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Letters to the Editor |
Johnson & Johnson Pharmaceutical, Research & Development L.L.C., Global Preclinical Development, Raritan, NJ
aAddress correspondence to this author at: Johnson & Johnson Pharmaceutical Research & Development L.L.C., Global Preclinical Development, PO Box 300, 1001 Route 202 North, Raritan, NJ 08869.
To the Editor:
Many studies have shown that determination of the blood concentrations of cardiac troponin I and T (cTnI and cTnT) offers higher clinical sensitivity and specificity for the diagnosis of cardiac injury than do the standard cardiac protein markers such as creatine kinase (CK) (1)(2). However, the release of cardiac troponins into the blood stream of laboratory animals in response to cardiac insults such as drugs is still not well defined. It was our objective to examine the release of cardiac troponins together with CK by repeat dosing in isoproterenol (ISO)-treated beagle dogs, a routinely used animal model in preclinical studies.
ISO is a sympathomimetic agent and ß-adrenergic receptor agonist that causes acute cardiac injury at high doses (3). In the present study, 1 male and 1 female beagle dog (9 months old) were given a 1 mg/kg subcutaneous injection of ISO on each of 2 consecutive days. Abnormal pathologic alterations, including severe coagulative necrosis, were observed in both animals. Blood was collected from the jugular vein into collection tubes (Beckon Dickinson) containing EDTA for the analysis of cardiac troponins and into the same type tubes with no additive for CK. The blood concentration of cTnI was measured by a modified Access® AccuTnITM immunoassay (Beckman-Coulter) performed manually on 96-well plates according to the manufacturer’s recommended experimental conditions. cTnT was determined by an ELISA containing a biotinylated monoclonal cTnT-specific antibody from the third-generation cTnT STAT immunoassay (Roche Diagnostics) and a pair-matched monoclonal antibody against human cTnT (clone 1A11) obtained from Hytest. The biotinylated antibody against cTnT in the cTnT-containing immunocomplex was quantified by peroxidase-labeled streptavidin and a chemiluminescent substrate, according to the manufacturer’s recommended conditions (Pierce Biotechnology). CK activity was measured with an N-acetylcysteine–activated test at 37 °C on an ADVIA® 1650 Chemistry System (Bayer), as developed by Szasz et al. (4).
Both troponin assays were validated by use of purified human and dog troponin calibrators. The calibrators were linear on dilution up to 50 µg/L. The cTnI assay specifically measured purified dog cTnI, but not purified dog cTnT, up to 50 mg/L. Similarly, the cTnT assay specifically measured purified dog cTnT, but not purified dog cTnI, up to 50 mg/L. In addition, the blood concentrations of cardiac troponins in untreated healthy dogs were very low (<0.7 µg/L) or not detectable.
The blood samples analyzed in the present study were collected before administration of ISO and at 1, 4, 8, 10, and 24 h after each daily dose. After the initial ISO administration, the blood concentrations of the cardiac troponins in both dogs increased quickly by 4 h after the first ISO administration, then peaked at 
8 h, followed by a slow disappearance of the cardiac troponins (panels A and B in Fig. 1
for the male and female dog, respectively). The blood concentrations of cTnI and cTnT were highly correlated in the male (r = 0.9326; P <0.0001) and female dogs (r = 0.9189; P <0.0001). The cTnI concentration remained increased at 24 h after the first ISO administration, whereas cTnT remained increased only in the male dog. Nevertheless, the blood content of cTnI and cTnT appeared to correlate with the pathology finding that the cardiac injury in the male dog was more severe than in the female dog. After the first administration, for example, the peak blood concentration of cTnI [mean (SD), 55.2 (11.2) µg/L] in the male dog was 17-fold higher than the blood concentration [3.2 (0.2) µg/L] in the female dog. Similarly, the peak blood concentration of cTnT [19.1 (0.6) µg/L] in the male dog was 16-fold higher than that in the female dog [1.2 (0.1) µg/L].
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Comparing the release of cardiac troponins after each of 2 administrations of ISO, the second administration triggered only a slight increase, but the concentrations still peaked at 8 h after the second administration (Fig. 1
). The mean (SD) cTnI and cTnT concentrations at the second peak [31.3 (7.6) µg/L cTnI and 12.4 (0.5) µg/L cTnT in the male dog, and 2.5 (0.1) µg/L cTnI and 0.7 (0.1) µg/L cTnT in the female dog] were markedly lower than the concentrations at the first cardiac troponin peak, which might be attributable to ß-adrenergic receptor internalization and desensitization (5).
To examine the possible relationship of CK with cardiac troponins, we used the collected blood samples from the same animals to determine total CK activity, a frequently used cardiac protein marker (6). As shown in Fig. 1
, the blood CK concentrations quickly increased in both ISO-treated dogs. Similar to the release of cardiac troponins, the increase started at 4 h after the initial administration. Similarly, the initial increase in CK was much more dramatic than the second increase. Differently, CK in the female dog showed maximal activity at 4 h, which was earlier than the release of cardiac troponins (Fig. 1B
) with a faster clearance as well. The correlation of CK activity with the release of cTnI differed between the male and female dogs, mainly in the times of their peak blood concentrations. In the male dog, cTnI release was correlated with CK activity (r = 0.7165; P = 0.0131), but in the female dog, we found no correlation of CK activity with the release of cTnI (r = 0.2748; P = 0.4135).
The possible relationship of cardiac troponins and CK has been examined in ISO-treated rats (7)(8). Similar to our findings in beagle dogs, the release of cardiac troponins in rats occurred within 4 h after ISO administration, but it peaked at 4 h and returned to baseline by 24 h. However, no significant increase in CK was observed in ISO-treated rats, suggesting that beagle dogs might represent a better experimental model for the investigation of drug-induced cardiac injury, at least for therapeutic agents with a mechanism of action to similar that of ISO.
In conclusion, the release of cardiac troponins and CK exhibited time-dependent and monophasic increases in ISO-treated beagle dogs. The increases in these protein markers on the second day was markedly lower, indicating limited additional release. Most importantly, CK decreased from peak concentrations faster than both cTnI and cTnT, indicating a narrower diagnostic window. The return to basal values of cardiac troponins and CK was delayed by repeat dosing, but was not inhibited. Thus, the limited additional release of cardiac troponins and CK does not prevent their use as indicators for cardiac injury. On the basis of the dynamic increases seen in the present study and the cardiac-specific origin, cardiac troponin concentrations in beagle dogs provide an ideal experimental measurement for cardiac injury, bridging preclinical and clinical studies to evaluate new therapeutic agents (2).
References
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), cTnT (
), and CK (•) in male (A) and female (B) beagle dogs treated with 1 mg · kg–1 · day–1 ISO.