Oxidative stress implication after prolonged storage donor heart with blood versus crystalloid cardioplegia and reperfusion versus static storage.

Several factors are known to limit cardiac transplantation, such as number of donors, quality of cardiac graft preservation, and ischemia-reperfusion injury. Some mechanisms of reperfusion injury are now recognized; they include oxygen free radical (OFR), white blood cells activation, changes in calcium influx, alteration of microvascular blood flow, and sympathetic activation. The goal of this study was to assess the effects of two types of cardioplegia with long-term storage, either static or continuous perfusion, in 30 isolated sheep hearts as a model for heart transplantation. We examined myocardial function, histology, ischemic damage, and markers of oxidative stress. Two types of cardioplegia and storage conditions using a Langendorff reperfusion were studied in a combined approach: crystalloid (CP) [groups I and III] or cold oxygenated autologous blood (BC) [groups II and IV], immediate storage during 8h in profound hypothermia (groups I and II), or reperfused with crystalloid (group III), or blood cardioplegia (group IV). All perfusate samples were drawn from the coronary sinus. Lactate levels increased progressively in groups I, II, and IV, but not in group III, as no significant elevation was shown [90 min: 13.6+/-1.7 versus 5.2+/-1.0 mmol/L (P<0.01)]. Arrhythmias were more frequent when using BC (n=5) than CP (n=0). For plasma thiobarbituric acid-reactive substances (TBARS) levels a significant difference was found between group III and the other groups since 15 to 90 min (P<0.05). Vitamin E concentration decreased significantly from 5 min for groups II and IV, 15 min for group I, and 30 min for group III, with a significant difference between groups II and IV (P<0.05) but not between groups I and III. CP followed by a reperfusion with the same solution showed a significantly lower ischemic injury and OFR production, less frequent ventricular arrhythmias while stable hemodynamic parameters carried on. However, this protocol did not act on the early postoperative contractile function.

Oxidative stress implication in a new ex-vivo cardiac concordant xenotransplantation model.

Xenotransplantation (XT) reveals a growing interest for the treatment of cardiomyopathy. The major barrier is an acute vascular rejection due to an acute humoral rejection. This pathogenesis is a difficult issue and in order to elaborate means for its prevention, we analysed the implication of oxidative stress (OS) on hearts from mini-pigs followed by reperfusion with either autologous or human blood in an attempt to simulate xenotransplantation. About 14 hearts were studied after a Langendorff blood reperfusion: allografts with autologous blood (n = 7) or xenografts with human blood (n = 7). Blood samples were drawn from the coronary sinus to assess ischemia and OS. In xenografts, arrhythmias occurred more frequently (p < 0.01, left ventricular systolic pressure decreased more significantly (p < 0.05), thiobarbituric acid-reactive substances concentrations increased at 30 min (0.7 +/- 0.1 vs. 2.4 +/- 0.3 mmol/l; p < 0.05) while vitamin A levels decreased (p < 0.05). XT was associated with a significant increase in ischemic injury and OS production. OS might play an eminent role in hyperacute humoral rejection.

Activation of PAF receptor by oxidised LDL in human monocytes stimulates chemokine releases but not urokinase-type plasminogen activator expression.

BACKGROUND: We investigated whether the increase of urokinase-type plasminogen activator (uPA) monocyte expression and chemokine releases induced by oxidised low density lipoproteins (LDL), which participate to vascular tissue remodeling and to atherosclerotic plaque rupture, involved proinflammatory phospholipid products having platelet-activating factor (PAF)-like activity via the PAF-receptor pathway. METHODS: uPA monocyte expression was stimulated by either copper ions-oxidised or O2*-/HO* free radical-oxidised LDL. The effects of PAF and oxidised LDL on the production of monocyte chemoattractant protein-1 and interleukin-8 were also examined. RESULTS: Synthetic PAF significantly enhanced chemokine releases (P<0.001) without modifying uPA expression. Copper-oxidised LDL, which exhibit a higher content in lysophosphatidylcholines than free radical-oxidised LDL, induced a significantly higher enhancement in uPA expression (P<0.05). By contrast, free radical-oxidised LDL were more efficient than copper-oxidised LDL to increase chemokine releases (P<0.01). Oxidised LDL-enhanced uPA expressions were not altered by the PAF-receptor antagonist SR27417, whereas increases in chemokine releases induced by oxidised LDL and by PAF were abolished. PAF-acetylhydrolase activity was rapidly and largely inhibited in free radical-oxidised LDL when compared to copper-oxidised LDL, suggesting that free radical-oxidised LDL would contain a higher content in PAF-like products than copper-oxidised LDL. CONCLUSION: Our results indicated that PAF-like oxidation products are responsible for the monocyte chemokine releases, but did not contribute to the enhanced monocyte uPA expression by oxidised LDL.

A different approach to analyzing age-related HbA1c values in non-diabetic subjects.

Using appropriate statistical tests and taking into account the analytical performance of hemoglobin A1c (HbA1c) measurements, is it useful to establish HbA1c age-related values in non-diabetic subjects? Non-diabetic subjects (n=135, 72 women and 63 men) from the neuromuscular department of the Pitié-Salpétrière Hospital (Paris) were involved in our study. Subjects were divided into two groups related to age: 51 patients under 50 years old and 84 subjects aged 50 years or more. Fasting plasma glucose and HbA1c measurements were respectively performed by enzymatic assay using the hexokinase method and high-performance liquid chromatography based on the ion exchange methodology with high precision. We first checked the normality of HbA1c distribution using the Kolmogorov-Smirnov test. Then we compared mean HbA1c in the two age subgroups using the Student's t test. Mean HbA1c was significantly (p<0.0001) higher in the subgroup aged 50 years or more (mean HbA1c=5.2%) than in younger subjects (mean HbA1c=5.0%). Then plots were drawn to check the relationship between HbA1c and age. Under the hypothesis of linearity, determination coefficients (R2) were calculated. However, considering their low values, this hypothesis must be rejected and other factors than age must be retained to explain HbA1c variability.

Protection of endogenous beta-carotene in LDL oxidized by oxygen free radicals in the presence of supraphysiological concentrations of melatonin.

This study was designed to evaluate the effect of high concentrations of melatonin on the peroxidation of human low density lipoproteins (LDLs) initiated by O(2)(*-) and ethanol-derived peroxyl radicals (RO(2)(*)) from water gamma radiolysis in the presence of ethanol. LDL (3 g/l; total LDL concentration) was oxidized in the absence of melatonin or in its presence at three concentrations (50 x 10(-6), 100 x 10(-6) or 250 x 10(-6) mol/l) in ethanol. Radiolytic yields (i.e. number of mole consumed or produced per Joule) of the markers of lipid peroxidation were determined (i.e. decrease in the endogenous antioxidants alpha-tocopherol and beta-carotene, formation of conjugated dienes and of thiobarbituric acid-reactive substances [TBARS]). Melatonin decreased the yields of lipid peroxidation products and delayed the onset of the propagation phase for conjugated dienes and TBARS in a concentration-dependent manner. Nevertheless, melatonin did not protect endogenous alpha-tocopherol against peroxyl-induced oxidation (probably due to a lower scavenging capacity than that of alpha-tocopherol towards peroxyl radicals), but delayed the consumption of LDL endogenous beta-carotene and decreased its rate of disappearance. The effect of melatonin seemed to be the highest for a melatonin concentration of 250 x 10(-6) mol/l.

Melatonin related compounds inhibit lipid peroxidation during copper or free radical-induced LDL oxidation.

This study was designed to evaluate the protective effect of two melatonin related compounds towards low density lipoproteins (LDL) oxidation initiated in vitro either by defined free radicals [i.e. superoxide anion (O2*-) and ethanol-derived peroxyl radicals (RO(2)(*))] produced by gamma radiolysis or by copper ions. The compounds studied were N-[2-(5-methoxy-1H-indol-3-yl)ethyl]-3,5-di-tert-butyl-4-hydroxybenzamide (DTBHB) and (R,S)-1-(3-methoxyphenyl)-2-propyl-1,2,3,4-tetrahydro-beta-carboline (GWC20) which is a pinoline derivative. Their effects were compared with those of melatonin at the same concentration (100 micromol/L). None of the three tested compounds protected endogenous LDL alpha-tocopherol from oxidation by RO(2)(*)/O(2)(*)- free radicals. By contrast, they all protected beta-carotene from the attack of these free radicals with GWC20 being the strongest protector. Moreover, melatonin and DTBHB partially inhibited the formation of products derived from lipid peroxidation (conjugated dienes and thiobarbituric acid-reactive substances or TBARS) while GWC20 totally abolished this production. As previously shown, melatonin (at the concentration used) inhibited copper-induced LDL oxidation by increasing 1.60-fold the lag phase duration of conjugated diene formation over the 8 hr of the experimental procedure, however, DTBHB and GWC20 were much more effective, because they totally prevented the initiation of the propagation phase of LDL oxidation. It would be interesting to test in vivo if DTBHB and GWC20 which exhibit a strong capacity to inhibit in vitro LDL oxidation would reduce or not atherosclerosis in animals susceptible to this pathology.

In vitro low-density lipoprotein oxidation by copper or *OH/O*(2)(-): new features on carbonylation and fragmentation of apolipoprotein B during the lag phase.

The aim of our study was to evaluate the carbonylation and the carbonylated fragmentation of apolipoprotein B upon low-density lipoprotein (LDL) oxidation induced in vitro by copper and *OH/O*(2)(-) free radicals generated by gamma-radiolysis. Therefore, we developed a very sensitive Western blot immunoassay using 2,4-dinitrophenylhydrazine which allows the revelation of the apolipoprotein B carbonylation and its carbonylated fragmentation. The main results of this study show that (i) apolipoprotein B carbonylation is present during the lag phase of LDL oxidation in the two oxidative processes and (ii) apolipoprotein B carbonylated fragmentation was not detected during the lag phase of copper-oxidized LDL but was detected during the propagation phase. By contrast, apolipoprotein B carbonylated fragmentation was detected in the lag phase of *OH/O*(2)(-) oxidized LDL.