THE OCCURRENCE AND MECHANISM OF THE HEXOSE MONOPHOSPHATE SHUNT IN RAT LIVER SLICES*

BY J. KATZ, S. ABRAHAM, R. HILL, AND I. L. CHAIKOFF

(From the Department of Physiology of the University of California School of Medicine, Berkeley, California)

(Received for publication, September 27, 1954)

It was recently shown by Bloom et al. (1,2) that, in liver, the 1st carbon of glucose is converted to CO2 to a larger extent than is the 6th carbon, and that the Cl402 derived from glucose-l-Cl4 exceeds that from glucose evenly labeled with CY4. These workers suggested that the hexose mono- phosphate shunt plays a significant rale in glucose metabolism in liver. A previous report from this laboratory dealt with equations for estimating the extent of the hexose monophosphate shunt in that tissue (3). In the present report, those equations have been extended, and the assumptions upon which they are based have been examined. The operation of this shunt in liver slices was studied, and some phases of its mechanism have been elucidated.

Materials and Methods

Preparation of Labeled Substrates-Glucose evenly labeled with Cl4 (glucose-E-C14) w&s prepared photosynthetically as described by Putman and Hassid (4). Glucose-l-Cl4 and ribose-i-Cl4 were purchased from the National Bureau of Standards. Glucose-6-Cl4 was prepared by the method of Sowden (5) as modified by Roseman (6). All sugars were checked chro- matographically for purity.

Incubation Procedure-Long-Evis rats weighing from 200 to 300 gm. were fed a high carbohydrate diet (7) for 3 days before they were sacrificed. The preparation of liver slices and the incubation techniques were described in previous reports (8).

Isolation of C14-Glucose from hcubation Mixture and Determinution of Cl4 Content of Its Individual Carbon Atoms-Glucose-Cl4 was isolated chromat.ographically on paper (8) and eluted with water. Carrier glucose was added, and the solutions were concentrated. The C14-glucose was degraded by fermentation with Leuconostoc mesenteroides which yields COz from carbon 1, ethanol from carbons 2 and 3, and lactate from carbons 4, 5, and 6 (9). The organisms were grown for 12 hours and harvested es- sentially as described by Gunsalus and Gibbs (9). The organisms were washed three times and tested for activity before use.

* This work was supported by a contract from the United States Atomic Energy Commission.

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854 HEXOSE MONOPHOSPHATE SHUNT

The type of flask used for fermentation has been described elsewhere (10). The fermentation mixture consisted of the cells from about 1 liter of growth medium, 0.4 mmole of the CY4-glucose, and 5 ml. of 0.5 M phosphate buffer (pH 6.0) in a final volume of 12 to 15 ml. The vessels were flushed with nitrogen, alkali was added to the center wells, and the vessels were capped and evacuated with a hypodermic needle. The fermentation was terminated after 1 hour, and the organisms were removed by centrifuga- tion. The recovery of CO2 was quantitative, but that of ethanol and lac- tate ranged from 60 to 70 per cent. The ethanol was obtained by distilla- tion of about one-half the volume of the neutralized medium, then oxidized to acetate with acid-dichromate (ll), and the acetate was recovered by steam distillation. The non-volatile fraction containing the lactate was acidified to pH 2, excess silica was added, and the fairly dry mixture was extracted on a Biichner funnel with a 1: 1 mixture of wet n-butanol-chloro- form. In some experiments, anhydrous sodium sulfate was used instead of silica, and the dry mixture was extracted with ether. Water was added to the extract, the lactate was then titrated with base, and the organic sol- vents were evaporated. The methods used for the degradation of lactate and acetate were described elsewhere (12).

When glucose-l-C14, -2-CY4, and -6-Cl4 were degraded by this procedure, about 2 to 5 per cent of the Cl4 w&s recovered in carbons other than 1, 2, and 6. This may be due in part to some side reactions of bacterial metabo- lism; hence, incorporation of the order of 5 per cent of the Cl4 in any 1 carbon is not considered significant.

Evaluation of Hexose Monophosphate Shunt in Liver Slices

The conversions of glucose-l-CY4, -6-CY4, and -E-V to Cl402 are com- pared in Table I. In each experiment, separate portions of a single liver were incubated with either glucose-1-CY4, -6-CY4, or -E-C!14. When glucose served as the sole substrate, the CY402 yields ranged from 2 to 13 per cent for glucose-1-C14, from 0.7 to 4.5 per cent for glucose-6-CY4, and from 2 to 5 per cent for glucose-E-CY4. It should be noted, however, that, in the case of each liver, the Cl402 recoveries from glucose-l-U4 were about the same as that from glucose-E-C” and were appreciably higher than those observed with glucose-6-CY4. This is reflected in the values for the ratio, U, i.e. (CY402 from glucose-E-CY4)/(CY402 from glucose-1-CY4), which ranged from 0.7 to 1.2 (except for a single value as high as 2), and the ratio, W, i.e. (C1402 from glucose-6-C14)/(C1402 from glucose-l-CY4), which ranged from 0.33 to 0.65.

The values for ratios U and W observed here in experiments in which glucose served as the sole substrate were higher than those reported by Bloom et al. (1, 2). These investigators, however, added 50 pmoles each of lactate, acetate, and gluconate to their incubation media. Table I

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TABLE I Oxidation of Variously Labeled CW&ucoae

250 mg. of liver slices were incubated in 2.5 ml. of Ringer-bicarbonate buffer at pH 7.3 (24) containing (unless otherwise stated) 55 @moles of labeled glucose. The addition referred to by + was a mixture of 50 ymoles each of acetate, lactate, and gluconate as their Na salts. Gas phase 95 per cent 01-5 per cent CO*. 3 hours incubation at 37”.

Rat No.

9

10

11

16

17

18

C-E C-l C-E C-l C-E C-l C-E C-l C-E C-l C-E C-l C-E C-l C-E C-l C-E C-l C-E C-l C-E C-l C-E C-l C-E C-l C-6 C-l C-6 C-l C-6 C-l C-6 C-l C-6 C-l C-6 C-l

4ddition to

medium

+

+

+

+

-t-

+

+

+

+

+

+

+

+

+

Per cent of added 0’ ecovered BP

C’Ot

5.0 2.6 4.1 4.3 4.8 4.7 4.6 4.7 3.3 3.8 2.9 3.6 0.8 2.6 2.9 4.2 1.0 2.7 2.8 2.3 0.8 1.4 4.5 4.4 0.7 2.2 4.0

11.2 1.9

10.3 4.5

12.3 1.5

10.9 3.4 5.3 1.2 5.3

.y = c?! C-l

2.0

0.95

1.02

0.98

0.87

0.81

0.31

0.76

0.37

1.2

0.57

1.2

0.32

-

I

-

Iv- g

0.36

0.12

0.36

0.14

0.65

0.21

Contribution of glycolysis to

:ot fomled

Woolf=

per cm;

98

88

89

89

86

85

47

83

57

92

76

89

50

89

69

90

73

97

81

GlUCO~ xtabolized

mt

pbl cc.d

94

70

72

72

67

64

23

61

31

78

49

72

25

75

42

75

45

91

58

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856 HEXOSE MONOPHOSPHATE SHUNT

TABLE I-Concluded -i-

Rat No. Addition

to medium r

--

-

U-C-E C-l

gl = e! C-l

_-

19

w

C-6 C-l

C-6 C-l C-E C-6 C-l C-E C-6 C-l

f +

5.1

13.1

3.0 17.7 2.0 0.7 2.1 3.3 1.6 4.8

0.39

0.18

0.96

0.69

- -

0.33

0.33

-

( fl

.I -

-

Contribution of glycolysis to

:Oz formed ram glucose

V3*

)a cc?&;

91

78

88

88

80 88

Glucose :atabolized

wt

per ccnl

77

54

71 71

57 71

* Calculated from Equation 1 or 2 with R = 2.7. t Calculated from Equation 3 or 4 with R = 2.7 and P = 3.1. $ In the experiments carried out n-ith the livers of these rats, only 3 rmoles of

labeled glucose were incubated in 2.5 ml. of the medium with 250 mg. of slices.

shows that, when these additions are made, there is a lowering of the values for both ratios. It should be noted, however, that the concentrations (0.02 M) of lactate, acetate, and gluconate used by us were twice those used by Bloom et al. (1,2) and that the COZ ratios obtained by us upon the addition of these compounds were lower than the ones reported by Bloom et al. These additions considerably depressed the CY402 yields from glu- case-6-Cl4 and glucose-E-Cr4, but had little or no effect on the C?402 yields from glucose-1-CY4.

In order to evaluate the relative significance of the glycolytic and non- glycolytic pathways of glucose metabolism in rat liver, Bloom et al. (1) made the following three assumptions: (1) that glucose is catabolized to COZ by only two pathways, namely, Embden-Meyerhof glycolysis plus Krebs cycle and the direct oxidative pathway, (2) that, via the oxidative pathway, only the aldehyde carbon of glucose contributes to the COZ, and (3) that, glycolytically, glucose yields 2 triose molecules whose carbons give rise to CO2 in the same proportion as do the respective carbons of added lactate. On the basis of these assumptions, equations relating the ratio U to the recoveries of C1402 from lactate-l-G4, -2-C14, and -3-Cl4 and the conclusion that a major portion of the COZ derived from glucose catabolism arose via the shunt were derived. A subsequent examination of these equations and calculations by Katz et al. (3) showed, however, that the amount of COz, produced from glucose by liver slices, via the direct oxida-

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J. KATZ, S. ABRAHAM, R. HILL, AND I. L. CHAIKOFF 857

tive patch, is small, and that the major portion of the COZ derived from glucose was formed glycolytically.

In the above considerations we have dealt only with the CO:! derived from glucose. We must also consider the fraction of the glucose molecules catabolized by each pathway. Let us designate the fraction of the CO2 derived glycolytically as E and the fraction of the glucose catabolized glycolyt- ically as F. Both E and F can be expressed as functions of the CY402 ratios U and W.

Expressions for E were derived previously (3) and are given in Equations 1 and 2.

B = mu - 1) --.- U(6R - 1)

(1)

6RW E = 1 + W(6R - 1)

where the parameter R is equal to (c + d + e)/3e and c, d, and e are the yields of the C1402 recovered from lactate-l-C14, -2-CY4, and -3-C14, respec- tively.

Expressions for F, t,he fraction of glucose catabolized glycolytically, can be derived if it is assumed that the carboxyl carbons of the phospho- pyruvate derived from glucose are completely oxidized to CO*. Then each ‘mole of glucose oxidized glycolytically yields 2(c + d + e)/c moles of COZ, and each mole of glucose oxidized via the shunt yields 1 mole of COZ. Let n be the moles of glucose catabolized, nF moles be t,he fraction pro- ceeding via glycolysis, and n( 1 - F) moles that proceeding via the shunt. Thus ((nF) 2(c + d + e))/c moles of CO2 will be formed via glycolysis, and n(1 - J’) moles of COZ will be formed via the shunt. For simplicit,y, let us represent 2(c + d + e)/c by P. Employing the expressions for t,he specific activity of the COZ as derived for Equations 1 and 2, we obtain the following expressions for F as functions of U and W.

whence

R(6U - 1)

F = R(P - 1) + U(6R - I’)

PF

w = n6R [ 1

PF n 6~ + (1 - F) 1

(3)

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858

whence

HEXOSE MONOPHOSPHATE SHUNT

F- 6RW

P(1 - W) + 6RW

The relation between E and F can be expressed by a simple and useful equation. Let m moles of CO2 be formed from glucose by both mecha- nisms, and let mE and m(1 - E) moles be formed by glycolysis and the shunt, respectively. Since each mole of glucose yields P moles of COz via glycolysis and 1 mole via the shunt, mE/P and m(1 - E)/l moles of glucose, respectively, will

4 A. 4.0 . . . . .._ _ (z

--- E (02)

2.6 - E (R=4)

2.2

be formed via these two paths. The fraction F

B.

IO 30 50 70 90 Ia3

%CO,irom Glucose via Glycolysis

% Glucose Co+ob?iired via Glycolysis

--..... f= --- E(R=2) - EW4)

IO 30 50 70 90 loo

%COefrom Glucge via Glycolysis

% Glucose Cotabolizcd via Glycolytis

FIQ. 1. Plot of Equations 1 to 4

can now be expressed by the equation

F= E/P

; + (1 - I.$) (5)

Equations 1 to 4 are plotted in Fig. 1, with values of 2 and 4 for R and 3 for P. These values were selected because they represent the range of the experimentally determined values for R and P (see Tables II and III). Figs. 1, A and 1, B show t,hat, when glycolysis predominates, a small in- crease in the extent of the direct oxidative pathway will cause a pronounced decrease in the values for t,he ratios U and IV. The values for E, as cal- culated from Equation 1, are not appreciably altered by changes in the parameter R. As shown in Fig. 1, A, doubling of the value for R over the

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J. KATZ, 5. ABRAHAM, R. HILL, AND I. L. CHAIKOFF 859

whole range of the curve results in only a small decrease in the value of E. On the other hand, in the case of Equation 2 (Fig. 1, B), an increase in R

TABLE II

Oxidation of Lactate-C’” and Calculation of Values for R and P

Each vessel contained 50 amoles of labeled dl-1actat.e and 50 Fmoles of glucose. Other conditions as in Table I.

Rat No.

6

7

8

8A*

dlel of lactate

-

I

C-l c-2 c-3 C-l c-2 c-3 C-l c-2 c-3 C-l c-2 c-3

Per cent of added 0 recovered BS

C”O2

47.8 (c) 10.5 (d) 8.0 (e)

48.5 (c) 11.1 (d) 0.8 (e)

48.2 (c) 10.2 (d) 7.4 (e)

40.3 (c) 8.2 (d) 7.3 (e)

I- R==c+d

3c

2.8

3.2

3.0

2.6

T p _ Z(c + d + e) c

2.8

2.7

2.8

2.8

* This rat was fasted for 24 hours prior to sacrifice.

TABLE III Comparison of Values for Parameters R and P Obtained from Various Sources

R P source No. of rats

Range Aversge Range AWIll@

Table II.. . 4 2.6-3.2 2.9 2.7-2.9 2.8 Bloom et al. (l)*. 5 1.7-2.3 2.0 3.1-3.4 3.2 Felts ‘I I‘ (7). 3 2.2-3.0 2.6 3.1-3.4 3.2 Strisowert . 4 3.2-3.8 3.4 3.2-3.8 3.3

Mean...................... 2.7 3.1

* The incubation medium contained lactate, acetate, and gluconate. t Unpublished experiments.

will cause an increase in the value of E, and, when the values for the ratio W are low, this effect becomes appreciable.

Values for R and P were calculated from experimental data obtained here and from those reported in the literature. These values are recorded in Tables II and III. The values for R ranged from 1.7 to 3.8, and those for P from 2.7 to 3.8. Although it is preferable to determine these param-

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860 HEXOSE MONOPHOSPHATE SHUNT

eters for each liver in which the glucose ratios are obtained, we have, for the purpose of subsequent calculations, chosen mean values of 2.7 and 3.1 (from Table III), respectively, for R and P.

Calculat,ed values for the contribution of glycolysis to (1) the glucose- derived COZ, i.e. E’, and (2) the total glucose catabolized, Le. F, are shown in the last two columns of Table I. When glucose was t,he sole subst,rate, the values for E ranged from 83 to 98 per cent. In other words, in some cases, practically all of the glucose-derived COZ arose via glycolysis. Un- der these same conditions, the values for F ranged from 61 to 94 per cent. When lactate, acetate, and gluconate were added to the medium in addition to the labeled glucose, t,he values for E and F were depressed and, as pre- viously noted, the CY402 yields from glucose-E-C4 and glucose-6-Cl4 were depressed to a much greater extent than those from glucose-1-CY4. From this it may appear that the additions to the incubation medium reduced the amount of glucose that was metabolized via glycolysis without affecting the operation of the direct oxidative pathway. It is also possible t,hat glucose catabolism via glycolysis was not affected, but t,hat the Cl4 activity was trapped in the large lactate pool.

It was also observed that the additions caused a doubling in the incor- poration of labeled glucose into liver glycogen. Thus, in the presence of these additions, the percentages of the labeled glucoses recovered in glyco- gen were increased from 4 to 5 per cent to 9 to 10 per cent.

It is significant that variations in the experimental conditions greatly affect the values for the ratios U and W. Whereas, under our conditions, glucose seems to be catabolized primarily via the Embden-Meyerhof and Krebs cycle, other procedures of incubaGon or keatment of t,he rats may yield different patterns.

The most critical assumption involved in the derivation of the equations is that glucose carbons 2 to 6 do not contribute to COZ. If, however, these carbons are oxidized to COZ, the extent of the shunt will be greater than that calculated from Equations 1 to 5, and the discrepancy between the true and calculated values will be large for low values of the ratios U and W.

With regard to liver slices, this assumption is valid if the pentose formed via the shunt is completely reconverted to glucose and mixes with the glucose pool. In liver slices, in which only a small fraction of t,he added glucose is oxidized, the contribution of the pentose moiety to COZ by secondary oxidation will not greatly affect the magnitude of the calculated values for E and F. On the other hand, in tissues in which glucose oxida- tion is large, the resynthesized glucose formed via the shunt will be exten- sively metabolized; in this case CO* format,ion from carbons 2 t,o 6 will be appreciable. As has been pointed out in the case of rat mammary gland slices which metabolize glucose rapidly (13) or in systems in which this

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J. KATZ, S. .4BRAHAM, R. HILL, AND I. L. CHAIKOFF 861

recycling of metabolized glucose is extensive, Equations 1 to 5 cannot be used.

In the calculation of t,he fraction of the glucose catabolized via glycolysis, F, another assumption was made, namely, that carbons 3 and 4 of glucose are completely converted to Cot. The validity of this assumption may be questioned, since Cl4 was always detected in lactate (see Fig. 2). The radioactivity incorporated into 1actat.e was variable but quite low. Under other experimental conditions (14), however, the CP incorporation into lactate may be larger, and in such cases the amount of glucose catabolized via the Embden-Meyerhof scheme will exceed that calculated by Equations 3 to 5. Thus when CY402 production is used to estimate the relative con- tributions of these two pathways, E provides a more reliable index than does F.

Concerning Mechanism of Hexose Monophosphate Shunt

Chromatographic Patterns Observed ifi Metabolism of Glucose-l -Ct4, -6-C14, and -E-Cl4

The products of the water-soluble fraction of the incubation mixture were examined by a chromatographic, radioautographic procedure similar to that reported for acetate-Cl4 (8). Typical chromatograms obtained in expe?iments with glucose-1-C14, glucose-6-CY4, and glucose-E-Cl4 are shown in Fig. 2. -4bout 90 per cent of the Cl* in the water-soluble fraction was present as glucose, and the remaining Cl4 was accounted for by six or seven compounds, the most active of which was lactate. Glutamic acid, glutamine, and alanine were also identified. With the level of Cl4 used in these experiments, incorporation into any compound of as much 5t9 0.5 per cent of the added Cl4 could be detected on the chromatogram. No signifi- cant differences in the chromat,ographic patterns were discernible when the experiments were carried out with labeled hexoses of high specific activity (Table IV). If the shunt did play a major r61e in glucose catabo- lism in liver, a considerable difference in the chromatographic patterns should have been detected.

The C1402 ratios (U and W) and the chromatographic data can be ex- plained on the basis of two possible mechanisms: (a) the exchange between C-l of glucose and CO2 and (b) the reconversion of the pentose moiety into glucose.

Exchange between C-1 of Glucose and CO-The reversibility of the de- carboxylation of phosphogluconate has been demonstrated (15), and the possibility that t,his reaction accounts for some COZ fixation was suggested. To test t,his possibility, liver slices were incubated witch CY4-labeled bicar- bonate in the presence of unlabeled glucose or ribose. The CY4-glucose formed during t,he course of the incubation was isolated chromatographi-

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862 HEXOSE MONOPHOSPHATE SHUNT

FIG. 2. Radioautographs of chromatograms obtained from rat liver slices incu- bated with variously labeled CW-glucoses. In A, B, and C, chromatograms obtained from the liver of the same rat (Rat 20, Table I). The 04 activities of the labeled glucoses incubated were not identical, and the radioautographs were intentionally overexposed to show the less active compounds. In D, chromatogram obtained with a glucose sample of considerably higher specific activity than used in the other ex- periments.

tally and degraded. The results are shown in Table V. Most of the 04 was equally distributed between carbons 3 and 4, and no significant fixation of COz into the aldehyde carbon occurred. Thus, the operation of an exchange mechanism in our system would appear to be excluded.

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TABLE IV Chromatographic Analysis of Water-Soluble Compounds Derived from

Metabolism of Glucose-E-C’4 and Glucose-l-04 by Rat Liver Slices

The experimental conditions were the same as those described in Table I, except that about 0.1 mg. of a labeled glucose containing IO+. was used as substrate. The chromatographic procedure is described in the text. The values for t,he glucose- 1-W experiment were det,ermined from the chromatogram represented in Fig. 2, D.

_.~..

Distribution of Cl4 on chromatogram in

Substrate

Glucose-E-(74. Glucose-l-Cl*.

TABLE V Distribution of 04 Recovered in Glucose Derived from Ribose-l-O4

250 mg. of rat liver slices incubat.ed with 3 pmoles of ribose-1-C’4. Conditions similar to those described in Table I. The additions of lactate, acetate, and glu- conate were the same as those described in Table I.

10 .+ 12* CY402 + unlabeled ribose 1 2 3 4 5 6

10a + 12a* Ribose-1-W 1 2 3 4 5 6

10b + 12b* Ribose-l-04 + additions 1 2 3 4 5 6

Rats 13-15* Ribose-l-Cl4 1 2 3 4 5 6

Substrate lucose carbon No.

* The glucose samples were pooled for purposes of degradation.

863

PI

--- :r cent of total C’” recovered in in-

dividual carbons ._-.__-

5

4 43 43 4 1

43 4

42 5 3 3

44 3

40 5 6 2

36 5

36 11 6 6

.-.

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864 HEXOSE MONOPHOSPHATE SHUNT

Ribose Metabolism by Rat Lker Slices-To test for the second mechanism, rat liver slices were incubat,ed w&h ribose-1-C14. It was catabolized to COZ and readily converted t,o glucose. As shown in Table VI, the CY402 yields varied from 3 to 10 per cent when ribose served as sole substrate. The CY402 recoveries were, however, markedly depressed by t.he addition of unlabeled lact,ate, acetate, and gluconate. When the aqueous extracts were examined chromatographically, the predominant CY4-labeled com- pound found was glucose, and very little Cl4 was detected in other com- pounds (Fig. 3). In one experiment (Rats 13,14, and 15), all of the added ribose was utilized, but in Experiments lOa, 12a, 20, and 21, the utilization of the ribose did not proceed to this extent. In Experiments lob and 12b,

TABLE VI

Oxidation of Ribose-1-P and Its Conversion to C14-Glucose by Rat Liver Slices

For the description of the experiments see Table V and the text.

Rat NO. Flask

a b

None

+ None

+ None

‘I “ “ ‘I

E

_.

Per cent of added C” re-

overed BS 0%

4.7 1.1 8.1 2.2 5.0

10.0 7.3 2.7 6.7

Distribution of Cl4 on cbromatogrsm

10

11

13 14 15 20 21

per cent per cmt per cent 77 21 2 65 34 1 79 20 1 68 31 1 81 0 19 80 0 20 76 0 24 45 48 7 56 38 6

in addition to U4-glucose and CY4-ribose, t,he only other labeled compound detected was lactate. In Experiments lOa, 12a, 20, and 21, some of the Cl4 was also found in alanine and glutamic acid. In the experiments with Rats 13, 14, and 15, in which no CY4-labeled ribose remained at the end of the incubation period, t,he Cl4 incorporation into compounds other than glucose amounted to about 20 per cent. Most of this 20 per cent was present in three unidentified compounds that were not observed in the other experiments, and the most prominent occupied a position overlapping that of urea.

Although the presence of ribokinase in microorganisms has been demon- strated (16), t,he existence of ‘ribokinase has not hitherto been reported in animal tissues. Our findings, in conjunction with the well described con- version of pentose phosphate to hexose phosphate (17), suggest the ex- istence of an active ribokinase in rat liver.

GhlCOSe Ribox Others

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J. KATZ, S. ABRAHAM, R. HILL, AND I. L. CHAIKOFF 865

The mechanism of the conversion of a pentose phosphate to a hexose phosphate has been extensively studied at the enzyme level, and the results

FIG. 3. Radioautographs of chromatograms obtained from rat liver slices incu- bated with ribose-I-C14. The numbers in parentheses refer to the rat numbers (see Table VI). Chromatograms A and C were exposed for 1 week; Chromatogram B for 2 weeks.

are discussed in several reviews (16, 18-21). Thus far, studies on this conversion in rat liver slices have not appeared. The conversion of ribose to hexose can be formulated by two schemes. These schemes merely

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866 HEXOSE MONOPHOSPHATE SHUNT

represent abbreviated reaction sequences elaborated by other workers. Only the carbon skeletons of the compounds involved are considered. The first scheme represents the conversion of 2 ribose molecules to 1 of glucose and 1 of t&rose (Scheme A).

1c 1c I I

2c 2c I I

2 3c 1c I I

4c 2c I I

5c -+ 3c I

4b I

5c

1c I

2c I

1c 2c I I

+ 3c --j 36 + 3i= I I I

4c 4c 4c I I I

5c 5c 5c

2 pentose + heptose + triose ---) hexose + tetrose

SCZIEME A

The tetrose can act as a glycolaldehyde acceptor, which is donated by a pentose molecule, giving rise to a hexose and a triose. Thus the over-all result can be expressed as presented in Scheme B.

It should be noted that, since the triose can also be resynthesized into glucose, Scheme B providea for the complete conversion of a pentose to a hexose.

1C 1c 1c I I I

2c 2c 2c I I I

3 3c 1C 2c I I I

4c --) 3c + 3c + 3c

3 pentose -+ hexose + hexose + triose

SCHEME B

According to Schemes A and B, with ribose-l-C4 as substrate, only car- bons 1 and 3 of the resulting glucose will be labeled. In the case of Scheme A, the ratio of Cl4 in carbon 1 of glucose to that in carbon 3 will be 1, and, in the case of Scheme B, the value for this ratio will be 2.

The results of the degradation of glucose formed from ribose-l-W, in

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J. KATZ, S. ABRAHAM, R. HILL, AND I. L. CHAIKOFF 867

three experimer&, are recorded in Table V. Most of the activity was found in carbons 1 and 3, and the observed ratio waa about 1. Thus, our finding served t,o support Scheme A.

The low C** activity of carbon 2 of glucose is of interest. If the 1,3-C’*- hexose phosphate formed from ribose-l-C’* is further metabolized via the shunt, hexose phosphate with C!** activity in carbon 2 will be formed by another turn of this cycle. The absence of significant activity in this carbon indicates that, in rat liver slices, t,he formation of free glucose from hexose phosphate, with subsequent mixing of such glucose with the general glucose pool, is much faster than the over-all operation of the shunt.

The C?*O2 yield from glucose-l ,3-C’* will be higher than that from glucose-l-c’*, and the observed Cl402 yields from ribose-l-C?* can be accounted for readily by the met.abolism of such labeled glucose.

Our results suggest the formation of a tetrose which does not serve as an acceptor for the glycolaldehyde derived from pentose or heptose. This was unexpected, since the results of Racker et al. (22) and of Horecker et al. (17) imply that the t&rose can act as an acceptor.

Horecker et al. (17) studied the conversion of ribose-5-phosphate-l-C’* into hexose phosphate by a rat liver enzyme preparation and fouhd 74 and 24 per cent of the Cl* in glucose carbons 1 and 3, respectively. This result was considered to be in fair agreement with Scheme B. Experiments with ribose-5-phosphate-2,3-U* also were in line with the operation of Scheme B; i.e., 3 pentose -+ 23 hexose. The results obtained here with rat liver slices apparently are at variance with those obtained with cell- free preparations.

We have found no evidence for the accumulation of a tetrose, and, because of the high efficiency of the conversion of the pentose to hexose in many systems (23), it is suggested that the tetrose in some way is con- verted to glucose. This problem is under further investigation.

SUMMARY

1. The catabolism of glucose-l-C!* and -6-C’* and evenly labeled glucose- Cl* of rat liver slices was studied.

2. The CY402 yields observed in experiments with glucose-E-C’* and -l-C’* were about equal and about 3 times those observed with glucose-6- 04.

3. The addition of a mixture of 50 pmoles each of lactate, acetate, and gluconate greatly depressed C1*02 formation from both glucose-E-C’* and -6-C’*, but had little effect on the PO2 derived from the glucose-l-C14.

4. Equations for calculating t,he extent of glucose metabolism in rat liver slices via the Embdcn-Meyerhof-Krebs route and the hexose mono- phosphate shunt are discussed. It was calculated, for rat liver slices,

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868 HEXOSE MONOPHOSPHATE SHUNT

that 83 to 98 per cent of glucose-derived COZ was formed glycolytically, and that 61 to 94 per cent of the total utilized glucose was catabolieed glycolytically. When lactate, acetate, and gluconate (0.02 M) were added, a greater portion of the glucose catabolized apparently proceeded via t.he hexose monophosphate shunt.

5. The products of the metabolism of glucose-1-U4, -6-C14, and -E-C?4 were examined by paper chromatography, and the metabolic patt,erns of all three labeled sugars were found to be similar. The significance of this finding is discussed.

6. Rat liver slices converted free ribose to glucose. It is therefore suggested that liver contains an active ribokinase. Glucose derived from ribose-l-Cl4 was isolated and degraded,,and the Cl4 distribution in its carbons was determined. The Cl4 was equally dist,ributed in carbons 1 and 3, with no significant incorporation in the other carbons. These results suggest t.hat the pentose formed by the decarboxylation of 6-phos- phogluconate is resynthesized into glucose by rat liver.

BIBLIOGRAPHY

1. Bloom, B., Stetten, M. R., and Stetten, D., Jr., J. Bid. Chem., 964, 681 (1953). 2. Bloom, B., and Stetten, D., Jr., J. Am. C&m. Sot., 76, 5446 (1953). 3. Katz, J., Abraham, S., Hill, R., and Chaikoff, I. L., J. Am. Chem. Sot., 76, 2277

(1954). 4. Putman, E. W., and Hassid, W. Z., J. Bid. Chem., 196, 749 (1952). 5. Sowden, J. C., J. Am. Chem. Sot., 74, 4377 (1952). 6. Roseman, S., J. Am.. Chem. Sot., 74, 4467 (1952). 7. Felts, J. M., Chaikoff, I. L., and Osborn, M. J., J. Biol. Chem., 191, 683 (1951). 8. Kat,z, J., and Chaikoff, I. L., J. Biol. Chem., 266, 887 (1954). 9. Gunsalus, I. C., and Gibbs, M., J. Biol. Chem., 194,871 (1952).

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297, 393 (1954). 18. Dickens, F., Brookhaven Symposia in Biology, No. 5, 134 (1953). 19. Horecker, B. T,., Brewers’ Digest, 96, 214 (1953). 20. Racker, E., Advances in h’nzgmol., 16, 141 (1954). 21. Dickens, F., Brit. Med. Bull., 9, 105 (1953). 22. Racker, E., de la Iiaba, G., and Leder, I. G., Arch. B&hem. and Biophys., 46,

238 (1954). 23. Glock, G. E., Biochem. J., 62, 575 (1952). 24. Umbreit, W. W., Burris, R. IT., and Stauffer, J. F., Manometric techniques and

related methods for the study of t.issue met,abolism, Minneapolis (1945).

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J. Katz, S. Abraham, R. Hill and I. L. ChaikoffSHUNT IN RAT LIVER SLICES

OF THE HEXOSE MONOPHOSPHATE THE OCCURRENCE AND MECHANISM

1955, 214:853-868.J. Biol. Chem. 

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