Myocardial enzyme activities in plasma after whole-heart irradiation in rats.

Plasma levels of myocardial enzymes present after local heart irradiation were studied in a rat model. The purpose was to investigate whether, within days after irradiation, these enzyme levels change to such an extent that they may be helpful in assessing the severity of cardiac damage after radiotherapy. Therefore, activities of creatine kinase (CK), lactate dehydrogenase (LDH), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alpha-hydroxybutyrate dehydrogenase (alpha-HBDH) were determined in the plasma and left ventricular myocardium of rats following local heart irradiation with a single dose of 20 Gy. A dose of 20 Gy is known to cause irreversible cardiac damage and to reduce survival times of the animals. Cardiac enzyme assays were performed directly after and twice daily for up to 2 weeks after radiation. Plasma CK, LDH, AST and alpha-HBDH levels were increased between 2 h and 24 h after irradiation. Plasma ALT levels remained unchanged. Myocardial enzyme levels, measured between 24 h and 16 days after radiation, did not differ between irradiated and control animals, although acute (first 12 h) reductions were observed in the irradiated group. The elevated enzyme levels in plasma appeared to correlate with the acutely reduced myocardial enzyme levels. Although irradiation with a dose of 20 Gy induced acute rises of cardiac enzyme levels in plasma, it is doubtful that fractionated radiation, as applied clinically for treatment of solid tumors, will induce plasma enzyme elevations that are large enough to indicate the extent of cardiac damage occurring acutely or chronically.

Cholesterol or triglyceride loading of human monocyte-derived macrophages by incubation with modified lipoproteins does not induce tissue factor expression.

Macrophages/foam cells localized in cholesterol- and triglyceride-rich regions of atherosclerotic plaques express high levels of tissue factor (TF), the essential cofactor and receptor of factor VIIa. It is not clear whether modified lipoproteins, for which several agonistic effects on macrophages have been described, are independent stimuli of TF expression in these cells. Therefore, we studied the effect of short-term (1 day) and long-term (4 to 7 days) incubation of human monocyte-derived macrophages cultured in suspension with modified and native LDLs or VLDLs on the expression of TF mRNA, antigen, and activity. We used native LDL or VLDL, moderately oxidized LDL or VLDL, severely oxidized LDL or VLDL, acetylated LDL, and beta-VLDL at a protein concentration of 100 microg/mL. Cholesterol loading occurred within 9 hours after the addition of acetylated LDL and continued during long-term incubation. Incubation of severely oxidized LDL for 7 days resulted in a slight increase in cholesterol content. Triglyceride loading was observed during short-term and long-term incubation with native and modified VLDLs. Neither cholesterol nor triglyceride loading resulted in expression of TF. Bacterial LPS still could induce TF expression in lipid-laden macrophages. Our results show that incubation with modified lipoproteins or lipid loading does not lead to TF expression in monocyte-derived macrophages cultured in suspension. This suggests that induction of TF expression in foam cells in the atherosclerotic lesion is triggered by additional or other components.

A placebo-controlled parallel study of the effect of two types of coffee oil on serum lipids and transaminases: identification of chemical substances involved in the cholesterol-raising effect of coffee.

In a randomized double-blind parallel study in 36 subjects the effect on serum cholesterol of a daily dose of 2 g lipid extracted from green Arabica and Robusta coffee beans was studied. Arabica oil elevated serum total cholesterol by 1.1 mmol/L (95% CI for the difference from placebo: 0.41, 1.73 mmol/L); the effect of robusta oil (+0.5 mmol/L) was not statistically significant (95% CI: -0.01, 0.92 mmol/L). Arabica oil also raised plasma triglycerides by 0.8 mmol/L (95% CI: 0.26, 1.25 mmol/L). The effect of robusta oil on triglycerides was +0.14 mmol/L and not significant (95% CI: -0.26, 0.42 mmol/L). In the 12 subjects taking arabica oil an average serum alanine amino-transferase elevation of 18 U/L (95% CI: 9.4, 28.4 U/L) was observed. Because only arabica oil contains kahweol and arabica coffee contains more cafestol than does robusta oil, this is further evidence for the role of diterpenes in the rise of serum cholesterol and alanine aminotransferase after consumption of boiled coffee.

Protection of myocytes against free radical-induced damage by accelerated turnover of the glutathione redox cycle.

The primary defence mechanism of myocytes against peroxides and peroxide-derived peroxyl and alkoxyl radicals is the glutathione redox cycle. The purpose of the present study was to increase the turnover rate of this cycle by stimulating the glutathione peroxidase catalysed reaction (2GSH-->GSSG), the glutathione reductase catalysed reaction (GSSG-->2GSH), or both. Neonatal rat heart cell cultures were subjected to a standardized protocol of oxidative stress using 80 mumol.l-1 cumene hydroperoxide (CHPO) for 0-90 min. The consequences of this protocol were described in terms of cellular concentrations of GSH, GSSG, NADPH and ATP, formation of malondialdehyde (MDA), release of GSSG and of ATP catabolites, depression of contraction frequency, cellular calcium overload, and enzyme release. Trolox-C, an analogue of vitamin E, accelerated the glutathione peroxidase reaction leading to lowering of GSH concentration and the GSH/GSSG ratio, less MDA formation, diminished negative chronotropy, delayed calcium overload, and less enzyme release. Glucose was used to accelerate the glutathione reductase reaction by supplying NADPH, leading to higher GSH concentration and a higher GSH/GSSG ratio, less MDA formation, diminished negative chronotropy, unchanged development of calcium overload, and less enzyme release. As a full turn of the glutathione redox cycle involves both the peroxidase and the reductase reactions, the combination of Trolox-C and glucose was superior to either of the two alone: 90 min following addition of CHPO together with Trolox-C and glucose, the GSH concentration and the GSH/GSSG ratio were almost normal, MDA formation was extremely low, calcium overload was markedly delayed, and enzyme release hardly occurred at all. Cells remained beating in the observation period of 30 min. We conclude that the capacity of the glutathione redox cycle to withstand oxidative stress can be increased by stimulation of either the peroxidase reaction or the reductase reaction, and that optimal redox cycling is achieved by stimulation of both reactions.

Desferrioxamine protects myocytes against peroxide-induced myocyte damage without affecting glutathione redox cycle turnover.

The primary defense mechanism of myocytes against peroxide-derived free radicals is the glutathione redox cycle. The purpose of the present study was to investigate whether desferrioxamine protects myocytes against peroxide-induced cell damage, and if so, whether the turnover rate of the glutathione redox cycle is involved in this protection. Neonatal rat heart cell cultures were subjected to a standardized oxidative stress by a 90 min incubation with 80 mumol/l cumene hydroperoxide. The consequences of this stress protocol were described in terms of cellular concentrations of GSH, GSSG, ATP, ATP-catabolites, and Ca2+, formation of malondialdehyde to quantify lipid peroxidation, and enzyme release to quantify the relative number of irreversibly injured cells. Following pretreatment of cell cultures with 10 mmol/l desferrioxamine mesylate for 1 h, 80 mumol/l cumene hydroperoxide caused less malondialdehyde formation (at 90 min: 0.34 v 2.35 nmol), less ATP depletion (at 60 min: 16.7 v 3.6 nmol), less Ca2+ overload (at 30 min: 40 v 1500 nM) and less enzyme release (at 90 min: 4.6 v 60.5% of the cells) compared to cell cultures subjected to cumene hydroperoxide without pretreatment. However, in desferrioxamine pretreated cell cultures cumene hydroperoxide caused cellular GSH depletion (at 60 min: 19.5 v 20.8 nmol) and GSSG efflux (at 60 min: 6.3 v 6.0 nmol) which was not different from cell cultures subjected to cumene hydroperoxide without pretreatment. Added to the finding that in a cell-free system cumene hydroperoxide is a substrate for glutathione peroxidase, we conclude that desferrioxamine, by chelating free iron ions (1), prevented the formation of cumene alkoxyl and peroxyl radicals associated with protection of the myocytes, and (2) did not diminish rapid glutathione redox cycling leading to GSH depletion and GSSG efflux.

Screening for familial defective apolipoprotein B-100 with improved U937 monocyte proliferation assay.

Frostegård et al. (J Lipid Res 1990;31:37-44) demonstrated that the proliferation of the human monocyte cell line U937 is critically dependent on the uptake of low-density lipoprotein (LDL) via the apo B, E (LDL) receptor, a characteristic that was used to detect patients with familial defective apolipoprotein B-100 (FDB). Here we applied this principle to develop a simple and reproducible assay for the detection of patients with functionally defective LDL. We added serum to U937 cells in cholesterol-free incubation medium and determined the increase in cell number after a 72-h incubation at 37 degrees C by using an electronic cell counter. Sera from 10 normolipidemic individuals and from 34 patients with type IIa hyperlipoproteinemia stimulated the growth of U937 cells in proportion to the exogenous cholesterol concentration (r = 0.83, P < 0.001) and the LDL-cholesterol concentration (r = 0.81, P < 0.001). However, sera from 16 patients with FDB stimulated less cell proliferation than did sera from patients with type IIa hyperlipoproteinemia with equal LDL-cholesterol concentrations. With a 15% reduction in growth as the cutoff value, this test had a sensitivity and specificity for diagnosis of FDB of 87.5% and 100%, respectively. The improved U937 monocyte proliferation assay can be used for screening hypercholesterolemic patients to detect individuals with functionally defective LDL.

Relationship between apolipoprotein E and low density lipoprotein particle size.

The relationships between the particle size of low density lipoproteins (LDL) and various lipid parameters, including apolipoprotein (apo) E concentration and apo E phenotype, were analyzed in plasma samples obtained from 196 apparently healthy 35-year-old males. The LDL particle size was determined by gradient gel electrophoresis. Using stepwise multiple regression analysis it was found that LDL particle size correlated negatively to the plasma concentrations of triglyceride (r = -0.497, P < 0.001), apo E (r = -0.415, P < 0.001), apo B (r = 0.395, P < 0.001) and cholesterol (r = -0.235, P < 0.001) and correlated positively to the plasma concentrations of apo A-I (r = 0.297, P < 0.001) and apo A-II (r = 0.145, P < 0.05). However, the LDL particle size did not differ significantly among the different apo E phenotypes. Indeed, when entered as a variable in the multiple regression analysis, the apo E phenotype was not correlated to the LDL particle size. It is concluded that the LDL particle size is related to the plasma concentrations of triglyceride, apo E, apo B, apo A-I, apo A-II and cholesterol and is not affected by the apo E phenotype in healthy 35-year-old males.

Buthionine sulfoximine reduces the protective capacity of myocytes to withstand peroxide-derived free radical attack.

Mammalian heart myocytes have a limited capacity to withstand the deleterious effects of free radical generating compounds. To assess the role of the glutathione redox cycle relative to this capacity, rat heart cell cultures were subjected for 90 min to 80 mumol/l cumene hydroperoxide (CHPO) without and with prior glutathione depletion by buthionine sulfoximine. Preincubation of cultures with 125 mumol/l buthionine sulfoximine for 2 h and 17 h caused a reduction of glutathione by 33% and 82%, respectively, without concomitant increase of glutathione disulfide. Subsequent incubation with CHPO for 90 min caused slowing of NADPH consumption (in the first 20 min 27 pmol vs 68 pmol without pretreatment with buthionine sulfoximine for 17 h), which indicates that glutathione depletion reduced the turnover rate of the glutathione redox cycle. Pretreatment with buthionine sulfoximine for 17 h exaggerated the negative chronotropic effects of CHPO: the time elapsed to 50% of baseline contraction frequency fell from 5.7 +/- 1.4 min without buthionine sulfoximine to 3.7 +/- 0.4 min after pretreatment with buthionine sulfoximine (P < 0.02). The severity of CHPO-induced lipid peroxidation as assessed by malondialdehyde formation (2.23 +/- 0.51 vs 0.99 +/- 0.05 nmol in the first 20 min; P < 0.05) was increased by buthionine sulfoximine pretreatment, as was the extent of cell necrosis as assessed by release of alpha-hydroxybutyrate dehydrogenase (39.5 +/- 5.1 vs 29.0 +/- 12.9% in the first 45 min). A "sublethal" dose of 10 microM CHPO for 60 min caused no substantial HBDH release, no formation of malondialdehyde, and no exhaustion of cellular GSH (35 nmol/U HBDHt = 0). However, following pretreatment with buthionine sulfoximine, 10 microM CHPO for 60 min produced 12% HBDH release and extensive lipid peroxidation (1.95 nmol malondialdehyde/U HBDHt = 0). As the deleterious effects of CHPO were aggravated by glutathione depletion, we conclude that the glutathione redox cycle plays a major role in the protection of myocytes against peroxide-induced free radical attack.

Effects of in vivo heart irradiation on myocardial energy metabolism in rats.

To investigate the effect of in vivo heart irradiation on myocardial energy metabolism, we measured myocardial adenosine nucleotide concentrations and mitochondrial oxygen consumption in left ventricular tissue of rats 0-16 months after local heart irradiation (20 Gy). At 24 h and 2 months no difference in myocardial adenosine nucleotide concentration was apparent between irradiated and control hearts. The total myocardial adenosine nucleotide concentrations in irradiated hearts compared to those of nonirradiated controls tended to be lower from 4 months onward. The rate of oxidative energy production (state 3 respiration) in irradiated hearts was significantly reduced compared with that of age-matched controls from 2 months onward. Moreover, as a result of aging, a time-dependent decrease in the rate of oxidative energy production was observed in both irradiated and control hearts (P < 0.001). The respiratory control index (RCI = oxygen consumption in state 3/oxygen consumption in state 4) in irradiated hearts was not different from the RCI measured in age-matched control animals. During the period of study the RCI diminished significantly with age in both groups (P < 0.005). The number of oxygen atoms used per molecule of ADP phosphorylated (P/O ratio) was not influenced by the irradiation. The P/O ratio for the NAD(+)-linked substrates remained unchanged at a value of about 3 during the period studied. At 6 months after irradiation activities of myocardial enzymes such as lactate dehydrogenase, creatine kinase, citrate synthase, and cytochrome c oxidase were reduced. The reduction in myocardial energy production and the changes in energy supplies provide a mechanism to explain impaired contractility after local heart irradiation.

Effects of glucose, Trolox-C, and glutathione disulphide on lipid peroxidation and cell death induced by oxidant stress in rat heart.

OBJECTIVE: The aim was to find effective protection of myocytes against peroxide induced damage in terms of preservation of contractile activity, protection against lipid peroxidation, and protection against cell death. METHODS: The components of the glutathione redox cycle, the production of malondialdehyde, cell contractions, and enzyme release from myocytes were measured in cultured neonatal rat heart cells before and after administration of cumene hydroperoxide, 80 mumol.litre-1. The protective action was tested of (1) glucose (10 mmol.litre-1) which stimulates the production of NADPH; (2) Trolox-C (0.16 mmol.litre-1) which is a water soluble analogue of alpha tocopherol and a scavenger of free radicals; and (3) GSSG (0.6 mmol.litre-1) which increases the intracellular concentrations of GSH and GSSG. RESULTS: Although the three substances tested were equally effective in reducing the formation of malondialdehyde, exogenous GSSG afforded only slight protection against cumene hydroperoxide induced cell death, whereas glucose and Trolox-C were highly effective protectors. The depressant effect of cumene hydroperoxide on beating frequency was not influenced by preincubation with GSSG, nor by coadministration of glucose, but Trolox-C was able to diminish the negative chronotropic action of cumene hydroperoxide. CONCLUSIONS: Effective protection against cumene hydroperoxide induced lipid peroxidation is not associated per se with effective protection against cumene hydroperoxide induced loss of beating frequency and cell death.

Myocardial creatine kinase-MB concentration in normal and explanted human hearts and released from hearts of patients with acute myocardial infarction.

Using an enzyme immunoassay of creatine kinase (CK)-MB concentration commercially available for diagnosis of acute myocardial infarction (AMI), we studied CK-MB concentrations in myocardium of subjects who died from noncardiac causes and in cardiac explants of patients with either coronary heart disease or cardiomyopathy who underwent cardiac transplantation. Secondly, CK-MB concentrations were measured in serial plasma samples of 93 patients with AMI. By calculation of cumulatively released amounts of CK-MB and cumulatively released activities of CK, aspartate aminotransferase (AST) and alpha-hydroxybutyrate dehydrogenase (HBDH), we obtained values of the proportions in which these quantities were released from the myocardium. Taking a myocardial HBDH activity of 152 U/g as a reference value, the released activities of CK and AST, and the released mass of CK-MB per gram of myocardium were calculated. These values were compared to the corresponding quantities in myocardium of normal hearts and in explanted myocardium. Normal hearts differ from explanted myocardium and from "infarcted" hearts with respect to CK-MB concentration, but not with respect to CK, AST and HBDH activities. The wide range of CK-MB concentrations in normal hearts (1-515 micrograms/g) suggests variable expression of the CK-MB gene. The presence of CK-MB is not confined to cardiac tissue. CK-MB concentration in 12 samples of human skeletal muscle equalled 27 +/- 1 micrograms/g (2.1 +/- 0.5% of total CK activity). In conclusion, the mean concentration of CK-MB in normal hearts is low (139 micrograms/g) with a high variation coefficient (127%), but is high (369 micrograms/g) with a small variation coefficient (31%) in explanted hearts.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of simvastatin on the apparent size of LDL particles in patients with type IIB hyperlipoproteinemia.

After 15 weeks of simvastatin therapy (20 mg/day), low density lipoprotein particle size in sera of 16 patients with type IIb hyperlipoproteinemia increased significantly from 233 +/- 5.0 A to 237 +/- 7.0 A (P less than 0.05), analyzed by 2-16% polyacrylamide gradient gel electrophoresis. Under simvastatin therapy the concentrations of total cholesterol, total triglyceride, very low density lipoprotein cholesterol and triglyceride, low density lipoprotein cholesterol and apolipoprotein B in serum fell significantly by 30%, 30%, 43%, 28%, 36% and 26%, respectively, and the concentration of high density lipoprotein cholesterol rose significantly by 14%. The changes of low density lipoprotein particle size induced by simvastatin therapy were correlated best with the changes of very low density lipoprotein triglyceride concentration (r2 = 0.438, P less than 0.01). Our results suggest that simvastatin therapy, additionally to a reduction of the serum cholesterol concentration, increases low density lipoprotein particle size which may contribute to reduction of the risk of coronary heart disease in patients with type IIb hyperlipoproteinemia.

Quantitation of cumulative release of lactate dehydrogenase isoenzyme-1 in plasma of patients with acute myocardial infarction using a commercially available test.

In 27 patients with acute myocardial infarction (AMI) we calculated cumulative release of alpha-hydroxybutyrate dehydrogenase (alpha HBDH) per liter plasma which is a routine procedure in our coronary care unit, and compared these values with calculated cumulative release of lactate dehydrogenase isoenzyme-1 (LDH-1) per liter plasma using a LDH-1 test that has become commercially available recently. Theoretically, myocardial (iso)enzyme release is more accurately determined with LDH-1 than with alpha HBDH, due to the higher cardiac specificity of LDH-1 compared to alpha HBDH. The only disadvantage of LDH-1 is its abundance in erythrocytes necessitating a correction by measurement of free hemoglobin (Hb) concentration in plasma. After division of cumulatively released activities (Q72) of alpha HBDH and LDH-1 by the activities per gram of normal myocardium (135 and 81 U/g, respectively), the values of Q72(alpha HBDH)/135 and Q72(LDH-1)/81 were compared per patient. Elevated alpha HBDH levels in the presence of normal creatine kinase levels in plasma samples taken on admission, as well as hemolysis gave rise to overestimation of cumulative release of alpha HBDH as compared to LDH-1, but hepatic congestion occurring secondary to AMI (48-72 h after onset of infarction) did not disturb the equality of Q72 (alpha HBDH)/135 and Q72(LDH-1)/81 values. In 16 patients showing none of the mentioned conditions, the relation between Q72(alpha HBDH)/135 and Q72(LDH-1)/81 coincided with the line of identity (r = 0.97). We conclude that the use of an easy and rapid plasma LDH-1 assay improves the assessment of enzymatic infarct size, provided free Hb levels are measured to correct LDH-1 activities for a contribution by erythrocytes.

A method to quantitate cell numbers of muscle cells and non-muscle cells in homogenised heart cell cultures.

Neonatal rat heart cell cultures are popular research models in cardiovascular investigations. A major disadvantage is the variable contribution of non-muscle cells to the cultures. As biochemical and pharmacological quantities are generally measured in homogenised cultures, we looked for a method to calculate numbers of muscle cells and non-muscle cells per culture after homogenisation. By means of a model based on the presence of one diploid nucleus per myocyte and per non-muscle cell, a nuclear DNA content of 7.5 pg, and a constant ratio of DNA content and sum of the lactate dehydrogenase isoenzymes LDH-4 and LDH-5 (DNA/LDH4+5 = 11.6 +/- 1.5 micrograms.U-1) in non-muscle cells, we calculated that in 21 neonatal rat heart cell cultures, cultured for 0-6 days, the number of muscle cells was 1.5 X 10(6) per culture, independent of time; and the number of non-muscle cells was low at day 0 (1.6 X 10(5) per culture), increasing to 4 X 10(6) per culture at day 6. Based on a time dependent increase in lactate dehydrogenase content per muscle cell we showed that muscle cells in culture underwent progressive hypertrophy: in 6 days myocyte volume increased fourfold. Thus, by measurement of DNA content and the activities of lactate dehydrogenase and its isoenzymes in a homogenised culture the cellular composition of the culture can be assessed quantitatively.

Comparison of myocardial changes between pressure induced hypertrophy and normal growth in the rat heart.

To evaluate differences in tissue composition between hearts with pressure overload hypertrophy and normal hearts of comparable weight, 30 rat hearts with aortic constriction of 4, 10 and 30 days, and nine hearts of sham operated controls were studied. Surgery was performed at age 70 days. Morphometric analysis of myocardial tissue sections revealed (1) myocyte hypertrophy in left ventricular myocardium of hypertrophic hearts was proportional to heart weight, and in normal growth myocyte volume increased in proportion to heart weight; (2) myocyte number in left ventricular myocardium was identical in hypertrophic and normal hearts; (3) non-muscle cell proliferation was proportional to heart weight identically in hypertrophic and normal hearts; (4) volume fractions of myocytes were significantly lower in hypertrophic hearts [0.76(SD 0.05)] than in normal hearts [0.82(0.04)]; (5) volume fractions of all nuclei, myocyte nuclei and non-myocyte nuclei were similar in hypertrophic and normal hearts; (6) measured ventricular DNA content increased with heart weight identically in hypertrophic and normal hearts, and equalled DNA content calculated using the data on tissue composition. Neither right ventricular weight nor right ventricular DNA content were affected by the presence of left ventricular hypertrophy. We conclude that left ventricular hypertrophy due to aortic constriction in the rat resulted in changes of myocardial tissue composition similar to the changes associated with normal growth. Tissue composition of hypertrophic rat hearts corresponds strikingly to that of normal rat hearts with comparable heart weight, although myocardial changes in hypertrophy develop considerably faster than in normal growth.

Myocardial (iso)enzyme activities, DNA concentration and nuclear polyploidy in hearts of patients operated upon for congenital heart disease, and in normal and hypertrophic adult human hearts at autopsy.

To investigate biochemical characteristics of hypertrophic myocardium of young and adult humans, we analysed myocardial biopsies obtained from 28 mainly young patients undergoing cardiac surgery for congenital heart disease and 41 autopsied hearts from 18 adult normal and 23 hypertrophic human subjects. Myocardial activities of the enzymes creatine kinase and lactate dehydrogenase were independent of age during childhood, but decreased significantly with hypertrophy at adult age. Myocyte nuclei showed increased polyploidization during childhood which was progressive with age, and in the adult stage polyploidization was correlated with heart weight. Nevertheless myocardial DNA concentration fell under both conditions, which is to be ascribed to the 'diluting' effect of myocyte hypertrophy. Before an age of 8 years DNA concentration in the child heart material studied has reached the value found in adult nonhypertrophic hearts, although at that time polyploidization of myocyte nuclei in child hearts was only half the value found in adult non-hypertrophic hearts. Biochemical measurement of DNA concentration in peroperatively taken myocardial biopsies may contribute to the in vivo diagnosis of ventricular hypertrophy in quantitative terms, in combination with radiology, echocardiography and histology.

Assessment of hypertrophy in myocardial biopsies taken during correction of congenital heart disease.

Myocardial biopsies were obtained from 27 patients undergoing corrective cardiac surgery for congenital heart disease. Normal hearts of 18 autopsied patients were used as reference. The biopsy material was assessed for desoxyribonucleic acid (DNA) concentration and ploidy profile of cell nuclei in order to quantitate myocardial hypertrophy at the time of operation. DNA-concentration decreased significantly with age (r = -0.76; p less than 0.001). Ploidy profile of myocardial nuclei correlated with age: the relative number of diploid nuclei decreased (r = -0.67; p less than 0.001), the relative numbers of tetraploid and octoploid nuclei increased with age (r = 0.58; p less than 0.01 and r = 0.77; p less than 0.001 respectively). At 8 years of age the patients with congenital heart disease reached myocardial DNA-concentrations comparable with those in normal adult hearts. At higher age the patients with congenital heart disease exceeded normal adult values for myocardial DNA-concentration. These findings are interpreted to represent rapid development of hypertrophy with an early onset, reaching at 8 years of age values observed in normal adult hearts. Quantitation of myocardial hypertrophy by DNA-concentration and ploidy profile of nuclei may offer a means to explain some of the factors of influence on the outcome of corrective cardiac surgery for congenital heart disease in relation to its timing. Our data stress the need for preventing irreversible myocardial damage by timely (surgical) therapy.

Enzymatic assessment of myocardial injury after infarction or cardiac surgery. Is isoenzyme analysis required?

Estimation of enzyme release in plasma requires knowledge of the fractional catabolic rate constant (FCR) for the elimination enzyme activity from plasma. However, the total plasma content of such enzymes usually consists of several isoenzymes with different values of FCR. Thus, the use of a single overall value for FCR may cause error. This problem was studied by determination of the plasma isoenzyme activities of creatine kinase, lactate dehydrogenase, aspartate aminotransferase and alpha-hydroxybutyrate dehydrogenase in patients after cardiac surgery and after acute myocardial infarction. Values of FCR and the cumulative release of activity in plasma are estimated for separate isoenzymes and for total enzyme activity. Results are compared with the enzyme content of myocardium, skeletal muscle and blood cells. It is concluded that isoenzyme separation is not required for the quantitative use of such data. The implications for the validation of enzymatic estimation of cardiac injury are discussed. The results indicate that local inactivation of enzymes after cardiac injury must be limited.

A comparative study on enzymatic data from myocardial biopsies and cardiac function in patients who underwent cardiac surgery.

Twenty-three patients underwent cardiac surgery for valve replacement, valve reconstruction, aorto-coronary bypass grafting, aneurysmectomy or combinations of these. Excised cardiac tissue was obtained from left ventricular (LV) papillary muscle (17 patients), LV outflow tract (3 patients), or LV aneurysms (3 patients). A total of 34 myocardial samples, collected from excised cardiac tissue, were analysed for creatine kinase (CK), CK-isoenzymes, cytoplasmic and mitochondrial isoenzymes of aspartate aminotransferase (cAST and mAST, respectively), and lactate dehydrogenase (LDH) isoenzymes. Myocardial CK activity correlated positively with preoperative LV ejection fraction (p less than 0.001), negatively with the preoperatively measured extent of LV wall motion abnormalities (p less than 0.001), and negatively with preoperative LVEDP (p less than 0.02). Myocardial CK activity was negatively correlated with preoperative validity class (p less than 0.005). However, no correlation existed between myocardial CK activity and postoperative validity. Excluding the biopsies from LV aneurysms, myocardial CK activity was positively correlated with the fraction H-subunits in LDH (p = 0.02), and was negatively correlated with the fraction mAST in total AST (p less than 0.005). While cAST activity was proportional to CK activity in the biopsies from VL papillary muscle and LV outflow tract, mAST activity declined only with 2.4 +/- 1.2% per 10% fall of CK. The increase of mAST/cAST ratio with decreasing CK, together with the decrease of LDH-H/LDH and CK-M/CK ratios with decreasing CK, indicated the presence of an adaptation process in a myocardium with low CK activity rather than a process of necrosis.(ABSTRACT TRUNCATED AT 250 WORDS)