Using an ultrasensitive troponin I assay, we were able to demonstrate a quantifiable rise in circulating troponin levels in response to stress testing, and the magnitude of that rise was proportional to the degree of ischaemia on perfusion imaging. Furthermore, a rise in the level of troponin I after stress testing was significantly associated with inducible ischaemia even after adjusting for traditional parameters such as limiting angina and ST depression.
Cardiac troponins are the preferred diagnostic biomarker for MI.1
Using current commercial assays, most healthy individuals have levels below the limit of detection and certainly below the cutpoint at which the CV is <10%.5
Any elevation in troponin in the appropriate clinical setting has been considered indicative of myocardial necrosis rather than ischaemia.4
Previous attempts to quantify rises in troponin in the setting of transient myocardial ischaemia have been unrewarding. Specifically, in prior studies in individuals undergoing stress testing, troponin levels following stress testing have either been undetectable, or barely detectable but within the normal range, and changes in troponin were either undetectable, below the cutpoint for <10% CV, or not associated with the presence of ischaemia during the test.10–15
In three studies, an increase in troponin was demonstrable after stress testing, but in only a minority of patients with ischaemia and at levels close to the 10% CV cutpoint.16–18
Now, using a novel troponin assay that is one to two orders of magnitude more sensitive than commercial assays, we have been able to demonstrate that brief periods of stress-test induced myocardial ischaemia are associated with quantifiable rises in the concentration of circulating troponin.
Definitive delineation of the cellular events responsible for the rise in circulating troponin that we observed is beyond the scope of this clinical study. Specifically, whether cardiac troponin can be released in the setting of pure myocyte ischaemia without necrosis remains controversial. An immunohistochemistry study in animals subjected to coronary occlusion found staining for troponin in necrotic, but not in non-necrotic, areas of myocardium.19
Conversely, another animal study demonstrated elevated levels of circulating troponin without electron microscopic evidence of irreversible myocyte necrosis.20
Furthermore, cytosolic pools of troponin have been demonstrated, by some estimates accounting for ~5% of total myocyte troponin.21
Troponin in these pools could egress from injured myocytes without the need for myofibril degradation. To that end, in specialized animal and human models, brief periods of myocardial ischaemia have been shown to lead to the release of troponin.22–24
Furthermore, in canine models, 15–20 min of sustained ischaemia is required to induce irreversible myocyte cell death;25
in contrast, the duration of myocardial oxygen supply–demand mismatch during stress testing was only a few minutes in our study, making necrosis unlikely. Nonetheless, although our findings support the concept that cardiac troponin may function as a biomarker of reversible myocardial injury, without pathological examination, the possibility of micronecrosis during stress testing cannot be excluded.
Our findings have several clinical implications. First, a more sensitive troponin assay that can detect transient myocardial injury would give clinicians the ability to seek biochemical data to support the diagnosis of unstable angina. Such objective evidence would be welcome in these cases, for which clinicians currently are forced to rely on a subjective history and transient ECG changes. Although the elevations seen in our study were subtle, and the range of values overlapped significantly between those with and without ischaemia, the duration and magnitude of ischaemia were, by design, brief. We would speculate that spontaneous ischaemia due to plaque rupture would generate more profound elevations in troponin. Clinicians might be able to use such elevations to diagnose ACS objectively. Of course, the prognostic implications and the utility in terms of guiding aggressiveness of care of such truly quantitatively minor elevations in patients will need to be studied.
Secondly, measuring biomarkers of myocardial injury (troponin) and wall stress (BNP) in patients undergoing stress testing with electrocardiography could potentially aid in the diagnosis of clinically significant epicardial coronary artery disease. Incorporation of measurement of these biomarkers along with assessment of traditional risk predictors could help obviate the need for initial myocardial perfusion imaging in a subset of patients. However, our goal in this proof-of-concept study was not to define the clinical utility of such measurements, but rather to study the release patterns of cardiac troponin in response to transient myocardial ischaemia. The ability of troponin or BNP levels to add to the diagnostic utility of stress testing needs to be confirmed in additional, larger prospective studies.
Thirdly, the clinical adoption of a troponin assay as sensitive as the one we used will likely necessitate a revised approach to reporting of troponin levels. No longer will individuals outside the setting of MI typically have undetectable levels. Rather, similar to most of the analytes clinicians measure, there will be a quantifiable reference range for the population. Using the current commercial troponin T assay, we have previously shown that elevated levels of troponin T are found in <1% of the population, but that those individuals typically have cardiovascular abnormalities including heart failure, diabetes mellitus, and left ventricular hypertrophy.26
In these patients, an elevated troponin may reflect chronic supply–demand mismatch. Using a more sensitive assay, it may be possible to better quantify and even track chronic myocardial ischaemia over time. To that end, using a troponin T assay 10-fold more sensitive than current commercial assays, elevated levels of troponin T were found in 92% of the chronic heart failure patients and higher levels were associated with a worse long-term prognosis.27
In accordance with that observation, in our study, we found that patients who manifested the most severe ischaemia on stress testing had slightly higher levels of troponin at baseline.
Our study has several limitations that must be considered. Not all subjects had blood samples successfully obtained at all times points; however, statistical comparisons of biomarker values between timepoints were performed by analysing the change in values within subjects, thereby guaranteeing a comparison across identical cohorts. We relied on nuclear imaging to quantify the degree of ischaemia. This remains an imperfect gold standard. Nonetheless, we think it superior to angiography, which can verify the presence of coronary atherosclerosis but cannot demonstrate inducible ischaemia. The magnitude in rise of troponin was relatively small, the IQRs wide, paired measurements were required to optimally discriminate between those who did and did not develop inducible ischaemia, and even then the sensitivity and specificity were modest. However, the accuracy was better than either ST deviation or limiting angina: two key parameters currently used to determine whether a stress test is indicative of ischaemia. Moreover, the duration of significant ischaemia during stress testing was brief, an average of only 2 min even in those with moderate-to-severe ischaemia. Furthermore, the cutpoint we used of a rise of 1.3 pg/mL corresponds to an increase of 30%, similar to the 25% threshold recommended when evaluating a patient with elevated levels of biomarkers at baseline.28
We applied a dichotomous cutpoint for the ultrasensitive troponin assay that was derived in the same data set and therefore is likely overly optimistic. Bootstrapping yielded an identical result with narrow IQR but wide 95% CI. Validation of this cutpoint, particularly in the setting of multivariable analysis, and consideration of modelling ultrasensitive troponin as a continuous variable in larger external data sets will be required. As is the case for any assay, before it can be adopted into routine clinical practice, it requires validation according to Clinical and Laboratory Standards Institute guidelines, including limit of quantitation across multiple lots of reagents.
In summary, by using a novel, ultrasensitive troponin assay that is at least an order of magnitude more sensitive than current commercial assays, we could detect changes in circulating troponin that were associated with the degree of transient stress-test induced myocardial ischaemia as detected using myocardial perfusion imaging. These findings pave the way for additional studies of the diagnostic and prognostic utility of troponin assays with markedly improved sensitivity.