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Technical Briefs |
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Cardiovascular Research Institute Maastricht and
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Department of Educational Development and Research, Maastricht University, 6200 MD Maastricht, The Netherlands; Departments of
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Clinical Chemistry and
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Cardiothoracic Surgery, University Hospital Maastricht, 6200 MD Maastricht, The Netherlands;
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Department of Pathology, Atrium Medical Center Heerlen, 6401 CX Heerlen, The Netherlands
aaddress correspondence to this author at: Cardiovascular Research Institute Maastricht, Maastricht University, PO Box 616, 6200 MD Maastricht, The Netherlands; fax 31-43-3670916, e-mail w.hermens{at}carim.unimaas.nl
In patients with acute myocardial infarction (AMI), the activity of creatine kinase isoenzyme MB (CKMB) in plasma consistently accounts for 
15% of the total CK activity (1)(2). By contrast, the CKMB content of cardiac tissue, although sometimes reported to be consistent with the 15% plasma activity of CKMB (2)(3), has also been reported to be negligible in healthy myocardium (4)(5). In these studies, the higher CKMB found in diseased hearts was thought to reflect cellular adaptation to disease. An alternative explanation for low CKMB is its limited thermostability and susceptibility to pH (6).
The objective of the present study was to examine whether inactivation of CKMB, either postmortem or during perimortal tissue acidosis, could explain the absence of CKMB in cardiac tissue at autopsy. The influence of tissue acidosis was studied by exposing heart tissue to pH values of 5.0–7.5. Finally, because myocardial ischemia and tissue acidosis attributable to AMI are located predominantly in the endocardium (7)(8), transmural differences in CKMB content were studied in the hearts of patients who died after AMI.
Slices (1 cm), midway between apex and base, were obtained from 20 hearts of patients (11 males and 9 females) who died from noncardiac causes and without history of cardiac complaints. Mean values for age, autopsy delay, and heart length and weight (± SD) were 71 ± 15 years, 29 ± 22 h, 167 ± 12 cm, and 467 ± 83 g, respectively. Similar heart slices were obtained from 6 patients who died within 6 h after AMI.
Tissue samples (133 ± 34 mg wet weight) were homogenized as described previously (9), and dry weight (dw) was determined by freeze drying the homogenate in a Leybold Heraeus GT2. CK, CKMBmass, CKMBact, and 
-hydroxybutyrate dehydrogenase (HBD) in the supernatant were measured and expressed per milligram of dw of tissue. CK and CKMBact were determined at 37 °C, CK with a N-acetyl-cysteine-activated test from Merck Diagnostics and CKMBact with the Isomune assay of Roche Diagnostics. HBD was measured at 25 °C with the optimized HBDH test from Roche Diagnostics. CKMBmass was measured with the Immulite Automated Analyzer (detection limit, 0.42 µg/L; Diagnostic Products Corporation).
The first nine non-AMI hearts were used for a detailed analysis of regional tissue protein content. The left ventricle was cut circumferentially into eight samples, each divided into epicardial and endocardial parts (9). Results were analyzed with variance component analysis (SPSS, Ver. 10.0; SPSS Inc.). Neither circumferential nor transmural location influenced protein content; only differences between hearts were significant (P <0.05). Therefore, single tissue samples were randomly taken from the remaining 11 non-AMI hearts. The six hearts from AMI patients were analyzed again in detail.
Linear regression analysis of the 20 non-AMI hearts showed no influence of sex, heart weight, or age on tissue protein content. A statistically insignificant tendency toward higher CKMBmass in hearts with longer autopsy delays was noted, with mean values of 0.18 (n = 4), 0.53 (n = 7), and 0.88 (n = 9) µg/mg in hearts with autopsy delays of <10 h, 10–25 h, and >25 h, respectively.
The high variability in tissue CKMB content, shown in the Table 1
, was explained by very low CKMB values in 10 of the 20 non-AMI hearts (CKMBmass, <0.1 µg/mg). Independently from these tissue data, total CK activity and CKMBmass were also measured in plasma samples obtained 6 h after onset of symptoms from 164 patients with AMI. As shown in Table 1
, the CKMBmass/CK ratio for this in vivo protein release is much higher and less variable than in tissue. These data suggest that the loss of tissue CKMB is caused by a variable perimortal effect, which is already completed in the first few hours after death. A variable degree of tissue acidosis, depending on the agonal phase, could be such an effect and could also explain its all-or-none character.
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Susceptibility of CKMB to tissue acidosis was studied in tissue samples (60–150 mg wet weight) incubated at 37 °C in buffered phosphate–citrate with 20 g/L bovine serum albumin. After incubation, samples were stored at -70 °C. Tissue samples were homogenized, and CK, CKMBact, CKMBmass, and HBD were assayed in the homogenates as well as in the incubation buffers. CKMBmass completely disappeared within 2 h at pH 5.0 and 5.5 (Fig. 1
). In contrast, HBD remained stable at all pH values, and CK remained stable for pH values down to 5.5, but was inactivated at pH 5.0. Because of the stability of total CK (predominantly CKMM) at pH 5.5, total inactivation of CKMBact at this pH may seem surprising. The Isomune assay, however, measures CK activity before and after blocking of M subunits and then subtracts the CK activity measured after the removal of all M units, including CKMB, from the sample. A CKMB molecule with an inactivated B subunit will not contribute to the activity in either fraction and thus will not be detected. We do not know if the M subunit activity remains.
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The transmural ratios (endocardial/epicardial) of HBD and CKMBmass were computed in only four of the first nine non-AMI hearts and in four of the six AMI hearts because of the very low CKMBmass content (<0.2 µg/mg) in the remaining hearts. Because of the short time (<6 h) between onset of symptoms and death, infarcted areas in the AMI hearts had physiologic HBD content (>0.4 U/mg dw) and endocardial/epicardial ratios of HBD were equal in non-AMI and AMI hearts (1.08 ± 0.04 and 0.98 ± 0.05, respectively; mean ± 95% confidence interval). For CKMBmass, however, these values were 0.92 ± 0.12 and 0.53 ± 0.16, respectively (P <0.005; Student t-test). Apparently, endocardial CKMBmass disappears from AMI hearts even before significant leakage of proteins from the infarcted tissue has occurred.
In conclusion, our study shows that the low and highly variable CKMB content of myocardial autopsies is not related to the influence of sample location or autopsy delay, but may be caused by perimortal tissue acidosis. The narrow range of pH values at which inactivation occurs, between 5.5 and 6.0, could explain the noted all-or-none aspect. Myocardial pH after AMI may indeed reach values below 5.5 (10) and, after death, will increase again because of termination of lactate production. The tendency toward higher CKMB for longer autopsy delays may then indicate that this CKMB inactivation is partly reversible. Lack of CKMB in apparently healthy hearts from traffic accident victims (4)(5) could be explained by serious tissue acidosis in the prolonged agonal phase of these victims who survived long enough to die after admission to hospital.
References
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, pH 5; 
, pH 5.5; 
, pH 6.5; +, pH 7.5.