Clinical Science and Molecular Medicine (1976) So, 401-407,

Turnover of plasma glucose and free fatty acids in patients on the first day after myocardial infarction I. A. NIMMO, R. H. SMITH, M. A. DOLDER"'

A N D M. F. OLIVER Department of Biochemistry, University of Edinburgh and the Coronary Care Unit and Department of Cardiology, Royal Infirmary, Edinburgh, Scotland

(Received 8 December 1975)

summary 1. The turnover of plasma glucose and free fatty acids was measured in ten patients within 24 h of the onset of symptoms of acute myocardial infarction and in two with symptoms of acute myocardial ischaemia. The measurements were repeated in seven of the patients 12-40 weeks after the acute episode, 2. Both for the patients with acute myocardial infarction alone and for all the individuals studied the turnover of glucose increased with plasma glucose concentration but was not related to the turnover of free fatty acids or the plasma concentrations of free fatty acids, insulin or total catecholamines. There was no obvious difference in the nature of the glucose turnover-concentration relationship between the patients with acute myocardial infarction, with acute myocardial ischaemia and on reexamination. 3. For all the individuals studied the turnover of free fatty acids increased with the concentration of these but was not related to the turnover of glucose or the plasma concentrations of glucose, insulin or total catecholamines. There was no obvious difference in the nature of the free fatty acids turnoverconcentration relationship between the patients with acute myocardial infarction, with acute myocardial ischaemia and on re-examination.

Key words: free fatty acids, glucose, myocardial infarction, turnover rate. Present address: Medizinische Kliik, Universitgt Inselspital, 3010 Bern, Switzerland. Correspondence: Dr I. A. Nimmo, Department of Biochemistry, University Medical School, Teviot Place, Edinburgh EH8 9AG, Scotland.

Introduction Acute myocardial infarction is accompanied by marked changes in body metabolism and in the concentrations of circulating hormones. Increased catecholamine concentrations, a rise in the plasma concentrations of free fatty acids and glucose, a reduction in plasma insulin concentration and impairment of glucose tolerance have all been demonstrated (Videbaek, Christensen & Sterndorff, 1972; Christensen & Videbaek, 1974; Oliver, Kurien & Greenwood, 1968; Allison, Chamberlain & Hinton, 1969; Datey 8c Nanda, 1967; Lebovitz, Shultz, Matthews & Scheele, 1969; Pearson, 1971). The time-course of the changes has also been fairly well documented (Vetter, Strange, Adams & Oliver, 1974), and the relations between the changes and the complications of myocardial infarction are being investigated. One suggestion is that the heart accumulates toxic amounts of free fatty acids from plasma and tissue lipolysis (Kurien & Oliver, 1970). A logical approach to therapy would then be the inhibition of adipose tissue and myocardial lipolysis, and recently this has been shown to be associated with a reduction in the incidence of serious ventricular arrhythmias (Rowe, Neilson & Oliver, 1975). Alternatively or additively, the myocardium may require glucose as a source of anaerobic energy so that the promotion of glucose utilization could be beneficial in the prevention of complications (Opie, 1972). These therapeutic approaches would be more firmly based if the rate of uptake of glucose and free fatty acids by the ischaemic myocardium were known. There does not appear to be any published data for turnover rates of glucose and free fatty acids

401

I. A . Nimmo et al.

402

which would indicate the rates at which the metabolites were being mobilized and utilized in the whole body in this situation. This paper reports estimates of the whole body turnover rates of these two components in the first 24 h after the onset of the symptoms of acute myocardial infarction and compares them with the same measurements made in the same patients some months after the acute episode. The relation between the turnover rates and the plasma concentrations of glucose, free fatty acids, insulin and catecholamines was also examined. A preliminary account of this study has already appeared (Nimmo, Smith, Dolder & Oliver, 1974).

Methods Clinical methods

Twelve patients, eleven male and one female, were studied within 24 h of the onset of the symptoms of acute myocardial infarction. In ten of them the clinical diagnosis was confirmed electrocardiographically, by WHO criteria (World Health Organisation, 1959), and by a significant rise in the serum activities of the enzymes creatine kinase, lactate dehydrogenase and aspartate aminotransferase. In the remaining two patients (G.B. and A.H.) there was no such confirma-

tion and a diagnosis of acute myocardial ischaemia (intermediate coronary syndrome, acute coronary insufficiency) was made. Two of the patients (P.T. and J.D.) had been taking clofibrate before the onset of symptoms. In one patient (W.M.) only glucose turnover was measured. The clinical data are summarized in Table 1. Of the twelve patients, seven were studied again between 12 and 40 weeks after the acute episode. To reduce diurnal variation all studies were carried out between 09.00 and 11.OO hours. In the acute situation none of the patients had eaten for more than 12 h and in the repeat situation they were studied after overnight fasting. All studies were carried out in a quiet uninterrupted atmosphere and in each case the informed consent of the patient was obtained before the study began. Under local anaesthesia, a 2 inch plastic cannula (Medicut, 18 gauge) was placed percutaneously into one brachial artery and a second into the median cubital vein of the opposite arm. Patency of the cannulae was maintained by periodic flushing with sodium chloride solution (1 54 mmol/l) without heparin. At least 10 min after the insertion of the cannulae the first arterial blood sample was taken and the preparation of radioactive glucose and palmitate (see 'Biochemical methods' below) was injected

TABLE1. Clinical data for the patients

Patient

Time between Time between symptoms and symptoms and Site of Weight initial study repeat study myocardial infarct (weeks) (kg1 (h)

Age (years)

Height (cm) I66 175 175

70 85

J.S.

59 56 62

A.M.

49

174

G.B.") J.H. A.H.'') W.H.

38 60 43 46

175

79 78

M.M.") J.M. W.M. G.H.

P.r. J.D.

-

6 24

Max. creatine kinase'') (unitsll)

-

22

Anterior Inferior Anterior Anterior

40

?

12

Anterior

36

Anterior

540 36 1060

22

-

420 990 660

59

21 12 8 14

1 70 179

73 108

9 11

68 52 50

163 161 175

64 61 74

13

-

21 11

17 13

Anterior Anterior Inferior

640 108

59

175

70

11

-

Anterior

800

-

19

~

-

1420 64

?

~~

154

~

Diagnosed as acute myocardial ischaemia. Female. (3) Normal range of creatine kinase: males, 30-200 units/l; females, 30-150 units/l.

(I)

Drugs other than cyclomorphine Clofibrate Clofibrate Amitriptylinr (Alcohol)

-

Lignocaine

-

Phenytoin, phenobarbitone Digoxin Propranolol Lignocaine, practolol Lignocaine, propranolol ~~

-

Glucose and fatty acids utter myocardial infaction

into the vein. Further arterial samples were taken at 2, 4, 6, 10, 15, 20, 30, 45, 60, 90 and 120 min after the injection. The circulating plasma volume was then measured with SI-labelled albumin. The total volume of blood withdrawn was about 200 ml. Biochemical methods

The injection comprised 10 pCi of sodium [1-I4C]palmitate bound to human albumin and 50 pCi of [2-3H]glucose (both radiochemicals from New England Nuclear Corp.). The palmitate was shown by gas-liquid chromatography to be more than 99% pure. An aliquot of injectate was retained for estimation of the content of glucose and palmitate per unit weight; the quantity injected was determined by weighing the syringe used for the injection before and after the injection. The blood sample for total catecholamine estimation was collected into 10 ml heparinid tubes containing 5 mg of sodium metabisulphite. All other samples were collected into 10 ml heparinid tubes, containing 6.4 mg of sodium fluoride, immediately placed in ice, and then separated by centrifugation at 4°C within 90 min of collection (there was no change in the concentrations of either glucose or free fatty acids during this period). Determinations of glucose and free fatty acids were begun within 4 h. Plasma for insulin assay was stored at - 20°C for up to 6 months. Determination of plasma free fatty acids. Concentration was estimated in duplicate by the procedure of Dole (1956) as modified by Trout, Estes & Friedberg (1960). The coefficient of variation of the method was about 5%. Radioactivity was estimated in duplicate by extracting the plasma lipids with chloroform-methanol, isolating the free fatty acids by thin-layer chromatography on Silica Gel G and finally counting their activity to 1 % SD by liquidscintillation spectrometry, all essentially as described by Boberg (1966). The presence of [2-3H]glucosein the plasma sample did not interfere. Determination of plasma glucose. Plasma (duplicate 1 ml aliquots) was deproteinid with 2 ml of ZnS04 (0.15 molll) and 2 ml of Ba(OH)z (0-15mol/l), the supernatant was evaporated to dryness at 75°C in DUCUO and then reconstituted with 1.5 ml of water. All the radioactivity in the reconstituted supernatant was assumed to be in glucose (Issekutz, Allen & Borkow, 1972; Issekutz & Borkow, 1973). A 1 ml

403

aliquot was mixed vigorously with Aquasol (New England Nuclear Corp.) and radioactivity was counted to 1% SD by liquid-scintillation spectrometry. Quenching was assessed by adding 100 pl (20 000 c.p.m.) of [2-3HJglucoseas internal standard. The concentration of glucose in 100 pl of reconstituted supernatant was determined by an automated glucose oxidase method as previously described (Nimmo, Kirby & Lassers, 1973), with standards which had also been ‘deproteinized’. No correction was made for the volume occupied by plasma proteins. The coefficient of variation of the method was about 2%. Computation of turnover rates. Turnover was measured by the single-injection technique (Atkins, 1969). An injection of labelled free fatty acids or glucose was given, and the turnover was computed from the time-course of disappearance of label from the plasma. For free fatty acids, a two-exponential function a = Xl exp(- llt ) + Xzexp(- l at ) was fitted to the radioactivity (&time ( t ) data for the fvst 45 or 60 min by the method of Atkins (1971), the points being weighted according to the reciprocals of their variances. The plasma pool size (Ql) was equated to the product of the plasma volume ( Vl)and the average arterial concentration for this period of time (Cl), The turnover rate (RI) was calculated from the relation (Atkins, 1969): R=

nlnz(xl + xzvlcl n 1 x z +lax1

For glucose, a two-exponential function was fitted as before to the radioactivity-time data for the full 120 min. The initial volume of distribution of the injected glucose (total radioactivity Z) was calculated from the relation Vl = Z/(Xl + X,)and the turnover rate from R1 = llLZC1/(AIXz +&XI). Hormone estimation. Total catecholamines were estimated in the first sample by a fluorimetric method (Carruthers, Taggart, Conway, Bates & Somerville, 1970) and the insulin in it by a single-antibodyradioimmune assay (Lind, de Groot, Brown & Cheyne, 1972). Statistical methods. The correlation and partial correlation coefficients given below were calculated by parametric methods (Snedecor & Cochran, 1967). Kendall’s rank correlation coefficient was also calculated (Siegel, 1956), but the values have not been given below because they were comparable with the parametric ones. Partial rank correlation coefficients

I . A . Nimmo et al.

404

were not calculated because, as their sampling distribution does not seem to be known (Siege], 1956; Kendall, 1970), no level of significancecan be attributed to them. Results During each study the plasma concentrations of glucose and free fatty acids fluctuated but by no more than f 11% (glucose) and f 18% (free fatty acids). These fluctuations cannot have been caused by methodological error alone and therefore show that the patients were not in a steady state. The turnover rates were calculated from the mean values of the concentrations observed at 0, 15, 30, 45, 60, 90 and 120 min and consequently represent an average value for this period. The two-exponential function always fitted the glucose data, but in seven instances it appeared to give a biassed fit to the results for free fatty acids (asjudged by the distribution of residuals). In these instances a three-exponential function fitted the data well. However, as the turnover rates estimated with the two sorts of function never differed by more than 20%, and as no attempt was made to analyse the data in terms of the number of mathematical or physiological compartments contributing to the plasma free fatty acid metabolism, all the turnover rates given below were computed from twoexponential fits.

*.

%

I"

. 8

0

8

0 Patient A.M.

I

I

400

800

/. I 1200

plasma free fatty acids, for patients with acute myocardial infarction ( 0 ,acute episode; 0 , control) and with acute myocardial ischaemia (W, acute episode; 0 , control).

0

4

0 .

Plasma free fatty ocid concn. (pnol/l)

. 0

.

0

FIG.2. Relation between turnover and concentration of

0 0

0

The relationship between glucose turnover and concentration is shown in Fig. 1. There is a positive correlation between the two variables both for all the data (r = 0.751, P 0.10). As for glucose, the data for the patients on re-examination seem to fall on the same line as those for the patients in the acute phase, and

405

Glucose and fatty acids after myocardial infarction TABLE 2. Metabolic and hormonal data for the patients

In each column the value found in the acute situation is given first and that in the follow-up study second. ~~

Plasma free fatty acids

Plasma glucose Patients

P.T. J.D.

J.S. A.M. G.B."'

J.H. A.H.'" W.H. M.M.'~) J.M. W.M. G.H.

Turnover (pmol min-' kg- 9 15.9; 12-6 6.1; 11.5; 8-6; 1 0 4 11.8; 13.7 16.7; 13.2 12.6; 14.2; 11.8 10.5; 1 6 8 ; 12.2 13.3; 14.7 20.1; -

-

Turnover (pmol min-'

Concentration (mmol/l)

kg-7 11.5; 5.7 11.8; 5.1; 6.0; 6.1 5.9; 8.2 1 0 7 ; 12.3 7.2; 7.3; 7.3 94; 9 1 ; 10.7 -; 9.7 9.6;

6 6 7 ; 444 454; 6.07; 3-37; 5.31 4.03; 5.42 6.34; 5.41 3-82; 8.08; 5.66 3.56; 6.12; 4 9 3 5.59; 5.48 10.94; -

-

Concentration @mol/O

insulin

Plasma total catecholamines

(munits/l)

(Pcsll)

980; 580 910; 650; 1130; 750 440; 620 970; 720 750; 710; 690 870; 840; 900 -; 830

6 8 ; 8.6 9.7; 13.7; 8.4; 6 5 3.4; 4.3 17.5; 4.9 14.0; 13.2; 11.2 6.2; 15.8; 6-4 18.2; 5.6

1-6; 1.0

Plasma

-

-

810;-

-

8.9; -

1.0;1.6; -

1.4; 1-2 1.4; 0.7 1.4; 0.7 08;1.8; 0.3 1.3; 0.5; 0 9 0.8; 1.0 0.9;

-

~~

(I) (2)

Diagnosed as acute myocardial ischaemia. Female.

have the narrower spread of concentration. One patient with acute myocardial infarction (A.M.) did not conform to the general pattern, having a high concentration but a low turnover rate. He had drunk a considerable quantity of alcohol a few hours before his attack, and his plasma samples were opalescent. He has nevertheless been included in the free fatty acids correlations, and has weakened their statistical significance. Had he been excluded, the correlation coefficient for the patients in the acute phase of myocardial infarction alone would have been significant (r = 0.942, P0.10) with the turnover of glucose, or with the concentrations of glucose, total catecholamines or insulin. (Note that in these analyses the number of degrees of freedom was small.) For neither the patients in the acute phase nor those being restudied was there a significant correlation between the concentrations of glucose and free. fatty acids (P>0.10). The metabolic data are summarized in Table 2. F

Discussion Patients with acute myocardial infarction are particularly at risk for the development of arrhythmias in the first few hours after the onset of symptoms, during which period there are changes in their metabolism partly reflected by alterationsin the concentrations of plasma substrates (Vetter et al., 1974). Practical difficulties prevented our examiningpatients until at least 6 h after the onset of symptoms, by which time they had survived the most dangerous period. Moreover, none of our patients had the really high concentration of free fatty acids (over 1200 pmol/l) that seems to be associated with an increased incidence of arrhythmias (Oliver et al., 1968). Consequently our data refer only to a sub-group of patients with relatively mild acute myocardial infarction. Our main conclusions are that the whole-body turnovers of both glucose and free fatty acids increase with plasma concentration, and that there are no marked differences in the nature of the relationships between patients with acute myocardial infarction and those with acute myocardial ischaemia, or between the patients in the acute phase and when they were re-studied. The data for free fatty acids are consistent with the idea that the elevated plasma concentration of free fatty acids seen after acute myocardial infarction implies there is enhanced adipose

406

I. A. Nimmo et al.

tissue lipolysis and uptake of free fatty acids by the body as a whole. As mentioned above, none of our patients had a concentration of more than 1200 mmol/l of free fatty acids. Had the concentration exceeded this value one would have expected the main binding sites of albumin to have become saturated and the concentration of weakly-bound free fatty acids to have increased disproportionately : thus the graph of turnover against concentration would have tended to curve upwards (Spector, 1968). Whereas on reexamination the patients all had similar concentrations of plasma glucose their turnover rates were much more widely scattered. This may merely reflect the difficulty of measuring turnover precisely, but it may also indicate that in the normal fasting state it is plasma concentration, as opposed to hepatic output (or tissue uptake), that is closely regulated. In contrast, the patients in the acute phase had a wider range of both concentration and turnover. The positive correlation between glucose turnover and plasma concentration suggests that the mild hyperglycaemia often seen after acute myocardial infarction is due to increased hepatic output, rather than diminished whole-body uptake. However, it does not necessarily follow that just because the wholebody turnover of glucose has increased, the uptake of this metabolite by every cellular structure has also risen; it is feasible, for example, that the glucose is being recycled in the liver. The decreased glucose tolerance of patients after acute myocardial infarction suggests that myocardial uptake is indeed diminished, at least in response to the glucose load, perhaps because the concentration of insulin is initially low compared with that of glucose (Vetter et al., 1974), and the change in it induced by the load is also less than normal (Allison et al., 1969). A similar situation may exist in patients with severe injury, who also show hyperglycaemia and decreased glucose tolerance but increased glucose turnover (Long,Spencer, Kinney & Geiger, 1971). The lack of a negative correlation for both the patients in the acute phase and when they were restudied between either the concentrationsor the turnovers of glucose and free fatty acids might seem at first sight to be at variance with the concept of Randle’s glucoselfatty acid cycle (reviewed by Newsholme & Start, 1973). One would have thought that, had the patients been metabolically normal when they were re-studied, the reciprocal relationships would have been observed; consequently they

may in fact not have returned to a metabolically normal steady state. The reciprocal relationship between the two concentrations would not necessarily be expected for the patients in the acute phase, as their hormonal balance was presumably acutely and variably disturbed, with marked increases in plasma catecholamines, cortisol (Vetter et al., 1974) and glucagon (Laniado, Segal & Brig, 1973). Further, the rate of oxidation of either metabolite may not be proportional to its turnover rate, so there could still be an inverse correlation between the two rates of oxidation. The turnover rates reported here refer to the body as a whole and not to the myocardiumper se. Therefore one cannot draw any definite conclusions about the uptake of glucose and free fatty acids by the heart. Even sampling coronary sinus blood is not particularly useful in this context, because its composition reflects an average of the metabolism of areas of the myocardium that are grossly underperfused (the infarct), overperfused (marginal hyperaemia), and more or less normal. This inability to be specific about the sites of uptake of metabolites in the blood is a fundamental weakness of all turnover studies. On the other hand, if the metabolites are added to the blood by a single organ or tissue, as we have assumed to be true for both glucose and free fatty acids, then one can legitimately draw conclusions about this structure. Acknowledgments We are grateful to the medical and nursing staff of the Coronary Care Unit and the technical staff of the Lipid Research Laboratory for their interest and help in this study; and to Dr T. Lind (Newcastle) for insulin, Dr R.A. Riemersma for free fatty acids and Dr R. C. Strange for catecholamineestimations. This research has been supported by the Medical Research Council. References ALLISON, S.P., CHAMBERLAIN, M.J.&. HINTON,P. (1969) Intravenous glucose tolerance, insulin, glucose, and free fatty acid levels after myocardial infarction. British Medical Journal, iv, 776-778. ATKINS,G.L. (1969)Multicompartment Models for Biological Systems, pp. 44-48. Methuen and Co. Ltd, London. G.L.(1971) A versatile digital computer program for ATKINS, non-linear regression analysis. Biorhimica et Biophysica Acta, 252, 405-420. BOBERO,J. (1966) Separation of labeled plasma and tissue lipids by thin-layer chromatography. A quantitative methodological study. Clinica Chimica Ada, 14, 325-334.

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