Rapid quantification of malondialdehyde in plasma by high performance liquid chromatography-visible detection.

BACKGROUND: Malondialdehyde (MDA) is one of the better-known secondary products of lipid peroxidation, and it is widely used as an indicator of cellular injury. The employment of the thiobarbituric acid reactive substances (TBARS) technique to measure MDA has received criticism over the years because of its lack of specificity. Thus, a specific and reliable method for MDA determination in plasma by high performance liquid chromatographic (HPLC)-VIS was validated; alkaline hydrolysis, n-butanol extraction steps and MDA stability were established. METHODS: The plasma underwent alkaline hydrolysis, acid deproteinization, derivatization with TBA and n-butanol extraction. After this, MDA was determined at 532 nm by HPLC-VIS. The method was applied to 65-year-old subjects from a retirement home. RESULTS: The assay was linear from 0.28 to 6.6 microM. The reproducibility of intra-run was obtained with CV%<4% and the inter run with CV%<11%. The accuracy (bias) ranged from 2 to -4.1%, and the recovery was greater than 95%. The limit of detection (LOD) and limit of quantification (LOQ) were 0.05 and 0.17 microM, respectively. For the stability test, every sample was stored at -20 degrees C. The plasma MDA was not stable when stored after the alkaline hydrolysis step, remained stable for 30 days after TBA derivatization storage and was stable for 3 days when stored after n-butanol extraction. The elderly subjects had MDA plasma levels of 4.45+/-0.81 microM for women and 4.60+/-0.95 microM for men. CONCLUSION: The method is reproducible, accurate, stable, sensitive, and can be used in the routines in clinical laboratories. Besides, this technique presents advantages such as the complete release of protein bound MDA with the alkaline hydrolysis step, the removal of interferents with n-butanol extraction, mobile phase without phosphate buffer and rapid analytical processes and run times.

Comparison of a beta-blocker and an If current inhibitor in rabbits with myocardial infarction.

AIM: We compared protective effects of a ss-adrenoceptor blocker (metoprolol; Met) and a If current (Ivabradine; Iva) in a rabbit model of myocardial infarction. METHODS: Experiments were performed on 44 adult New-Zealand-White (NZW) rabbits. The effects of either metoprolol or ivabradine were assessed 15 min after experimental occlusion of a coronary artery (CAO), 28 days after CAO (drug gavage), and in vitro hearts (Langendorff apparatus). The results were compared with sham and placebo hearts. RESULTS: Metoprolol (0.25 mg/kg) slightly reduced heart rate and left ventricular systolic function. Ivabradine (0.25 mg/kg) reduced heart rate significantly (P<0.05) (18% vs control). Both drugs provided advantages over placebo: mortality was significantly (P<0.01)smaller (6/13 Pla animals died, 2/10 Met animals, and 3/11 Iva animals), left ventricular function was better preserved after 28 days (external power; Pla; Met; Iva=56%; 76%; 74%), and dilatation (BNP) was reduced (P<0.05). In the Pla group, the ST segment was significantly (P<0.05) elevated by 0.35 mV after CAO and exhibited in 50% of the animals Q waves after 28 days, while after ivabradine or metoprolol, ST displacement and Q waves had disappeared. The uneconomic myosin isoenzyme V3 predominated in Met hearts and Iva hearts (V3/V1: 63/37% and 62/38%), while it was further increased in Pla hearts (78/21%). External efficiency was lowest in Pla hearts (1.00+/-0.50 a.u.; P<0.05) and was significantly higher both in Met hearts (4.0+/-1.8 a.u.) and in Iva hearts (3.3+/-1.6 a.u.). CONCLUSIONS: Met and Iva seem suited for the treatment of chronic myocardial infarction.

Electrophoretic separation of ventricular myosin isoenzymes using a native polyacrylamide minigel system.

A method is presented to separate rabbit cardiac ventricular myosin isoenzymes (V(1), V(2), V(3)), which are large and important contractile proteins. This polyacrylamide gel electrophoresis--using a slab minigel format--does not involve preparation of an acrylamide gradient or denaturing conditions. The isoenzyme migration order was confirmed through identification with an anti beta-myosin heavy chain in cardiac ventricles (i.e., V(3)) antibody. Extracts from atrial and soleus muscle were used as positive control for V(1) and V(3), respectively. The relative quantification was obtained densitometrically and analyzed via TINA/Software. The reproducibility of method was additionally tested. The procedure employs Coomassie blue staining and is rapid and reproducible. Thus, the method permits easy and economic analysis of myosin isoenzymes under native conditions.

Effects of a bradycardic agent on postischemic cardiac recovery in rabbits.

Decreasing heart rate might be beneficial for improvement of myocardial energetics and could reduce the severity of myocardial ischemia. We examined the contribution of heart rate reduction by cilobradine (DK-AH 269), a direct sinus node inhibitor, on left ventricular function and peripheral vasomotion in anesthetized rabbits with experimental myocardial infarction. The rabbits were randomized to receive either placebo (n=10) or cilobradine (n=7). Cilobradine decreased significantly heart rate from 163 +/- 33 to 131 +/- 13 bpm, p< 0.05, without any inotopic or vascular effects. After 60 min coronary occlusion and 30 min reperfusion, both systolic and diastolic ventricular function were more reduced in the cilobradine group; i.e. maximal left ventricular pressure significantly decreased to 62 +/- 11 mmHg, p < 0.05 (placebo: 77 +/- 9 mmHg); dP/dt(min) significantly decreased to -904 +/- 247 mmHg, p < 0.05 (placebo: -1106 +/- 242 mmHg). However, infarct size in the cilobradine group was significantly smaller compared with the placebo group. In conclusion, cilobradine reduced heart rate without any negative inotropic effect and reduced infarct size. On that account, this bradycardic agent might open a promising therapeutical avenue to treat postischemic dysfunction.

Reduction in beta-myosin heavy chains in stunned myocardium as assessed by nondenaturing gel electrophoresis.

Myosin plays a key role in the structure and function of cardiac muscle. Three myosin isoenzymes (V(1), V(2), and V(3)) with different ATPase activities have been identified in mammalian ventricles based on their heavy chain constituents. The relative amount of myosin isoenzymes changes under physiological and pathological conditions. Until now, myosin isoenzymes have frequently been determined using either tube gel (nondenaturing) polyacrylamide gel electrophoresis (PAGE), or gradient or uniform sodium dodecyl sulfate (denaturing) PAGE. Both methods have disadvantages, e.g., a long running time. We developed, therefore, a uniform, nondenaturing PAGE with slab minigel format for analyzing the myosin isoenzymes in normoxic and stunned rabbit hearts. In normoxic hearts of adult rabbits, V(3) predominated over V(1) (46 vs 41%). In turn, in the stunned hearts, V(1) predominated over V(3) (70 vs 30%), and the heterodimeric V(2) was not anymore detectable. This alteration appears to result from a selective loss of myosin heavy chain (MHC)-beta. In parallel, the biochemical markers troponin I and creatine kinase were increased in the stunned hearts. We suggest that alterations of myosin isoenzymes in stunned myocardium can be monitored with native PAGE. The present analysis of myosin isoenzyme appears thus as a new tool for evaluating defects in MHC dimer formation in postischemic hearts.