Real-time measurement of nitric oxide by luminol-hydrogen peroxide reaction in crystalloid perfused rat heart

The objective of this study was to develop an assay system that allows continuous monitoring of nitric oxide (NO) released from crystalloid perfused hearts. We utilized chemiluminescence reaction between NO and luminol-H(2)O(2) to quantify the NO level in coronary effluent. Isolated rat hearts were subjected to ordinary Langendorff's perfusion, and the right ventricle was cannulated to sample coronary effluent. After equilibration, the coronary flow rate was set constant and the hearts were paced at 300 bpm. Coronary effluent was continuously sampled and mixed with the chemiluminescent probe containing 0.018 mmol/l luminol plus 10 mmol/l H(2)O(2). Chemiluminescence from the mixture of coronary effluent and the probe was continuously measured. NO concentration was calibrated by various concentrations (0.5-400 pmol/l) of standard NO solution. The lower detection limit of NO was 1 pmol/l. Basal NO release from isolated perfused rat heart was 0.41 +/- 0.17 pmol/min/g of heart weight, and that was significantly suppressed by 0.1 mmol/l of L-NAME to 0.18 +/- 0.10 pmol/min/g of heart weight (n = 7). Application of 0.1 and 0.3 micromol/l acetylcholine increased NO level in the coronary effluent, in a concentration-dependent manner, from 6.6 +/- 1.7 in a baseline condition to 16.3 +/- 7.4 and 30.3 +/- 16.1 pmol/l at each peak, respectively. Thrombin at 1 and 10 U/ml also increased NO level from 17.6 +/- 4.3 in control to 35.5 +/- 10.4 and 48.7 +/- 8.7 pmol/l at each peak, respectively (n = 7). Thus, this assay system is applicable to the continuous real-time measurement of NO released from crystalloid perfused hearts, and it may be useful for the study of physiological or pathophysiological role of NO in coronary circulation.