The Flux of Pyruvate in Perfused Rat Heart

Eur. J. Biochem. 36,362-368 (1973)

The Flux of Pyruvate in Perfbed Rat Heart

John MOWBRAY and James H. OTTAWAY Biochemistry Department, University Medical School, Edinburgh

(Received February 1, 1973)

1. It was possible to find a steady-state lactate concentration in a closed-circuit heart perfusion containing added glucose. Insulin but not growth hormone (4 pglml) increased the equilibrium concentration in perfusate by 200 Ole. Intracellular lactate also remained constant and was about twice that in perfusate: this distribution is difficult to explain whether ionic or undissociated lactate is the permeating species.

2. Pyruvate accumulated linearly in perfusate, reaching a peak after 30 min perfusion and then declined non-linearly. Insulin increased the peak concentration and growth hormone slowed the decline after the peak. The intracellular pyruvate concentration stayed constant and, except in the first few minutes, was lower than the perfusate concentration.

3. Pyruvate addition a t zero time could inhibit pyruvate accumulation but altered neither the non-linear decline, the intracellular pyruvate level nor the lactate equilibrium.

4. The [NAD]/[NADH] ratio, after 10 min of pre-perfusion on open circuit, was 23. During 2 h of perfusion the ratio rose slowly and then declined. If either insulin or growth hormone were present in the medium, the ratio fell to about 11 during the 6rst few minutes of perfusion and remained low for 90 min. These values could not easily be correlated with either the varying extracellular, or the constant intracellular [lactate]/[pyruvate] ratios.

5. The glucose and oxygen consumption of the hearts during perfusion was measured, and the change in glycogen concentration in the tissue. It was then possible to draw up a balance sheet which indicated that the control hearts oxidied little other than carbohydrate for the 60 min of observation, but both insulin and growth hormone-treated hearts oxidised significant quantities of fatty acid during the first 15 min. It appears that growth hormone, like insulin, has an immedi- ate effect on heart metabolism. The addition of pyruvate a t zero time appeared to stimulate carbohydrate oxidation in hearts perfused with insulin, even though the hormone had been present in the pre-perfusion fluid.

6. Consideration of the variation in the extracellular [lactate]/[pyruvate] ratio with length of perfusion, and its lack of relationship to the intracellular [lactate]/[pyruvate] ratio, together with similar reports of other workers, leads us to conclude that neither lactate nor pyruvate establish a concentration gradient across the heart cell membrane which is based on passive diffusion. The discrepancy is particularly marked with respect to pyruvate. Our results strongly suggest that the extracellular [lactate]/[pyruvate] ratio cannot be used as an index of the cyto- plasmic redox potential in perfused heart.

7. A tracer study of pyruvate metabolism in the reproducible non-steady-state conditions described can offer an explanation for the behaviour of the observed metabolic parameters : this study is reported in the following papers.

The experiments which are reported in this and succeeding papers were designed to produce data that could be used to test a new method of obtaining rate constants from isotopic tracer measurements.

Enzymes. Phosphoenolpyruvate carboxykinase or GTP : oxaloacetate carboxy-lyase (transphosphorylating) (EC 4.1.1.32); lactate dehydrogenase (EC 1.1.1.27); pyruvate dehydrogenase is complex of pyruvate decarboxylase (EC 4.1.1.1); lipoate acetyltransferase (EC 2.3.1.12); lipoamide dehydrogenase (EC 1.6.4.3).

Although this method would be an improvement on the usual curve-peeling or analytical minimisation methods (see for example [I]), it was still orginally conceived for the solution of problems that could be expressed in terms of first-order differential equations with constant coefficients. The biological system to produce the data had therefore to be in a steady- state with respect to material fluxes.

The system that was chosen for study was the disposition of pyruvate by the isolated perfused rat

Vol.36, No.2, 1973 J. MOWBRAY and J. H. OTTAWAY 363

heart. There were several reasons for this: first, it is easy to take multiple samples from perfusion systems, and on the other hand, it is easy to measure the concentrations and specific activities of metabo- lites a t the end of the perfusion by the “freeze-stop” technique [2], especially when an organ from a small animal is perfused. Secondly, cardiac muscle both produces and oxidizes lactate [3], so that there were good grounds for supposing a priori that it would be possible to determine a concentration of lactate in the perfusate at which utilisation just balanced production, i.e. a steady-state, for any given set of experimental conditions. Thirdly, the almost complete absence of phosphoenolpyruvate carboxykinase from cardiac muscle [4] meant that analysis of the tracer data would not be complicated by recy- cling of the label from the citric acid cycle to pyruvate or a precursor. Experiments which confirmed this assumption are reported in a later paper. Thus the movement of tracer should be analysed by a three- compartment mammillary system [5] as shown below, where the label should exchange reversibly among the three compartments labelled lactate, pyruvate, and alanine, and be lost irreversibly from the system by conversion of pyruvate to acetyl-CoA.

CHO

Lactate + Pyruvate + Alanine

Acetyl-CoA

I I

C’j,

In view of the known instability of pyruvate [63, and because it was thought that there would be difficulty in adding labelled pyruvate to the perfusate without disturbing the equilibrium, it was decided to add the tracer to the lactate pool, as ~-[W]lactate (prepared enzymically). The lactate was labelled in the 2-position, in order to get as much extra information as possible from the labelling patterns in citric- acid-cycle intermediates. Because of this decision, attention was concentrated on finding the conditions for a steady concentration of lactate in the perfusate. It was only later realised that, although it was possible to keep the concentration of lactate constant, it was not possible simultaneously to keep the p p - vate concentration in the perfusate constant, without altering the experimental arrangements more drasti- cally than was contemplated. The original purpose of the experiments (to test a mathematical hypothesis) had therefore to be abondaned, but at the same time it was realised that the kinetics of the tracer movement, particularly in the pyruvate compartment were capable of providing a great deal of information about the organisation of enzymes affecting pyruvate metabolism in intact cardiac tissue. The experiments were therefore carried to completion.

The work has been divided for simplicity into three parts: the establishment in the presence and absence of insulin and growth hormone of conditions for a steady state with respect to lactate and for a reproducible non-steady state with respect to pyruvate together with the evaluation of the material fluxes in this paper; the tracer experiments and the models of cell metabolism devised to explain these in the following paper ; and the theoretical basis behind the modelling on the last paper.

EXPERIMENTAL PROCEDURE The perfusion apparatus of O’Brien [7] was

modified for closed circuit use [8]. 0, concentration was monitored by Clark electrodes (Yellow Springs Instrument Co., Ohio) placed before and after the heart chamber, and the flow rate through the heart was estimated by the rate of travel of an injected bubble along a calibrated tube. An injected tracer (15 mg nicotinamide in 0.1 ml) was uniformly di- stributed in the liquid phase (50ml perfusate + heart water) within 2 min after injection. Hearts were removed under ether anaesthesia from 220- 260 g fasted male albino rats. The hearts were cooled, cleaned, weighed and washout preperfused for 10 min with oxygenated Krebs-Ringer medium [S]. After perfusion the heart was frozen with Wollenberger tongs cooled in liquid N, and homogenised in 1 M HC10, in 25O/, (v/v) ethanol-H,0 a t - 10 “C. The volume of extract removed with the protein and the KC10, after neutralisation was estimated by drying the precipitates to constant weight. All perfusate samples were rapidly heated to boiling point to denature enzymes, particularly lactate dehydrogenase, which they often contained.

Since a large number of estimations had to be carried out an a small volume of extract, it was essential that the method used to determine the volume of contaminating perfusate should give reliable results on a small sample. Chloride assayed by the method of White [S] proved a suitable “extracellular” marker. This method involves correcting for intracellular chloride concentration which we found to be 26.5*/, of extracellular using rafkose as a marker. This agrees very closely with the results of Zachariah (using raffinose) [lo] and Gilbert (using inulin) [Ill. Gilbert also found insulin does not affect the distribution. The intracellular fluid per g fresh tissue was 0.506 =F 0.019 [27] ml/g and in a direct comparison of this method with the raffinose method we found the calculated contamination agreed to within 301, of an average volume of 0.65 ml trapped perfusate per heart.

CO, production was calculated from the fall in pH (approx. 0.13 unit/h) measured by an electrode inserted in the system. The respiratory quotient calculated (i.e. volume of CO, volume produced/

Eur. J. Bioohem. 364 The Flux of I’yruvate in Perfused Rat Heart

volume oxygen used) averaged0.91 for all experiments. Standard methods were used to assay glucose [12], glycogen [13], lactate [la], pyruvate [15] and pyri- dine nucleotides [16].

Growth hormone (lot No. NIH-GH-Bll) was a gift from the Endocrinology study section of N.I.H. It was repurified using steps 10-12 of the method of Wilhelmi [17] and freeze dried. 200 pg dissolved in a drop of 0.01 N NaOH was added to 50 ml perfusate. It was not added to preperfusion fluid. Crystalline insulin (B.D.H., batch No 2444) with specific activity of 23.44 units/mg was dissolved in N/300HC1 and added to both perfusate and preperfusate to give 100 mU/ml.

RESULTS AND DISCUSSION

Establishment of a Xteudy-Xtate Lactate Concentration Preliminary perfusion experiments in which small

amounts of lactate were added successively to perfusate containing 1 mg/ml glucose demonstrated that a steady-state lactate concentration could be found. The value was around 1 mM hearts from fasted rats and about 2 mM for those from fed rats. In order to establish the equilibrium concentration more precisely, lactate was added to the medium before the heart was inserted, a t a concentration slightly above that of the steady-state. Fig. 1 shows that an equilibrium was quickly reached and maintained for periods longer than 90 min. In subsequent experiments it was found that the steady-state concentration tended to vary slightly with the time of year but was always easily determined for a particular group of rats.

The steady-state concentration of lactate was found to depend on heart weight (see [S]) and indi- cates that perfusate concentration is proportional to the total amount of lactate in the organ.

Although somewhat surprising, it is possible to suggest situations in which this would be the expected result. The simplest hypothesis would be where the lactate is not free but bound in the cell so that one is observing an equilibrium between perfusate solution lactate and and bound heart lactate. Alternatively, the results of this study indicate that the transverse- tubular system, which penetrates the heart cell in that region where lactate dehydrogenase appears to be bound, may be involved in the exchange of perfusate and cell lactate and pyruvate. Since cardiac hypertrophy in adult rats normally involves an increase in myofibrillar content and not in cell number [18], the amount of myofibril exchanging with a given T-tubular space increases, so that a higher concentration of metabolite is required at equilibrium in the T-tubule.

In our experiments the intracellular lactate concentration was 0.63 0.064 pmol/g tissue for the control group, 0.64 f 0.11 pmol/g for the growth

.0.5 1 0 30 60 90 120

Perfusion time (min)

Fig. 1. Maintenance of a steady lactate Concentration during perfusion of hearts from fmted rats. (A) Control series (7 hearts); (B) growth hormone (4 pgglml) in perfusate (6 hearts) ; (C) insulin (4 pg/ml) in perfusate (6 hearts). Points are means

f S.E.M.

hormone group, and 1.67 & 0.25 pmol/g for the insulin group. If these values were divided by the estimated cell water/g heart they would be exactly equivalent to the true heart lactate as defined by the formula used by Opie and Mansford [19]. We have not reported any of our results in this form because evidence in the following papers demonstrates that pyruvate is held in more than one compartment in the myocardium, and perhaps lactate is also (cf. [ZO]). However, if such a calculation is made, the ratio intracellular [lactate]/extracellular[lactate] is found to be 2.00 for the control and growth hormone groups, and 1.67 for the insulin group. Application of the simple form of the Donnan relationship predicts that the ratio ought to be 0.25.

On the other hand, if undissociated lactic acid is the only permeating species, application of the distribution equation of Waddell and Butler [2l] would predict a ratio of about 2, assuming that the intracellular pH is about 7.6 (cf. [22]). However, this would mean that the rate of diffusion in and out of the muscle cell would be very small, since, using Rashevsky’s [23] diffusion equations and the values for the diffusion constant of lactic acid calculated by Eggleton et al. [24], we estimate that the rate of permeation per g heart of the undissociated lactic acid would be 0.2 pmol/min from 1 mM solution at pH 7.4. A more sophisticated approach using the Goldman equation [25] does not a t present appear possible because the relevant permeability coefficient for lactate has not been determined. Opie and Mans- ford [19] have reported that lactate production in

Vo1.36, No.2,1973 J. MOWBRAY and J. H. OTTAWAY 365

I-! L z k 0.05 (z

al

m u) c

I I I I

V I 1 I I 0 30 60 90 120

Perfusion t ime (min)

Fig. 2. Pyruvate concentration in the perfusate during perfusion of hearts from fasted rats with a steady lactate concentration. (A) Control (6 hearts); (B) growth hormone (6 hearts);

(C) insulin (6 hearts)

working hearts can be as much as 1.8pmol xmir-lxg-l. They also found that in their conditions (fed rats) the ratio of intracellular to extracellular lactate was much higher than we found; on the average 5-6. In our own experiments we found that a t low lactate concentrations (about 0.2mM) the rate of utilisation could be 1 pmol x min-1 x g-l and we have also found that equilibra- tion of W-labelled lactate is extremely rapid [26]. We conclude that the distribution ratio is not maintained either by the impermeability of lactate ion, or by the Donnan equilibrium.

In common with Opie and Mansford [19], we do not feel that this relatively high intracellular lactate concentration is evidence of anoxia. For one thing the 0, consumption of the hearts was independent of heart weight, and moreover the results reported in this and subsequent papers all relate to hearts in which a steady-state was maintained over the perfusion period. We occasionally had preparations in which the perfusate lactate concentration rose steadily from the beginning of the perfusion, in association with tell-tale purple pete- chiae on the ventricle or in which there was a sudden 24 Eur. J. Biochem., Vo1.36

Perfusion t ime (rnin)

Fig.3. Effect of addition of pyruvate at zero time.( A) Pyruvate concentration in perfusate during perfusion with steady lactate concentration. ( O d ) , No pyruvate initially present; (0-o), 0.1 mM pyruvate present at zero time. (B) Lactate concentrations in perfusate (o-), No pyruvate initially present; (0-o), 0.1 mM pyruvate

piesent a t zero time

rise in lactate after an hour or so of perfusion because of blockage in a major coronary blood vessel. The results from these experiments, which were discarded, could clearly be distinguished from those shown in Fig. 1.

Variation of Perfusate-Pyruvate Concentration with Time

Pyruvate, originally absent from the medium, first accumulated during the perfusion, reached a maximum a t about 30 min and subsequently declined. Fig.2 shows the results for three groups of six hearts. The intracellular pyruvate concentration did not change significantly during the perfusion.

Since we were primarily interested in obtaining a system in a completely steady-state, our efforts were chiefly directed to preventing the fall in pyruvate concentration after 30 min perfusion.

Prolonged perfusion was without effect and although addition of 2 mM oleate (on serum albumin) suppressed pyruvate accumulation almost completely it unfortunately also suppressed pyruvate oxidation [27]. Neither were we able to relate the pyruvate

366

h - 0.4- 3

3 - 01

01 . H E c

2 0.2 > z) L

a aJ a = c L 01

- a 0 -

The FIux of Pyruvate in Perfused Rat Heart

-

-

-

Em. 3. Biochem.

T

I I I I

0 30 60 90 120 Perfusion t i m e ( rn in )

Fig.4. Effect of mruvate additiim to the perfusate on the time course of pyruvate ou tp t when insulinwas added to the perfusate and [lactate] remained steady at 2.2mN. (A-A) No pyruvate initially present; (e--a) 0.12 mM added at zero time; (0-0) 0.2 mM added at zero time. Four hearts in

each group

behavior to the appearance or deliberate addition of traces of glycerol, alanine [28], acetoacetate or 3-hydroxybutyrate [29] or CO,.

Deliberate addition of pyruvate a t zero time had some effect. With control hearts the pattern of i d u x to, and efflux from, the perfusate appeared to be superimposed on the zero-time level (Fig. 3A), a point we shall return to. Very noticeably pyruvate addition had no effect on perfusate lactate concentration (Fig. 3B) emphasising again that the extracellular pyruvate/lactate couple do not reach a free equilibrium catalysed by intracellular myocardial lactate dehydrogenase. The addition of pyruvate with growth hormone gave results like Fig.3 except that the peak was lower and the two curves superimposed sooner. In the presence of insulin, pyruvate addition progressively depressed the peak and eventually abolished it altogether (Fig. 4). The linear rate ofpyruvate accumulation in the first 15-30min when pyruvate was not present a t zero time is not consistent with inhibition of pyruvate efflux by pyruvate. Thus it is possible that pyruvihte is protecting its own utilisation mechanisms (e.g. pyruvate dehydrogenase against ATP inhibition [30]) although intracelIular pyruvate concentration is not markedly affected by extracellular pyruvate.

Lest substantial changes in redox potential lay behind perfusate pyruvate behaviour, “AD]/ [NADH] ratio was measured directly (Fig.5). Al- though the ratio might with difficulty offer an explanation in the control group, not so in the hormone groups. The drop in ratio in the presence of insulin agrees with the data of Williamson and Kreisberg [31] and could help to explain the higher lactate concentration required for a steady-state. That explanation cannot however hold for growth hormone, and thus these overall ratios probably reflect largely mitochondria1 NADH concentra-

50[ A T

I \ o . . A

0 0 15 30 45 60 75 90

Fig.5. [NAD]/[NADH] ratio in hearts perfused with a steady lactate concentration. All animals were fasted for 24 h. (0-0) Mean values for a group of 8 control hearts; (A, 0) individual values for hearts to which insulin and growth hormones, respectively, had been added to the medium.

Metabolism was arrested by the freeze-stop technique

Perfus ion t i m e (m in )

tions. Finally it should be added that the intracellular lactate and pyruvate concentrations which were measured in this series of experiments were the same for each group whether perfusion was continued for 15, 60 or 90 min.

Balance Xheet of Carbohydrate Disposition during Perf usion

Glucose uptake, glycogen change and oxygen uptake data are presented in Table 1. Glucose uptake was linear over a 2-hour perfusion time, for all groups.

In agreement with Williamson [3], lactate appears t o have reduced the degree of insulin stimulation and the data are similar to those reported by Randle et al. [32]. Extended preperfusion did not affect glucose uptake by controls. The glycogen content remained constant in controls [3], and increased in the presence of insulin and growth hormone [33,34]. The O2 utilisation was very close to that reported by Fisher and Williamson [35]. 0, uptake in the presence of growth hormone was not measured but since this hormone has been shown to change the respiratory quotient of whole animals or tissue slices without affecting oxygen uptake [36], we feel justified in assuming it lies between 4.5 and 4.7 [35] pmol x min-l x g-l.

The rate of catabolism of carbohydrate is shown in Table 2. If the reasonable assumption is made that the only other oxidisable nutrient of any significance in heart, in these conditions, is fatty acid, the total flux through the tricarboxylic acid cycle can be calculated by means of the equation:

where ro2 is the fiux (min-lxg-l) of oxygen; THex ia the flux from glucose units to pyruvate; rpyr,

Vo1.36, N0.2,1973 J. MOWBBAY and J. H. OTTAWAY 367

Table 1. Glucose consumption, glycogen synthesis and oxygen consumption in perfwed rat hearts Initial glucose concentration 5.5 mM; lactate 0.7 mM (control and growth hormone), 2.2 mM insulin. Concentration of hormones,

when present 4 pg protein/ml. All values are quoted & S.E.M. with number of observations in parentheses

Value for Parameter Time period

Control Insulin Growth hormone

h pmol x g-' x min-I Glucose uptake 2 0.62 f 0.031 (6) 0.74 f 0.034 (8) 0.66 f. 0.013 (4)

pmol x g-l Net glycogen 2 -0.67 f 0.63 (5) $0.417 f 0.65 (5)

synthesis 1 +4.51 f 1.06 (4)

pmol x g-'

Oxygen uptake 1 4.5 f 0.14 (4)s 4.6 & 0.38 (4)b

* Mean flow rate 15.1 ml x min-'x g-'. b Mean flow rate 15.0 ml x min-l x g-l.

Table 2. Estimates of the rates of hexose degradation (we=), pymvate (rPvr) and lactate (r& removed from the system and the total pymvate oxidimd, in control, insulin-treated and growth-hormone-treated hearts

The 0, consumption necessary to oxidize completely this glucose, pyruvate and lactate (for equation see text), and the percent- age of the total 0, consumption of the heart which this represents is shown. The last column gives the calculated flux of acetyl units from fatty acids to the tricarboxylic acid cycle. All values are in pmol x g wet wt-I x min-I. Where indicated pyruvate was

added before zero time O1 required

oxidized ~~~~~~, O1 uptake m e r my. + 2 ).me pyruvate for complete Fz:$:f ).FA

Pyruvate Heart added treatment Time

min pmol x g-' x min-' pmolx g wet @I. wt-'x min-'

0-15 - Control 0.63 +0.15 1.41 4.36 97 0.04 - Insulin 0.68 -0.365 0.995 3.36 73 0.41 - Growth 0.63 -0.39 0.87 2.8 62 0.59

hormone 0-15 + Control 0.63 +0.03 1.29 4.0 89 0.16

Insulin 0.68 +0.03 1.39 4.15 90 0.15 + Growth 0.63 -0.12 1.14 3.5 76 0.36 +

hormone 30 - 60 - Control 0.63 +0.09 1.35 4.0 89 0.17

- Growth 0.63 +0.045 1.305 3.9 85 0.23 Insulin 0.68 +0.105 1.465 4.35 94 0.09

hormone

T L ~ ~ , T F A are the fluxes from pyruvate, lactate and fatty acid (acetyl units) to acetyl-CoA respectively ; and TTCA is overall rate of oxidation of acetyl units in the cycle.

These calculations (Table 2) demonstrate su- prisingly (cf. [37]) that a considerable amount of fatty acid is being oxidised in the presence of insulin and that pyruvate addition to perfusate supresses this fat oxidation despite intracellular lactate and pyruvate remaining constant. Two further points are notable; the heart metabolism is changing con- siderably in the first 30 min; and growth hormone was instantly effective.

CONCLUSION An experimental system with steady-state lactate

but non-steady-state pyruvate metabolism has 24'

been investigated whose varameters confirm and amplify several independent revorts in the literature. A reproducible non-steady state appears to exist in the 30-60min period which makes the prepara- tion suitable for detailed investigation of some reac- tions involving pyruvate in the intact organ. For further discussion see the following paper [26].

REFERENCES 1. Atkins, G. L. (1969) Multiwmpartment Models for Bio-

logical Systems, Methuen, London. 2. Wollenberger, A., Ristau, 0. & Schoffa, G. (1960)

Pfliigers Arch. gesamte Physiol. Menachen Tiere, 270,

3. Williamson, J. R. (1962) Biochem. J . 83, 377-383. 4. Scrutton, M. C. & Utter, M. F. (1968) Annu. Rev. Bio-

5. Sheppard, C. W. (1962) Basic Principles of the Tracer

399-412.

chem. 37,249-302.

Method, John Wiley & Sons, New York.

368 J. MOWBRAY and J. H. OTTAW.4Y: The Flux of Pyruvate in Perfused Rat Heart Eur. J. Biochem.

6. Korff, R. W. (1965) J . Biol. Chem. 240, 1351-1358. 7. O’Brien, J. A. (1969) Ph. D. Thesis, Edinburgh Uni-

8. Mowbray, J. (1969) Ph. D. Thesis, Edinburgh University. 9. White, D. S. (1961) Mikrochim. Acta, 3, 449-456.

10. Zachariah, P. (1961) J. Physiol. (Lond.), 158, 59--72. 11. Gilbert, J. C. (1963) Ph. D. Thesis, Edinburgh Uni-

12. Hill, J. B. & Cowart, D. S. (1966) Anal. Biochem. 16,

13. Good, C. A., Kramer, H. & Somogyi, M. (1933) J. Biol. Chem. 100, 485-491.

14. Lundholm, L., Mohme-Lundholm, E. & Vamos, N. (1963) Acta Physiol. S c a d . 58, 243-249.

15. Bucher, Th., Czok, R., Lamprecht, W. & Latzko, E. (1963) in Methods in Enzymic Analysis (Bergmeyer, H. U., ed.), pp. 253-259, Academic Press, New York.

16. Lowry, 0. H., Passonneau, J.V., Schulz, D. W. & Rock, M. K. (1961) J. Biol. Chem. 236, 2746-2755.

17. Wilhelmi, A. W. (1955) in The Hypophyseal Growth Hormone: Nature and Actions, (Smith, R. W., Gaebler, 0. H. & Long, C. N. H., eds) pp. 59-104, MoGraw- Hill, New York.

18. Young, V. R. (1970) in Mammalian Protein Metabolism (Munro, H. N. ed.) vol. 4, pp. 600-605, Academic Press, New York and London.

19. Opie, L. H. & Mansford, K. R. L. (1971) Eur. J. clin. Invest. I , 295-306.

20. Henderson, A. H., Craig, R. J., Gorlin, R. & Sonnen- blick, E. J. (1969) Am. J . Physiol. 217, 1752-1756.

versity.

versity.

327-337.

21.

22.

23.

24.

25. 26.

27.,

28. 29. 30. 31.

32.

33.

34. 35.

36.

37.

Waddell, W. J. 6 Butler, T. C. (1969) J. Clin. Invest. 38, 720-729.

Waddell, W. J. & Bates, R. G. (1969) Physiol. Rev. 49, 285-329.

Rashevsky, N. (195)O in Medical Physics, (Glasser, O., ed.) vol. 2, pp. 164-127, The Year Book Publishers, Inc., Chicago.

Eggleton, G. P., Eggleton, P. & Hill, A. V. (1928) Proc. Roy. 8oc. Lond. B Biol. Sci. 103, 620-628.

Goldman, D. E. (1943) J . Gem. Physiol. 27,37-60. Mowbray, J. & Ottaway, J. H. (1973) Eur. J. Biochem.

Garland, P. B., Newsholme, E. A. & Randle, P. J. (1962)

Ottaway, J. H. (1969) &. J. Exp. Physiol. 54,56-59. Krebs, H. A. (1970) Adv. Enzyme Regul. 8,335-353. Reed, L. J. (1969) Curr. Top. Cell Regul. 1, 233-251. Williamson, J. R. & Kreisberg, R. A. (1965) Biochim.

Randle, P. J., Newsholme, E. A. C Garland, P. B. (1964)

36, 369-379.

Nature (Lond.), 195, 381-383.

Biophys. Acta, 97, 347-349.

Biochem. J. 93. 652 - 665. Adrouny, G. H. & Russell, J. A. (1956) Endocrinology

Russell, J. A. (1957) Am. J. Clin. Nutr. 5.404-416. 59, 241-251.

Fisher, R . B. ‘& Williamson, J. R. (1961) J . Physiol.

Ketterer, B., Randle, P. J. & Young, F. G. (1957)

Williamson, J. R. (1964) Biochem. J. 93, 97-106.

(LMzd.), 158, 102-112.

Ergebn. Physiol. 49, 126-211.

J. Mowbray’s permanent address : Department of Biochemistry, University College, Gower Street, London, England, WClE 6BT

J. H. Ottaway, Department of Biochemistry, University Medical School, Teviot Place, Edinburgh, Scotland, EH8 9AG

Documents

The Flux of Pyruvate in Perfused Rat Heart