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The purpose of this study was to develop a quantitative method for myocardial blood flow (MBF) measurement that can be used to derive accurate myocardial perfusion measurements from contrast-enhanced multidetector computed tomography (MDCT) images with bolus tracking and helical scanning. Nine canine models of left anterior descending artery (LAD) stenosis were prepared and underwent MDCT perfusion imaging during adenosine infusion (0.14-0.21 mg/kg/min) to study a wide range of flows. Neutron-activated microspheres were injected to document MBF during adenosine infusion. Six animals underwent dynamic MDCT perfusion imaging, and K1 and k2, which represent the first-order transfer constants from left ventricular blood to myocardium and from myocardium to the vascular system, respectively, were measured using a compartment model. The results were compared against microsphere MBF measurements, and the extraction fraction (E) of contrast agent and the mean value of K1/k2 were calculated. Six animals then underwent helical MDCT perfusion imaging during adenosine infusion. Prior to discontinuation of the adenosine infusion, neutron-activated microspheres were injected to document MBF during adenosine infusion. For each animal, by using the results of E and K1/k2 measurements, time registered helical MDCT myocardial attenuation data, and arterial input function data, myocardial CT value versus MBF tables were simulated for various MBF values to create look-up tables from the myocardial CT value to MBF. The results of helical MDCT-derived MBF were compared to microsphere MBF measurements. The extraction fraction in the dog as a function of flow (F) was E = (1 - exp(-(0.3396F + 0.3193)/F)). There was a strong linear association between the helical MDCT derived MBF and the microsphere MBF (y = 1.0648x - 0.6159, R2 = 0.8383). In conclusion, regional MBF can be measured accurately and noninvasively usin- - g the combination of bolus tracking and time-registered CT attenuation data from contrast enhanced helical MDCT scanning during adenosine stress. The helical CTA-derived K1 and MBF could be used to distinguish between the stenotic and remote territories under the condition of adenosine stress.