Methylglyoxal induces cardiac dysfunction through mechanisms involving altered intracellular calcium handling in the rat heart.

Methylglyoxal (MGO) is an endogenous, highly reactive dicarbonyl metabolite generated under hyperglycaemic conditions. MGO plays a role in developing pathophysiological conditions, including diabetic cardiomyopathy. However, the mechanisms involved and the molecular targets of MGO in the heart have not been elucidated. In this work, we studied the exposure-related effects of MGO on cardiac function in an isolated perfused rat heart ex vivo model. The effect of MGO on calcium homeostasis in cardiomyocytes was studied in vitro by the fluorescence indicator of intracellular calcium Fluo-4. We demonstrated that MGO induced cardiac dysfunction, both in contractility and diastolic function. In rat heart, the effects of MGO treatment were significantly limited by aminoguanidine, a scavenger of MGO, ruthenium red, a general cation channel blocker, and verapamil, an L-type voltage-dependent calcium channel blocker, demonstrating that this dysfunction involved alteration of calcium regulation. MGO induced a significant concentration-dependent increase of intracellular calcium in neonatal rat cardiomyocytes, which was limited by aminoguanidine and verapamil. These results suggest that the functionality of various calcium channels is altered by MGO, particularly the L-type calcium channel, thus explaining its cardiac toxicity. Therefore, MGO could participate in the development of diabetic cardiomyopathy through its impact on calcium homeostasis in cardiac cells.

Ticagrelor Prevents Endothelial Cell Apoptosis through the Adenosine Signalling Pathway in the Early Stages of Hypoxia.

BACKGROUND: Several studies have reported the beneficial effects of anti-platelet drugs in cardioprotection against ischaemia-reperfusion injuries. To date, no studies have focused on the indirect cytoprotective effects of ticagrelor via adenosine receptor on the endothelium. METHOD: By evaluating cell viability and cleaved caspase 3 expression, we validated a model of endothelial cell apoptosis induced by hypoxia. In hypoxic endothelial cells treated with ticagrelor, we quantified the extracellular concentration of adenosine, and then we studied the involvement of adenosine pathways in the cytoprotective effect of ticagrelor. RESULTS: Our results showed that 10 µM ticagrelor induced an anti-apoptotic effect in our model associated with an increase of extracellular adenosine concentration. Similar experiments were conducted with cangrelor but did not demonstrate an anti-apoptotic effect. We also found that A2B and A3 adenosine receptors were involved in the anti-apoptotic effect of ticagrelor in endothelial cells exposed to 2 h of hypoxia stress. CONCLUSION: we described an endothelial cytoprotective mechanism of ticagrelor against hypoxia stress, independent of blood elements. We highlighted a mechanism triggered mainly by the increased extracellular bioavailability of adenosine, which activates A2B and A3 receptors on the endothelium.

Complementary Role of P2 and Adenosine Receptors in ATP Induced-Anti-Apoptotic Effects Against Hypoxic Injury of HUVECs.

BACKGROUND: Vascular endothelial injury during ischemia generates apoptotic cell death and precedes apoptosis of underlying tissues. We aimed at studying the role of extracellular adenosine triphosphate (ATP) on endothelial cells protection against hypoxia injury. METHODS: In a hypoxic model on endothelial cells, we quantified the extracellular concentration of ATP and adenosine. The expression of mRNA (ectonucleotidases, adenosine, and P2 receptors) was measured. Apoptosis was evaluated by the expression of cleaved caspase 3. The involvement of P2 and adenosine receptors and signaling pathways was investigated using selective inhibitors. RESULTS: Hypoxic stress induced a significant increase in extracellular ATP and adenosine. After a 2-h hypoxic injury, an increase of cleaved caspase 3 was observed. ATP anti-apoptotic effect was prevented by suramin, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), and CGS15943, as well as by selective A2A, A2B, and A3 receptor antagonists. P2 receptor-mediated anti-apoptotic effect of ATP involved phosphoinositide 3-kinase (PI3K), extracellular signal-regulated kinases (ERK1/2), mitoKATP, and nitric oxide synthase (NOS) pathways whereas adenosine receptor-mediated anti-apoptotic effect involved ERK1/2, protein kinase A (PKA), and NOS. CONCLUSIONS: These results suggest a complementary role of P2 and adenosine receptors in ATP-induced protective effects against hypoxia injury of endothelial. This could be considered therapeutic targets to limit the development of ischemic injury of organs such as heart, brain, and kidney.

Drowsiness and uncommon fever in a child after cannabis ingestion.

Trivialization of cannabis consumption goes hand in hand with a growing exposure of children and the number of cannabis poisoning cases is steadily increasing. As clinical presentation can be different from what is currently seen in adults, added to the fact that it is not always suspected, diagnosis of cannabis intoxication in children is often delayed or missed. A 16-month-old girl was admitted to the pediatric emergency unit for an important drowsiness combined to moderate fever. After elimination of infectious causes, a toxic origin was considered and biological analyses led to the diagnosis of involuntary acute cannabis intoxication. In conclusion, cannabis intoxication in child has uncommon presentations compared to that seen in adults. In this context, biological analyses have a great importance for a rapid diagnosis and also for the understanding intoxication circumstance. This is of paramount importance because it may lead to consider child protection measures.

Therapeutic drug monitoring of mitotane: Analytical assay and patient follow-up.

Adrenocortical carcinoma (ACC) is an aggressive malignancy of the adrenal gland. Mitotane (o,p'-DDD) is the most effective chemotherapy for ACC. According to the literature, mitotane plasma trough concentrations within 14-20 mg L-1 are correlated with a higher response rate with acceptable toxicity. Therapeutic drug monitoring (TDM) of mitotane is therefore recommended. The aim of this study was to propose a robust and simple method for mitotane quantification in plasma. The validation procedures were based on international guidelines. Sample preparation consisted of a single protein precipitation with methanol using 100 µL of plasma. The supernatant was submitted to liquid chromatography coupled with ultra-violet detection at 230 nm. Mitotane retention time was 7.1 min. The limit of detection was 0.1 mg L-1 and the limit of quantification was 0.78 mg L-1 . The assay demonstrated a linear range of 0.78-25 mg L-1 with correlation coefficients (r2 ) at 0.999. Inter- and intra-assay precision was <4.85%. Evaluation of accuracy showed a deviation <13.69% from target concentration at each quality control level. This method proved easy and rapid to perform mitotane TDM and required a small volume of sample. It was successfully applied to routine TDM in our laboratory.

False negative on a proven involuntary intoxication in two children with lorazepam.

Children aged between 1 to 4 years are the most at risk of unintentional poisonings. Benzodiazepines are the most medicine often cause of the poisoning. Among the twenty-two most prescribed benzodiazepines in France, lorazepam ranks fifth behind zolpidem, alprazolam, bromazepam and zopiclone. However the automated assay currently available does not allow to detect and/or to quantify lorazepam. The alternative to the immunoassay is the liquid chromatography coupled with mass spectrometry (HPLC/MS). This technique, highly sensitive and specific, requires a pre-treatment phase and a good technical proficiency, justifying specialized staff. The clinical cases presented here illustrate the major interest of availability to this type of technology in routine and 24h/24h.

Current knowledge on the role of P2Y receptors in cardioprotection against ischemia-reperfusion.

During ischemia, numerous effective endogenous extracellular mediators have been identified, particularly, nucleosides such as adenosine as well as purinergic and pyrimidinergic nucleotides. They may play important regulatory roles within the cardiovascular system and notably as cardio-protectants. Indeed, the distribution of the P2Y receptors in mammalian heart includes several cellular constituents relevant for the pathophysiology of myocardial ischemia. Beside the well-known cardioprotective effect of adenosine, the additional protective role of P2Y receptors has emerged. However, interpretation of experimental results may be sometimes perplexing. This is due to the variability of: the experimental models, the endpoints criteria, the chemical structure of agonist and antagonist ligands and their concentrations, the sequences of drug administration with respect to the model used (before and/or during and/or after ischemia). The net effect may be in the opposite direction after a transient or a prolonged stimulation. Nevertheless, the overall reading of published data highlights the beneficial role of the P2Y2/4 receptor stimulation, the useful and synergistic role of P2Y6/11 receptor activation and even of the P2Y11 receptor alone in cardioprotection. More, the P2Y11 receptor could be involved in counter-regulation of profibrotic processes. Paradoxically, transient P2X7 receptor stimulation could contribute to the net cardioprotective effect of ATP. Recently, experimental data have shown that blocking the P2Y12 receptor after ischemia confers cardioprotection independently of platelet antiaggregatory effect. This suggests for P2Y receptors an important role in primary prevention and as a therapeutic target in myocardial protection during ischemia and reperfusion.

Homocitrulline as marker of protein carbamylation in hemodialyzed patients.

BACKGROUND: Homocitrulline (HCit) is a carbamylation-derived product (CDP) that has been identified as a valuable biomarker of morbidity and mortality in patients with chronic kidney disease (CKD). The aim of this study was to determine whether initiation of hemodialysis therapy (HD) could induce variations of HCit concentrations in CKD patients. METHODS: Serum HCit concentrations were determined by LC-MS/MS in CKD patients (n=108) just before (M0) and six months (M6) after the initiation of HD therapy. RESULTS: Mean HCit concentrations reached 1000µmol/mol Lysine before initiation of HD therapy and decreased by 50% within 6months after HD onset. HCit concentrations remained stable over time as assessed during a 24-months follow-up period. HCit was mostly found in its protein-bound form in HD patients. HCit concentrations obtained at M0 were positively correlated with urea (r=0.58) and carbamylated hemoglobin (r=0.41), and are likely to be promising predictive markers of mortality. However, no correlations were found between HCit concentrations and Kt/V values, suggesting that HCit is not a marker of HD efficiency. CONCLUSION: HCit concentrations reflect the intensity of protein carbamylation and are stable over time during HD treatment, making HCit a reliable biomarker in the follow-up of CKD patients.

Analytical interference in the therapeutic drug monitoring of methotrexate.

High-dose of methotrexate chemotherapy is used in the treatment of some tumors. It presents several side effects that required therapeutic drug monitoring, which is commonly performed on 24, 48 and 72h after the beginning of the methotrexate infusion. Treatment of overexposure to methotrexate is based on injection of carboxypeptidase G2, which specifically degrades methotrexate into inactive metabolite: DAMPA. FPIA immunoassay on TDx automated analyzer (Abbott™) was used for therapeutic drug monitoring of methotrexate. This immunoassay presented a significant cross-reactivity between methotrexate and DAMPA, which widely overestimate the residual concentration compared to the gold standard HPLC/MS. TDx automated analyzer was substituted by a new immunoassay on Architect automated analyzer (Abbott™). However, this immunoassay has the same cross-reactivity, which needs to be careful when monitoring methotrexate after an injection of carboxypeptidase G2. In order to determine the most suitable assay for the therapeutic drug monitoring of methotrexate, the knowledge of injection of carboxypeptidase G2 remains essential.

Validation of a fast UPLC-MS/MS method for quantitative analysis of opioids, cocaine, amphetamines (and their derivatives) in human whole blood.

BACKGROUND: Conventional methods for analysis of drugs of abuse require multiple assays which can be both expensive and time-consuming. This work describes a novel, rapid, simple and sensitive method for the quantification of 14 illicit drugs and their metabolites in whole blood. Results/methodology: This method employed a rapid liquid-liquid sample extraction of whole blood followed by UPLC-MS/MS analysis. Calibration curves were validated for analysis of appropriate concentrations. Inter- and intra-assay variations were <14.8%. Deviation of accuracy was <14.9% from target concentration for each quality control level. CONCLUSION: This work described the development and the full validation of a precise, sensitive and accurate assay. After validation, this new assay was successfully applied to routine toxicological analysis.

[Clinical and analytical toxicology of opiate, cocaine and amphetamine]. / Toxicologie clinique et analytique des opiacés, de la cocaïne et des amphétamines.

In several circumstances, determination and quantification of illicit drugs in biological fluids are determinant. Contexts are varied such as driving under influence, traffic accident, clinical and forensic toxicology, doping analysis, chemical submission. Whole blood is the favoured matrix for the quantification of illicit drugs. Gas chromatography coupled with mass spectrometry (GC-MS) is the gold standard for these analyses. All methods developed must be at least equivalent to gas chromatography coupled with a mass spectrometer. Nowadays, new technologies are available to biologists and clinicians: liquid chromatography coupled with a mass spectrometry (LC/MS) or coupled with a tandem mass spectrometer (LC/MS/MS). The aim of this paper is to describe the state of the art regarding techniques of confirmation by mass spectrometry used for quantification of conventional drugs except cannabis.

Population pharmacokinetics of nefopam in elderly, with or without renal impairment, and its link to treatment response.

AIMS: Nefopam is a nonmorphinic central analgesic, for which no recommendation exists concerning adaptation of regimen in aged patients with or without renal impairment. The objective was to describe the pharmacology of nefopam in aged patients to obtain guidelines for practical use. METHODS: Elderly patients (n = 48), 65-99 years old, with severe or moderate renal impairment or with normal renal function, were recruited. Nefopam (20 mg) was administered as a 30 min infusion postoperatively. Simultaneously, a 1 min intravenous infusion of iohexol was performed, in order to calculate the glomerular filtration rate. Blood samples were drawn to determine nefopam, desmethyl-nefopam and iohexol plasma concentrations. Nefopam and desmethyl-nefopam concentrations were analysed using a nonlinear mixed-effects modelling approach with Monolix version 4.1.3. The association between pharmacokinetic parameters and treatment response was assessed using logistic regression. RESULTS: A two-compartment open model was selected to describe the pharmacokinetics of nefopam. The typical population estimates (between-subject variability) for clearance, volume of distribution, intercompartmental clearance and peripheral volume were, respectively, 17.3 l h(-1) (53.2%), 114 l (121%), 80.7 l h(-1) (79%) and 208 l (63.6%). Morphine requirement was related to exposure of nefopam. Tachycardia and postoperative nausea and vomiting were best associated with maximal concentration and the rate of increase in nefopam plasma concentration. CONCLUSIONS: We identified the nefopam pharmacokinetic predictors for morphine requirement and side-effects, such as tachycardia and postoperative nausea and vomiting. In order to maintain morphine sparing and decrease side-effects following a single dose of nefopam (20 mg), simulations suggest an infusion time of >45 min in elderly patients with or without renal impairment.

Validation of a fast method for quantitative analysis of elvitegravir, raltegravir, maraviroc, etravirine, tenofovir, boceprevir and 10 other antiretroviral agents in human plasma samples with a new UPLC-MS/MS technology.

Therapeutic drug monitoring (TDM) of antiretrovirals requires accurate and precise analysis of plasma drug concentrations. This work describes a simple, fast and sensitive UPLC-MS/MS method for determination of the commonly used protease inhibitors such as amprenavir, atazanavir, darunavir, indinavir, lopinavir, ritonavir, saquinavir and tipranavir, tenofovir a nucleoside reverse transcriptase inhibitor (NRTI), the non-NRTI such as efavirenz, nevirapine, etravirine, the CCR5 antagonist maraviroc as well as the more recent antiretrovirals, the integrase inhibitors such as raltegravir, elvitegravir and the new direct acting anti-HCV boceprevir. Adapted deuterated internal standard was added to plasma aliquots (100µl) prior to protein precipitation with methanol and acetonitrile. This method employed ultra-performance liquid chromatography coupled to tandem mass spectrometry with electrospray ionization mode. All compounds eluted within 4.2-min run time. Calibration curves were validated, with correlation coefficients (r(2)) higher than 0.997, for analysis of therapeutic concentrations reported in the literature. Inter- and intra-assay variations were <15%. Evaluation of accuracy shows a deviation <15% from target concentration at each quality control level. No significant matrix effect was observed for any of the antiretroviral studied. This new validated method fulfills all criteria for TDM of 15 antiretrovirals and boceprevir drugs and was successfully applied in routine TDM of antiretrovirals.

Intracellular NAADP increase induced by extracellular NAADP via the P2Y11-like receptor.

The aim of the study was to identify a signalling pathway allowing NAADP-induced intracellular NAADP increase and involving the P2Y11-like receptor. P2Y11-like and ß-adrenergic receptors may play important regulatory roles within the cardiovascular system. Both receptors have been shown to be involved in triggering myocardial preconditioning. Using a Langendorff model we report a positive inotropic response induced by extracellular NAADP via P2Y11-like receptor stimulation. In cardiomyocyte cultures, P2Y11-like receptor stimulation by extracellular NAADP ([NAADP]e) increased intracellular cADP-ribose and NAADP concentration as evidenced by direct measurements. NF546, a new selective P2Y11 receptor agonist, increased intracellular cAMP, cADP-ribose and NAADP concentration confirming the involvement of the P2Y11-like receptor in this signalling pathway. NF157, a P2Y11 receptor antagonist, suppressed the increase in intracellular cADPr, NAADP and NAAD induced by either [NAADP]e or NF546. The response profile for intracellular cADP-ribose and NAADP concentration following P2Y11-like stimulation with NF546 was similar to reported data relating ß-adrenergic stimulation with isoprenaline. This response represents the signature of the Gs/ADP-ribosyl cyclase activity. Moreover, this study provides a signalling pathway: intracellular NAADP increase induced by extracellular NAADP via metabotropic activity of P2Y11-like receptor. This pathway implying P2Y11-like could take part in the intracellular calcium rise reported for extracellular NAADP.

Extracellular NAADP affords cardioprotection against ischemia and reperfusion injury and involves the P2Y11-like receptor.

BACKGROUND AND PURPOSE: Extracellular nucleotides may play important regulatory roles within the cardiovascular system and notably in cardioprotection. We aimed to look for a possible pharmacological preconditioning effect of extracellular NAADP ([NAADP]e) against ischemia/reperfusion injury. [NAADP]e has been recently reported to be a full agonist of the P2Y11 receptor. Therefore, we characterized the involvement of the P2Y11-like receptor in mediating ischemic/reperfusion tolerance induced by [NAADP]e. EXPERIMENTAL APPROACH: The cardioprotective effects of [NAADP]e were evaluated in a model of ischemia/reperfusion carried out on Langendorff perfused rat hearts. This model was also instrumented with a microdialysis probe. Furthermore, using isolated cardiomyocytes, we assessed cAMP, inositol phosphate accumulation and prosurvival protein kinases activation induced by [NAADP]e pretreatement. RESULTS: Pretreatment with 1µM [NAADP]e induced cardioprotective effects with regards to functional recovery, necrosis and arrhythmogenesis (p<0.05). These effects were completely suppressed with NF157, an antagonist of the P2Y11 receptor. Moreover, global ischemia induced a time-dependent increase in interstitial concentration of adenosine, NAADP and UTP. In cardiomyocyte cultures, NF157 suppressed cAMP and inositol phosphate accumulation induced by [NAADP]e. [NAADP]e induced phosphorylation of ERK 1/2, AKT and its downstream target GSK-3ß (p<0.05). These activations were also suppressed by NF157. CONCLUSIONS: Evidence suggests that NAADP signalling at the P2Y11-like receptor affords significant cardioprotection against ischemia/reperfusion injury. Besides adenosine and UTP, microdialysis study supports a potential endogenous role of [NAADP]e.

Lead levels in fur of rats treated with inorganic lead measured by inductively coupled argon plasma mass spectrometry.

The aim of this study was to investigate the relationship between continuous lead exposure and the concentration of this metal in fur. The two main questions we wanted to answer were: 1) Are the fur lead concentrations different according to exposure level? 2) Is the kinetics of lead concentration linear in different compartments?For 12 weeks, 6 rats were force-fed with water containing lead acetate in the following quantities: 0.5 and 50 µg/day. Furs were sampled every two weeks. The lead content of the samples was measured by inductively coupled argon plasma mass spectrometry (ICP-MS).There was a statistical difference (p<0.0001) between fur lead concentration and the three groups (control, low level exposure and high level exposure), between fur lead concentration and time exposure (p<0.0001), and between fur lead concentration and each exposure group at different time exposure (p<0.0001). Thus the level exposure factor and the time exposure factor have an effect on fur lead concentration. Since the determination coefficients were weak for the two exposed groups (0.032 and 0.032), a linear correlation cannot be concluded. The kinetic curves of fur lead concentration are similar for all the exposition groups. Two peaks (at 2 and 8 weeks of exposure) were noted for the two exposed groups.This experimental study cannot conclude a linear relationship to exist between fur lead concentration and exposition duration. It highlights the lack of understanding of mechanisms involved in hair incorporation of metals and raises the question of a cyclic accumulation in hair. A better understanding of the kinetic incorporation of lead in body growths is required.

Involvement of P2Y receptors in pyridoxal-5'-phosphate-induced cardiac preconditioning.

Using an isolated non-working rat heart model, this study investigated the mechanisms of pharmacological pre-conditioning (PC) induced by P2Y receptor stimulation with pyridoxal-5'-phosphate (PLP). After 6-hydroxydopamine pretreatment and a 15-min stabilization period, isolated rat hearts were perfused for 25 min then subjected to 40 min of global ischemia and 30 min of reperfusion (I/R); exposed for 15 min to 0.05 microM PLP bracketed for 25 min with broad-spectrum P2 antagonists (suramin or PPADS) or with more specific P2Y antagonists (AMPalphaS or MRS2578), 1 microM each, followed by a 5-min PLP-free perfusion before I/R; treated during 25 min with either glybenclamide (GLY, 1 microM), 5-hydroxydecanoic acid (5-HD, 100 microM), U73122 (0.5 microM), H89 (1 microM), or KN93 (1 microM), with an infusion starting 5 min before PLP. The main endpoints were the rate-pressure product (RPP), creatine kinase (CK) release and area necrosis. Recovery of RPP, measured 5 min after reperfusion, was rapidly improved by PLP, blocked by the P2 antagonists, and decreased with the different inhibitors. Fifteen minutes after the end of ischemia, CK release reached maximal values in all groups. PLP provided significant protection, whereas the P2 antagonists, 5-HD, a mitochondrial selective K(ATP) antagonist and GLY a non-selective K(ATP) channel blocker, suppressed the protective effect on myocardial injury. The suppression of the cardioprotective effects of PLP by AMPalphaS, the PKA inhibitor (H89), and phospholipase C blocker (U73122) is in agreement with the P2Y11 receptor as a receptor for PLP-induced PC. The suppression of the cardioprotective effects of PLP by MRS2578 and U73122 is in agreement with the P2Y6 receptor as a receptor for PLP-induced PC. Pre-ischemic exposure to nanomolar concentrations of PLP is protective against I/R. P2Y11 and P2Y6 represents the most likely candidate receptors for PLP-induced cardiac PC.

Determination of articaine in human plasma by liquid chromatography-mass spectrometry and its application in a preliminary pharmacokinetic study.

A specific liquid chromatography-mass spectrometric (LC-MS) method using an ion trap spectrometer was developed for the quantitation of articaine in human plasma. Articaine and the internal standard (trazodone) were extracted in a single step with diethyl-ether from 0.5 mL of alkalinized plasma. The mobile phase consisted of acetonitrile with 0.1% formic acid (40:60, v/v). It was delivered at a flow rate of 0.3 mL/min. The effluent was monitored by MS in positive-ion mode. Ionisation was performed using an electrospray ion source operating at 200 degrees C. Articaine was identified and quantified in SIM mode at m/z 185. Calibration curves were linear over the concentration range of 78.1-5000 ng/mL with determination coefficients>0.996. This method was fast (total run-time<3 min), accurate (bias<16%), and reproducible (intra-assay and inter-assay precision<14%) with a quantitation limit of 78.1 ng/mL. The good specificity and sensitivity achieved by this method allowed the determination of articaine plasma levels in patients following a submucosal infiltration injection of articaine in the patients undergoing a third molar surgery.

Binding of elastin peptides to S-Gal protects the heart against ischemia/reperfusion injury by triggering the RISK pathway.

Elastin peptides (EPs) generated by hydrolysis of elastic fibers by elastinolytic enzymes display a wide spectrum of biological activities. Here, we investigated their influence on rat heart ischemia-mediated injury using the Langendorff ex vivo model. EPs, i.e., kappa elastin, at 1.32- and 660-nM concentrations, when administered before the ischemia period, elicited a beneficial influence against ischemia by accelerating the recovery rate of heart contractile parameters and by decreasing significantly creatine kinase release and heart necrosis area when measured at the onset of the reperfusion. All effects were S-Gal-dependent, as being reproduced by (VGVAPG)3 and as being inhibited by receptor antagonists, such as lactose and V14 peptide (VVGSPSAQDEASPL). EPs interaction with S-Gal triggered NO release and activation of PI3-kinase/Akt and ERK1/2 in human coronary endothelial cells (HCAECs) and rat neonatal cardiomyocytes (RCs). This signaling pathway, as designated as RISK, for reperfusion injury salvage kinase pathway, was shown to be responsible for the beneficial influence of EPs on ischemia/reperfusion injury on the basis of its inhibition by specific pharmacological inhibitors. EPs survival activity was attained at a concentration averaging that present into the blood circulation, supporting the contention that these matrikines might offer a natural protection against cardiac injury in young and adult individuals. Such protective effect might be lost with aging, since we found that hearts from 24-month-old rats did not respond to EPs.