Enzyme pattern and lipid peroxides in endomyocardial biopsies from patients with cardiomyopathy and myocarditis

Clinica Chimica Acta, 173 (1988) 193-200 Elsevier

193

CCA 04123

Enzyme pattern and lipid peroxides in endomyocardial biopsies from patients with cardiomyopathy and myocarditis Irmbild

Lehmarm a, B&be1 Papies b, Rassouli Akhawan Parsi ‘, d, Ingolf Schimke b, Elke Parsi ’ and Marie-Luise K&rig b

Paul Romaniuk

a Haus der Gesundheit, Berlin, b Institut ftirPathologische und Klinische Biochemie, ’ Klinik ftir Innere Medizin and a Institut ftir Kardiooasculiire Diagnostik des Bereiches Medizin der Humboldt-Universitiit zu Berlin, Berlin (DDR) (Received

17 March 1986; revision received

Key words: Enzyme pattern;

Lipid peroxide;

1 September

1987; accepted

Endomyocardial

biopsy;

after revision

(Charite)

2 January

Cardiomyopathy;

1988)

Myocarditis

Methods have been developed for measuring several biochemical parameters (isoenzymes of LDH and ASAT, glycogen phosphorylase, lipid peroxides) in extremely small tissue samples (0.2-1.8 mg) taken using a left ventricular biopsy technique. Endomyocardial biopsies from patients with dilative and hypertrophic cardiomyopathy (CMP) and with myocarditis were investigated and compared with a reference group without actual functional and morphological evidence of chronic heart disease. Patients with myocarditis showed the highest activities of LDH and its isoenzymes, ASAT, ASATm and glycogen phosphorylase and the highest concentration of lipid peroxides. In patients with hypertrophic CMP increased activities of glycogen phosphorylase and decreased activities of ASAT and ASATm have been found. In patients with dilative CMP slightly elevated ASAT and ASATm activities have been observed. The results obtained in this study suggest that the parameters investigated could be useful in differentiating between cardiomyopathies and myocarditis.

Correspondence and requests Allee 3, Berlin 1020, DDR.

0009-8981/88/$03.50

for reprints

to: Dr. Irmhild

0 1988 Elsevier Science Publishers

Lehmann,

Haus

B.V. (Biomedical

der Gesundheit,

Division)

Karl-Marx-

194

Introduction The diagnosis of cardiomyopathies has, until now, been almost exclusively based on clinical and morphological data. There is very little information in regard to alterations in heart metabolism especially in human cardiomyopathy. Cardiomyopathy as well as myocarditis are characterized by a more or less pronounced hypertrophy of the heart, which causes limitation of the oxygen and substrate supply to the myocardium and leads to adaptive changes in myocardial metabolism recognized by, among others, stable alterations in the enzyme and isoenzyme pattern of the cells [l-3]. The isoenzyme pattern of lactate dehydrogenase (LDH; EC 1.1.1.28) as well as glycogen phosphorylase (EC 2.4.1.1) are considered to be sensitive indicators of the oxygen supply to the heart [3,4]. The isoenzymes of Aspartate Aminotransferase (ASAT; EC 2.6.1.1; mitochondrial isoenzyme ASATm, cytosolic isoenzyme ASATc) are related to the protein turnover in the myocardium. Oxygen deficiency as well as alterations in the metabolism of catecholamines described in cardiomyopathy [S] could result in the stimulated production of oxygen radicals and hence in an increased peroxidation of unsaturated membrane lipids. An increased concentration of lipid peroxides in inflammatory heart diseases like myocarditis might also be expected because of the known importance of oxygen radicals in the inflammatory process [6]. It was the aim of the present study to investigate the pattern of LDH and ASAT isoenzymes and glycogen phosphorylase as well as of lipid peroxides in endomyocardial biopsy specimen from patients with dilative and hypertrophic cardiomyopathies and myocarditis in order to evaluate the potential usefulness of these parameters for the differential diagnosis of cardiomyopathies and myocarditis. Materials and methods Tissue samples (0.2-1.8 mg wet wt.) were taken, using a left-ventricular endomyocardial biopsy technique, from 49 patients with the tentative diagnosis cardiomyopathy, respectively, myocarditis. None of the patients showed atherosclerotic alterations of the coronary vessels. Patients were divided into 4

TABLE

I

Number

and age of patients

Dilative CMP Hypertrophic CMP Myocarditis Reference group

n

Age in yr (mean * SD)

11 12 14 12

39.5* 6.8 40.85 8.7 41 .o f 10.5 37.1* 10.4

49

Male

Female

8 8 7 5

3 4 I I

28

21

195 TABLE

II

Essential

criteria

for the classification

of patients

Dilative CMP Clinical

symptoms

Dilation Diastolic left ventr. volume Systolic left ventr. volume Left ventricular heart mass Left ventricular systol. function Ejection fraction Left ventricular enddiast. pressure Hypertrophy of myocardial cells Fibrosis Inflammatory cells

Hypertrophic CMP

+ ++ t t t

Myocarditis

+ normal

Reference group

(+I (f) (11 (?I

+ normal normal normal

normal normal

(L) (11

normal normal

t

t

CT)

normal

+ + _

++ +

+ + +

_ _ _

groups (Table I) according to clinical, paraclinical and morphological criteria (Table II). Tissue samples were homogenized using a special glass-Teflon-Homogenizer in 0.005 mol/l Na-/3-glycerophosphate/O.OOl mol/l EDTA, pH 7.5. For samples 6 1 mg, 250 ~1 of the homogenizing medium were used. Samples > 1 mg were homogenized l/250. No influence of the different ratios was found. For the analysis of LDH and ASAT isoenzymes, which was performed using kinetic methods [7,8], a 40 ~1 portion of the homogenate was diluted l/16 with 0.03 mol/l sodium phosphate buffer, pH 7.5. Forty microliters of the homogenate were used for the determination of glycogen phosphorylase [9], another 40 ~1 for protein determination which was performed according to Lowry et al [lo]. One hundred microliters of the homogenate was used for the analysis of lipid peroxide concentration [ll]. All results were expressed as mean f SEM. Statistical data analysis was performed using the U test by Mann-Whitney. Results In endomyocardial tissue from patients with myocarditis significantly higher activities of LDH compared with other groups were measured. The higher LDH-activity resulted from increased activities of both LDH-H and LDH-M. Because of the larger increase in LDH-M the ratio LDH-H/LDH-M was decreased (Fig. 1). LDH-activity as well as LDH isoenzyme pattern in the myocardium of patients with both forms of CMP did not differ significantly from each other nor from the reference group. Both the total activity of ASAT and the activity of its mitochondrial isoenzyme were markedly higher in tissue of patients with myocarditis compared to

196

6-

5c ‘E ‘, ii 9

4-

3-

z G %

2-

l-

LDH-H LDH

LDH-H

LDH-M

LDH-M

Fig. 1. Activity of LDH isoenzymes (x, p 5 0.05; xx, p 5 0.01). 0, reference hypertrophic CMP and E, myocarditis.

group;

&I, dilative

CMP; q,

all other groups (Fig. 2). Myocardium of patients with dilative CMP revealed a slightly elevated activity of ASAT and ASATm in comparison to the reference group, whereas ASAT activities in myocardium of patients with hypertrophic CMP were considerably lower than those observed both in the dilative form of CMP and

ASAT-M ASAT

ASAT-C ASAT-C

Fig. 2. Activity of ASAT isoenzymes hypertrophic CMP and q , myocarditis.

ASAT-M

(xxx,

p s 0.001).

0,

reference

group;

~4, dilative

CMP;

q,

197

A Q-

o-

-xGlycogen

phosphorylase

Fig. 3. Activity of glycogen and E, myocarditis.

Lipid

phosphorylase.

0, reference

group;

H, dilative

peroxides

Fig. 4. Concentration

of lipid peroxides.

E!, CMP and E , myocarditis.

CMP;

198

in the reference group. The glycogen phosphorylase showed the highest activity in endomyocardial tissue of patients with myocarditis. Increased activity has also been found in tissue of the patients with hypertrophic CMP (Fig. 3). There was no difference in the lipid peroxide concentrations between dilative and hypertrophic CMP. Data obtained ih CMP have therefore been pooled and compared to myocarditis (Fig. 4). It can be seen that both groups were different in regard to their lipid peroxide concentration with higher values in the myo~dium of myocarditis patients. DiscusSion

In the past ten years, endomyocardial biopsies have been increasingly used, above all for morphological investigations. The value of such studies for the differential diagnosis of chronic cardiac disease is now positively judged by the majority of investigators [12-S]. The number of biochemical studies on human endomyocardial biopsy specimens, above all enzyme activities, is relatively small compared to morphological data [16-181. On the one hand this might be due to methodological problems, on the other hand, due to the fact that there is not complete agreement concerning the problem of the representability of such a small tissue sample for alterations in the whole myocardium. In systematic investigations on human heart tissue taken intraoperatively Van der Laarse et al [19] found no differences in the activity of various enzymes between the right and left ventricle nor between different regions of the left ventricle. Our own experience on human endomyocardial biopsies taken from various regions of the left ventricle confirm this results [20]. Evaluation of biochemical findings requires data from the myocardial tissue of a reference group. Because it is impossible to obtain myocardial tissue from healthy persons our reference groups includes patients with clinical signs of chronic cardiac disease which could not be confirmed either from paraclinical or from morphological investigation (see Table II). Patients with myocarditis showed a markedly elevated total activity of LDH. Due to the stronger increase of LDH-M the ratio LDH-H/LDH-M was distinctly lower in myocarditis patients in comparison with both forms of CMP and the reference group. The alteration of the LDH isoenzyme pattern in myocarditis could be an expression of an elevated share of fibrotic tissue and inflammatory cells in the myocardium. In granulocytes and lymphocytes predominantly LDH-M has been demonstrated [21]. On the other hand the lower ratio LDH-H/LDH-M might also be regarded as an adaptation to a regional oxygen deficiency resulting from a decreased capilla~ation of the subend~ar~um caused by the often pronounced hypertrophy in these patients. It is known that the number of capillaries in the subendocardial region is diminished in hypertrophic hearts [22]. An increased proportion of M-subunits in the total activity of LDH has been observed in human heart tissue with morphological evidence for a limited perfusion and increased muscle mass [3]. Limitation of oxygen supply to the subendocardium due to hypertrophy could at the same time be a reasonable explanation for the elevated activity of glycogen phospho~lase. Increased activities of this enzyme paralleled by

199

a high glycogen content have been found in various animal species near the endocardium and interpreted as adaptation to the lower vascularization and hence greater dependence on anaerobic energy production in these regions [4]. The most marked changes in the isoenzyme pattern of ASAT have again been observed in myocardial tissue of patients with myocarditis. However there were also discrete differences between dilative and hypertrophic CMP. The relative preponderance of the cytosolic ASAT in hypertrophic CMP could favour the preferential utilization of amino acids for protein synthesis and hence the development of hypertrophy. Patients with myocarditis showed higher levels of lipid peroxides compared to cardiomyopathy. It is suggested that this reflects an increased production of oxygen radicals by inflammatory cells, or might be related to the subendocardial lack of oxygen. In the present study more or less pronounced differences in the isoenzyme pattern of LDH and ASAT, in the activity of glycogen phosphorylase as well as in the concentration of lipid peroxides could be demonstrated between myocarditis and cardiomyopathies, on the one hand, as well as between both forms of cardiomyopathy on the other hand. Alterations in the myocardial tissue of myocarditis patients were particularly striking. In order to evaluate these results one has to take into consideration that within each group patients were heterogenous in regard to age, stage of the disease, degree of hypertrophy and impairment of left ventricular function. The planned correlation of the biochemical data with other clinical and functional parameters, among others with the degree of hypertrophy and the severity of impairment of left ventricular function should give more detailed information especially in regard to the differentiation of both forms of cardiomyopathy. References 1 Meerson FZ, Alekhina GM, Alexandrov PN, Bazardjan AG. Dynamics of nucleic acid and protein synthesis of the myocardium in compensatory hyperfunction and hypertrophy of the heart. Am J Cardiol 1968;22:337-347. 2 Sobel BE, Henry PD, Ehrlich BJ, Bloor CM. Altered myocardial lactic dehydrogenase isoenzymes in experimental cardiac hypertrophy. Lab Invest 1970;22:23-27. 3 Ballo IM, Messer IV. Lactate dehydrogenase isoenzymes in human hearts having decreased oxygen supply. Biochem Biophys Res Commun 1968;33:487-491. 4 Jedeikin LA. Regional distribution of glycogen and phosphorylase in the ventricles of the heart. Circulation Res 1964;14:202-211. 5 Jacobsson B. Receptor changes in heart failue. British Heart Foundation, Workshop on Heart Rejection, October 1983. 6 Babior BM, Kipnes RS, Cumutte JT. The production by leucocytes of Superoxide, a potential bactericidal agent. J Clin Invest 1973;52:741-744. 7 Graubaum HJ, Friedemann H, Nack B, Wagenknecht C, Zinsmeyer J, Egger E. Ein kinetischer LDH-Isoenzymtest-Methodik und altersabhangige Normalwerte. Zbl Pharm 1972;111:917-931. 8 Graubaum HJ, Ehrenberg K, Wagenknecht C. Eine einfache kinetische Methode zum Nachweis von zytoplasmatischer und mitochondrialer GOT ohne vorherige Auftrennung. Acta Bio Med Germ 1974;32:293-297. 9 Cori GT, Illingworth B. The effect of epinephrins and other glycogenolytic agents on the phosphorylase A content of muscle. Biochim Biophys Acta 1956;21:105-110.

200 10 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951;193:265-275. 11 Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analyt Biochem 1979;95:351-358. 12 Kaltenbach M, Loogen F, Olsen EGJ. Cardiomyopathy and myocardial biopsy. Berlin-HeidelbergNew York: Springer-Verlag, 1978. 13 Kawai C, Matsumori A, Kawamura K. Myocardial biopsy. Ann Rev Med 1980;31:139-157. 14 Yutani C, Okano K, Kamiya T, et al. Histopathological study of right endomyocardial biopsy. Br Heart J 1980;43:589-592. 15 Sekiguchi M, Hiroe M, Take M, Hirosawa K. Clinical and histopathological profile of sarcoidosis of the heart and acute idiopathic myocarditis. Concepts through a study employing endomyocardial biopsy II. Myocarditis Jpn Circ J 1980;44:264-273. 16 Peters TJ, Wells G, Oakley CM, et al, Enzymatic analysis of endomyocardial biopsy specimens from patients with cardiomyopathies. Br Heart J 1977;39:1333-1339. 17 Richardson PJ, Atkinson L. Enzyme activities in endomyocardial biopsy samples from patients with cardiomyopathy. In: Bolte HD, ed. Myocardial biopsy-diagnostic significance. Berlin-Heidelberg-New York: Springer Verlag, 1980;97-101. 18 Schultheiss HP, Bolte HD, Cyran J. Lactate dehydrogenase isoenzyme pattern in endomyocardial biopsies of patients with congestive cardiomyopathy and with alcoholic cardiomyopathy-clinical and experimental results. In: Bolte HD, ed. Myocardial biopsy - diagnostic significance. Berlin-Heidelberg-New York: Springer Verlag, 1980;102-115. 19 Van der Laarse A, Dijkshoom NJ, Hollaar L, Caspers T. The (iso) enzyme activities of lactate dehydrogenase, a-hydroxybutyrate dehydrogenase, creatine kinase and aspartate aminotransferase in human myocardial biopsies and autopsies. Clin Chim Acta 1980; 104:381-391. 20 Wagenknecht C, Papies B, Lehmann, et al. Das Verhalten von Enzymaktivitlten im Herzgewebe und Blutplasma von Patienten mit chronisch ischamischer Herzkrankheit (IHK). 2 Med Labor-Diagn 1982;23:312-320. 21 Roman W. Quantitative estimation of lactate dehydrogenase isoenzymes in serum I. Review of methods and distribution in human tissues. Enzymologia 1969;36:189-219. 22 Rakusan K, Moravec J, Hatt PY. Regional capillary supply in the normal and hypertrophied rat heart. Microvascular Res 1980;20:319-326.