let us take as an example the distribution of isotope in the liver glyco- gen in Experiment 6 (Table III). The hexose chain may be pictured

0.30 0.42 0.07 0.07 0.42 0.30

thus, C-C&C-C-C-C, in which notation the superscripts are to be taken as representing absolute amounts of Cl3 in each position in the molecule, rather than isotope concentrations.6 The two types of isotopic glucose from which the final product was constituted may be

0.12 0.12 0.30 0.30 0.07 0.07 0.30 0.30

represented as C-C-C--C-C-C and C-C--C-G-G-C. Thus

4 The symbols indicate the relative isotope concentration, * > 0. 6 For purposes of comparison, the absolute amounts of isotope in the different

positions of the glucose may be represented by the values for concentration, since atom per cent excess Cl3 X (mM of glucose)/100 = mh4 excess Cl3 for any given posi- tion in the glucose, and the factor (mM glucose)/100 is common to the calculation for all positions.

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LORBER, LIFSON, WOOD, SAKAMI, AND SHREEVE 527

it appears that the amount of Cl3 that has been involved in the re- actions of the tricarboxylic acid cycle prior to deposition in glycogen is about 5 times as great (1.34 to 0.24) as that which has gone directly to glycogen via glycolysis. If the Cl4 data from the same experiment are handled similarly, a ratio of the order of 10: 1 is obtained. It should be borne in mind that the differences in isotope content between carbons 1,6 and 2,5, on which these ratios are based, are not very large com- pared to the errors involved in estimating the isotope concentrations.

While the above figures may give an approximate indication of the relative amount of isotope that entered glycogen by different routes in the present studies, they need not indicate the relative amount of glyco- gen constituted from precursors coming over the different paths, since the degree of isotope dilution may not be the same for the different paths. Indeed, it seems quite reasonable to assume a lesser degree of dilution for isotope traversing the direct pathway of Reactions 2 and 1, from which it follows that the calculated ratios give a minimum value for the relative amount of glycogen formed from precursors that have been involved in the tricarboxylic acid cycle compared with those that have taken the direct path. In other words, a molecule of pyruvate introduced into the liver “pyruvate pool” should have, on the average, at least five chances to enter the reactions of the tricarboxylic acid cycle for every chance for entry into the glycolytic reactions (assuming that the reactions of glycolysis are essentially one way in the direction of glycogen deposition, under the circumstances obtaining). The recent paper of Topper and Hastings (15), in which the distribution of isotope in glycogen deposited in liver slices, with a-labeled pyruvate as substrate, is reported, is con- firmatory, in general, of the corresponding experiments performed in the intact animal, as described in the present study and in a preliminary report (1). Topper and Hastings have calculated that involvement of pyruvate in CO2 fixation and subsequent reactions occurs 4 times as rap- idly as does entry into the reactions leading from pyruvate to glycogen. The presentation of the r&e of the tricarboxylic acid cycle in the distri- bution of isotope in glycogen is somewhat incomplete, since there is no dis- cussion of the possibility that the cycle may act as a pathway, in addition to COz fixation, for introducing isotope into positions 3 and 4 of the glucose. Also the possibility that there may be differences in the degree of isotope dilution due to side reactions occurring on the different meta- bolic pathways is not considered. It may be, of course, that this latter factor is of little importance in determining the results in experiments of this type. Its role, however, remains to be evaluated.

It should be pointed out that the foregoing discussion has assumed, implicitly, a homogeneous metabolic system in which mutual interaction

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528 LACTATE CONVERSION TO GLYCOGEN

between the different metabolic pathways could occur freely. This is obviously not the case in the intact rat. Nevertheless, it seems likely that, in the present experiments, the isotopic composition of liver glyco- gen reflects, in the main, metabolic events within the liver cell, where the assumption of homogeneity may more nearly apply. The isotopic composition of the respiratory COZ, on the other hand, must result from the summed activity of all the tissues of the animal’s body.

Finally, it should be pointed out that the general argument, presented in terms of current metabolic schemes, has been conducted in a rather dogmatic fashion in order to limit the discussion to a reasonable length. The general agreement between the results presented and these schemes enhances the likelihood of their reality in the intact animal, but cannot be considered as establishing them as the main or sole metabolic pathway involved in the present studies, as might be inferred if some sections of the discussion are construed too literally.

SUMMARY

CH3.C3HOH.COONa, C13Hs.C13HOH.COONa, and CY4H3.C13HOH.- COONa have been fed to rats and the resulting liver glycogen isolated and degraded, and the distribution of isotope in the glucose chain de- termined. The results have been interpreted in terms of the reactions of glycolysis and the tricarboxylic acid cycle, with which schemes they appear to be in general agreement. The results with lactate labeled in the 01- or P-carbon indicate that of the administered lactate carbon that was deposited as liver glycogen probably less than one-sixth entered the glycogen directly via the reactions of glycolysis, without prior passage through reactions involving randomization of isotope between the LY- and P-carbons.

BIBLIOGRAPHY

1. Lorber, V., Lifson, N., Wood, H. G., and Sakami, W., Am. J. Physiol., 165, 452P (1948).

2. Wood, H. G., Cold Spring Harbor Symposia Qua&. Biol., 13, 291 (1948). 3. Buchanan, J. M., Hastings, A. B., and Nesbett, F. B., J. Biol. Chem., 160, 413

(1943). 4. Lifson, N., Lorber, V., Sakami, W., and Wood, H. G., J. Biol. Chem., 176, 1263

(1948). 5. Cramer, R. D., and Kistiakowsky, G. B., J. Biol. Chem., 137, 549 (1941). 6. Osburn, 0. L., Wood, H. G., and Werkman, C. H., Ind. and Eng. Chem., Anal.

Ed., 8, 270 (1936). 7. Barker, S. B., and Summerson, W. II., J. Biol. Chem., 138, 535 (1941). 8. Wood, H. G., Lifson, N., and Lorber, V., J. Biol. Chem., 169, 475 (1945). 9. Vennesland, B., Solomon, A. K., Buchanan, J. M., Cramer, R. D., and Hastings,

A. B., J. Biol. Chem., 142, 371 (1942).

by guest on May 15, 2018

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LORBER, LIFSON, WOOD, SAKAMI, AND SHREEVE 529

10. Henriques, F. C., Jr., Kistiakowsky, G. B., Margnetti, C., and Schneider, W. G., Ind. and Eng. Chem., Anal. Ed., 18, 349 (1946).

11. Lorber, V., Lifson, N., Sakami, W., and Wood, H. G., J. Biol. Chem., 183, 531 (1950).

12. Stern, J. R., and Ochoa, S., J. Biol. Chem., 179, 491 (1949). 13. Ogston, A. G., Nature, 162, 963 (1948). 14. Potter, V. R., and Heidelberger, C., Nature, 164, 180 (1949). 15. Topper, Y. J., and Hastings, A. B., J. Biol. Chem., 179, 1255 (1949).

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Bordeaux and Margaret Cookand With the technical assistance of Julien

Wood, Warwick Sakami, Walton W. Shreeve Victor Lorber, Nathan Lifson, Harland G.

STUDIED WITH ISOTOPIC LACTATEGLYCOGEN IN THE INTACT RAT,

CONVERSION OF LACTATE TO LIVER

1950, 183:517-529.J. Biol. Chem. 

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