Parinam S. Rao

Role of xanthine oxidase inhibitor as free radical scavenger: A novel mechanism of action of allopurinol and oxypurinol in myocardial salvage

Xanthine oxidase (XO) has been hypothesized to be a potential source of oxygen-derived free radicals during reperfusion of ischemic myocardium based on the fact that allopurinol, a XO-inhibitor, can reduce reperfusion injury. In this communication we report that both allopurinol and oxypurinol, the principle metabolite of allopurinol, prevent the reperfusion injury in isolated pig heart. However, we found that neither pig heart nor pig blood contain any XO activity. Our study showed a direct free radical scavenging action of these XO-inhibitors during ischemia and reperfusion, as judged by the reduction of free radical signals when compared using an Electron Paramagnetic Resonance Spectrometer. Using a Luminometer, we also confirmed that both allopurinol and oxypurinol can scavenge ClO2, HOCl, and significantly inhibit free radical signals generated by activated neutrophils. These XO-inhibitors, however, failed to scavenge O2. and OH. radicals. Our results suggest that these XO-inhibitors salvaged the ischemic-reperfused myocardium by scavenging free radicals, and not by inhibiting XO in the pig heart.

Sustain inhibition of nitric oxide by NG-nitro-l-arginine improves myocardial function following ischemia/reperfusion in isolated perfused rat heart

It has been postulated nitric oxide (NO) can react with superoxide anion (·O − 2 ) to generate hydroxyl (·OH) radical. If this is correct inhibition of NO synthesis could attenuate ·OH radical mediated ischemia/reperfusion injury. Therefore we studied the effects of N G -nitro- l -arginine ( l -NNA), a competitive inhibitor of the NO synthase enzyme on ischemia/reperfusion injury in isolated perfused rat hearts. Three groups of rats ( n = 12–15) were studied. Group I: Untreated ischemia/reperfusion control (37.5 min of global ischemia follows by 20 min reperfusion); Group II: ischemia/reperfusion with 25μm N G -nitro- l -arginine; and Group III: ischemia/reperfusion in the presence of l -NNA and 2m m l -arginine, the substrate for NO synthase. Coronary flow (in ml/min) and ventricular developed pressure +dP/dt and -dP/dt were measured 5 min prior to ischemia and at the end of reperfusion. Baseline preischemic developed pressure was significantly lower in l -NNA perfused hearts than controls (76.8±5.9 v 97.6±2.9 mmHg, P l -NNA perfused hearts (57.4±7.4 v 20.8±6.4 mmHg in control). This protective effect was reversed by the addition of l -arginine. Preischemic coronary flow was decreased significantly in the l -NNa group (6.4±0.5ml/min) compared to controls (11.6±0.7ml/min). The duration of sinus rhythm was significantly improved from 3.8±1.2 min in controls to 15.1±0.8 min in l-NNA perfused hearts. A corresponding significantly lower incidence of arrhythmias was observed (10.2±1.5 in ischemia/reperfusion group v 1.7±0.8 min with l -NNA). Again, hearts perfused with l -NNA plus l -arginine had more arrhythmias and a shorter duration of sinus rhythm. The results show that despite the reduction of myocardial contractility and coronary flow prior to ischemia l -NNA significantly preserved myocardial contractility and reduced arrhythmias following reperfusion. Electron spin resonance or cytochrome c reduction assay demonstrated that l -NNA did not scavenge ·OH nor ·O − 2 radical directly. These results suggest that ischemia/reperfusion injury observed in this model may in part be due to ·OH radical formed as a result of NO interaction with ·O − 2 and that inhibition of this pathway by l -NNA leads to recovery of myocardial function.

High-performance liquid chromatographic detection of hydroxylated benzoic acids as an indirect measure of hydroxyl radical in heart: its possible link with the myocardial reperusion injury

The present report describes a method suitable for the indirect assay of hydroxyl radical (OH.), which is likely to be produced during reperfusion of ischemic myocardium. Isolated rat heart perfused by the Langendorff technique was subjected to 30 min of ischemia, followed by 30 min of reperfusion. Salicylic acid (2 mM) was added to the perfusion circuit to trap any OH. radical generated during the experiment. 2,5- and 2,3-dihydroxybenzoic acids (hydroxylated products of salicylic acid) were identified by authentic standards as well as by pure OH.-generating system using high-performance liquid chromatography with electrochemical detection. In addition to serving as a chemical trap for the detection of OH., salicylate attenuated myocardial reperfusion injury as evidenced by reduced formation of creatine kinase, decreased lipid peroxidation, and improved myocardial contractile functions during reperfusion. These results thus provide direct evidence for the presence of OH. in heart and link it to the myocardial reperfusion injury.

Serial changes in renal function in cardiac surgical patients

Cardiopulmonary bypass is widely believed to be injurious to renal function. The low incidence of renal dysfunction with modern techniques of bypass led us to reexamine this concept by monitoring urine output and creatinine clearance in 18 adult patients undergoing nonpulsatile, hemodilution cardiopulmonary bypass for coronary artery bypass grafting (12 patients) or valve procedures (6 patients). Samples were taken before, during (mean duration of bypass, 105 +/- 26 minutes [+/- standard deviation]), and every two hours after bypass for 24 hours. Urine output (42 +/- 37.7 mL/h) and creatinine clearance (57 +/- 40.4 mL/min) were surprisingly low in the period before cardiopulmonary bypass (all values normalized to a body surface area of 1.73 m2). Urine volumes rose to 305 +/- 149.6 mL/h and creatinine clearance to 252 +/- 176.9 mL/min during bypass and decreased to stable values after eight hours in the postoperative unit (urine output, approximately 60 mL/h, and creatinine clearance, approximately 75 mL/min). Renal dysfunction did not develop in any patient. Nine patients who required loop diuretics for low urine output 18 hours postoperatively had a sustained increase in both urine output and creatinine clearance lasting up to six hours. We conclude the following: modern techniques of cardiopulmonary bypass are not injurious to renal function; urine output and creatinine clearance are decreased before cardiopulmonary bypass, probably because of preoperative dehydration; and loop diuretics in the postoperative period increase both urine output and creatinine clearance for as long as six hours after administration.

A specific sensitive HPLC method for determination of plasma dopamine

SummaryWe describe here a sensitive, selective and rapid method to quantitate plasma catecholamines, especially dopamine, using high-performance liquid chromatography with electrochemical detection. This method requires a 10-minute run time and has a threshold for detection of 2 picograms, (10pg/ml).A number of commonly employed mobile phases for catecholamine analysis have been tested and have failed to detect dopamine in biological samples. Neither acetonitrile (3–7%) or methanol, (5–8%) in the mobile phase has produced consistently interpretable data either due to inability to detect or interference from co-eluting substances. Optimal detection was achieved with a mobile phase containing sodium acetate (6.8g), citric acid (5.9g), EDTA (48mg), di-n-butylamine (270μl), Na-1-octane sulfate (850mg), methanol (100 ml) (amounts refer to 1 liter aqueous solution) (pH 4.3). The mobile phase was passed through a Waters 5μ resolve C18 column using a Waters 590 pump and m460 electrochemical detector and 740 data module, Flow rate was 0.9ml/min. Using this method, normal values in human and swine left ventricular myocardium and human and swine plasma have been established for norepinephrine, epinephrine, and dopamine.

Isocratic high-performance liquid chromatography-photodiode-array detection method for determination of lysine- and arginine-vasopressins and oxytocin in biological samples

A simple, isocratic, sensitive (1 ng), and specific high-performance liquid chromatographic (HPLC) method based on photodiode-array detection (PAD) is described for simultaneous quantitation of the bioactive peptides, lysine vasopressin (LVP), arginine vasopressin (AVP) and oxytocin (OXY). Acidified pig plasma and left ventricular (LV) tissue samples were first extracted with Sep-Pak C18 columns, and the bioactive peptides were eluted with methanol, then dried at 37 degrees C and reconstituted with HPLC mobile phase. The bioactive peptides were separated by HPLC on a Dynamax 3009-A C8 column with a mobile phase of 0.1% trichloroacetic acid-50 mM heptanesulfonic acid-30mM triethylamine-20% acetonitrile in water, pH 2.5 and identified with a Waters 990-PAD system (spectrum index plots in the range 200-400 nm). Standards of LVP, AVP and OXY and their mixtures showed a linear increase in the range 5 to 100 ng and were eluted at 6.1, 6.9 and 4.6 min, respectively. Spectrum analysis showed a distinct absorption peak at 280 nm, corresponding to peptide bonds. The reproducibility of the method coefficient of variation for standards is 6.9, 5.8 and 4.7% for LVP, AVP and OXY, respectively. In plasma and tissue it is much higher: 12.9% (LV tissue) and 18.6% (plasma) for LVP. Pig plasma contains negligible amounts of AVP and OXY; LVP is much higher (0.28 +/- 0.19 ng/ml). In pig tissue, LVP predominates (6.95 ng/g wet weight) compared to AVP (1.45) and OXY (1.50). Spectral analysis is necessary to identify the bioactive peptide peaks among interfering substances and to increase the sensitivity four-fold. The method described here is useful for the simultaneous determination of LVP, AVP and OXY in the nanogram range and can be extended to picogram levels by employing PAD spectral analysis techniques.

Reduction of myocardial injury with verapamil before aortic cross-clamping

The effect of verapamil administered before aortic cross-clamping was assessed in 40 patients undergoing elective coronary artery bypass grafting. Myocardial protection consisted of cold blood potassium cardioplegia, topical ice slush, and moderate (28 degrees C) systemic hypothermia. Patients were randomly divided into two groups: group 1 (18 patients) received verapamil (0.1 mg/kg up to 10 mg) intravenously three to five minutes before aortic cross-clamping; group 2 (22 patients) did not (control). Myocardial injury was assessed by cumulative release of the cardiac-specific isoenzyme of creatine kinase (CK-MB) after release of the aortic cross-clamp. Release of CK-MB was significantly lower in the verapamil group (44.9 +/- 6.2 versus 72.2 +/- 9.0 IU at 24.5 hours, p = 0.005). Calculated total infarct size was also lower in the verapamil group (6.0 +/- 0.9 versus 8.9 +/- 1.0 g-Eq, p = 0.035). Individual CK-MB release curves showed either one or two peaks. The two-peak pattern was more frequent in control patients (18 of 21 control patients versus 6 of 18 verapamil patients, p = 0.001) and was associated with a larger infarct size. Atrioventricular pacing was not required in any verapamil patient, but was needed in 1 control patient. We conclude that verapamil administered before aortic cross-clamping protects against myocardial injury during coronary artery bypass grafting with no increase in the incidence of atrioventricular block.

High-performance liquid chromatographic method for the direct quantitation of oxy radicals in myocardium and blood by means of 1,3-dimethylthiourea and dimethyl sulfoxide

A direct, sensitive (50-200 ng), simple and specific high-performance liquid chromatographic (HPLC) method is described for the quantitation of oxy radicals by means of the consumption of dimethyl sulfoxide (DMSO) by the hydroxy radical and dimethylthiourea (DMTU) by hydrogen peroxide. The specific scavengers catalase and L-methionine were used to quantitate hydrogen peroxide and OH, respectively. The DMSO and DMTU peaks were separated and identified by HPLC on a Waters C18 Resolve 10-microns Radial-Pak column with an isocratic mobile phase (5% aqueous methanol) at 2 ml/min with UV detection (DMSO, 214 nm; DMTU, 240 nm). The OH concentrations were extrapolated by a luminol chemiluminescence technique. A linear relationship was obtained for DMTU consumption by hydrogen peroxide in the range 0.25-0.40 mM with a coefficient of variation (C.V.) of 8.8 +/- 2.1% and for DMSO consumption by hydroxy radicals in the range 0.1-3.2 microM OH, with a C.V. of 9.6 +/- 3.6%. The limits of detection for this method were 50 ng of hydrogen peroxide for DMTU and 200 ng of OH for DMSO. Hydrogen peroxide averaged 10.5 +/- 3.6 nmol/ml in blood and 56.4 +/- 5.3 mumol/g wet weight in left ventricular (LV) tissue. The hydroxy radical concentration was 0.1 microM in blood and 0.3 microM in LV tissue.

Myocardial salvage by the free radical scavenging actions of xanthine oxidase inhibitors.

It is well established that oxygen-derived free radicals play a crucial role in the pathogenesis of ischemic-reperfusion injury.1 However, the source of these radicals remains unknown. McCord2 has proposed that oxygen radicals are generated during reoxygenation of ischemic myocardium by the action of xanthine oxidase on hypoxanthine. A number of investigators supported this hypothesis based on their observations of allopurinol-mediated reduction of reperfusion injury.3 Recently, the beneficial action of allopurinol to salvage ischemic-reperfused myocardium has been challenged by Reimer et al.4 In addition, most of the mammalian hearts, with the exception of rat heart, have been found to contain negligible or no xanthine oxidase activity at all.5 The purpose of the present study was, therefore, to investigate the role of two xanthine oxidase inhibitors, allopurinol and oxypurinol, on the ischemic-reperfused heart and to explore their mechanisms of action. Our results suggest that these xanthine oxidase inhibitors salvage the ischemic-reperfused myocardium by scavenging the free radicals.