A carnosine analog mitigates metabolic disorders of obesity by reducing carbonyl stress.

Sugar- and lipid-derived aldehydes are reactive carbonyl species (RCS) frequently used as surrogate markers of oxidative stress in obesity. A pathogenic role for RCS in metabolic diseases of obesity remains controversial, however, partly because of their highly diffuse and broad reactivity and the lack of specific RCS-scavenging therapies. Naturally occurring histidine dipeptides (e.g., anserine and carnosine) show RCS reactivity, but their therapeutic potential in humans is limited by serum carnosinases. Here, we present the rational design, characterization, and pharmacological evaluation of carnosinol, i.e., (2S)-2-(3-amino propanoylamino)-3-(1H-imidazol-5-yl)propanol, a derivative of carnosine with high oral bioavailability that is resistant to carnosinases. Carnosinol displayed a suitable ADMET (absorption, distribution, metabolism, excretion, and toxicity) profile and was determined to have the greatest potency and selectivity toward α,ß-unsaturated aldehydes (e.g., 4-hydroxynonenal, HNE, ACR) among all others reported thus far. In rodent models of diet-induced obesity and metabolic syndrome, carnosinol dose-dependently attenuated HNE adduct formation in liver and skeletal muscle, while simultaneously mitigating inflammation, dyslipidemia, insulin resistance, and steatohepatitis. These improvements in metabolic parameters with carnosinol were not due to changes in energy expenditure, physical activity, adiposity, or body weight. Collectively, our findings illustrate a pathogenic role for RCS in obesity-related metabolic disorders and provide validation for a promising new class of carbonyl-scavenging therapeutic compounds rationally derived from carnosine.

Regulatory landscape of AGE-RAGE-oxidative stress axis and its modulation by PPARγ activation in high fructose diet-induced metabolic syndrome.

BACKGROUND: The AGE-RAGE-oxidative stress (AROS) axis is involved in the onset and progression of metabolic syndrome induced by a high-fructose diet (HFD). PPARγ activation is known to modulate metabolic syndrome; however a systems-level investigation looking at the protective effects of PPARγ activation as related to the AROS axis has not been performed. The aim of this work is to simultaneously characterize multiple molecular parameters within the AROS axis, using samples taken from different body fluids and tissues of a rat model of HFD-induced metabolic syndrome, in the presence or absence of a PPARγ agonist, Rosiglitazone (RGZ). METHODS: Rats were fed with 60% HFD for the first half of the treatment duration (21 days) then continued with either HFD alone or HFD plus RGZ for the second half. RESULTS: Rats receiving HFD alone showed metabolic syndrome manifestations including hypertension, dyslipidemia, increased glucose levels and insulin resistance, as well as abnormal kidney and inflammatory parameters. Systolic blood pressure, plasma triglyceride and glucose levels, plasma creatinine, and albuminuria were significantly improved in the presence of RGZ. The following molecular parameters of the AROS axis were significantly upregulated in our rat model: carboxymethyl lysine (CML) in urine and liver; carboxyethyl lysine (CEL) in urine; advanced glycation end products (AGEs) in plasma; receptor for advanced glycation end products (RAGE) in liver and kidney; advanced oxidation protein products (AOPP) in plasma; and 4-hydroxynonenal (HNE) in plasma, liver, and kidney. Conversely, with RGZ administration, the upregulation of AOPP and AGEs in plasma, CML and CEL in urine, RAGE in liver as well as HNE in plasma and liver was significantly counteracted/prevented. CONCLUSIONS: Our data demonstrate (i) the systems-level regulatory landscape of HFD-induced metabolic syndrome involving multiple molecular parameters, including HNE, AGEs and their receptor RAGE, and (ii) attenuation of metabolic syndrome by PPARγ modulation.

Activated partial thromboplastin time correlates with methoxyhydroxyphenylglycol in acute myocardial infarction patients: therapeutic implications for patients at cardiovascular risk.

BACKGROUND/AIM: Acute myocardial infarction (AMI) is associated with increased coagulation which in the presence of unstable atheroma or endothelial damage leads to occlusive coronary vessel thrombosis. AMI is usually characterized by increased levels of catecholamines. It is possible there may be a link between catecholamines and hypercoagulation, but this still remains to be determined. In the current study we sought to verify whether hypercoagulation is associated with hypersympathetic activity in AMI patients, and whether there is a correlation between increased Methoxyhydroxyphenylglycol (MOPEG) levels (a metabolite of catecholamines) and shorter APTT (a marker of hypercoagulation). RESULTS: Shorter APTT values were detected in the plasma of AMI patients, which had also increased MOPEG levels. A linear correlation between APTT and MOPEG values was observed. High levels of the coagulation marker prothrombin (fragments 1+2) were also found. CONCLUSION: Shortened APTT demonstrates hypercoagulation and high MOPEG levels indicate increased catecholamine metabolism. A direct correlation between APTT and MOPEG was found herein, demonstrating a link between catecholamines and the process of coagulation. Catecholamines may interact with the α2-adrenergic receptors located on platelets and convert factor XII to XIIa or through the kallikrein-kinin system, they may activate factor XII. The activation of factor XII initiates the intrinsic coagulation pathway, which is monitored by APTT. It is suggested to control patients with a shortened APTT and increased sympathetic activity with the aim of preventing secondary coagulation and cardiovascular accidents by administering anti-thrombotic and anti-adrenergic agents.

Ellagitannins from Rubus berries for the control of gastric inflammation: in vitro and in vivo studies.

Ellagitannins have shown anti-inflammatory and anti-Helicobacter pylori properties; however, their anti-inflammatory activity at gastric level was not previously investigated. The aim of this research was to evaluate the effects of ellagitannins from Rubus berries on gastric inflammation. Ellagitannin enriched extracts (ETs) were prepared from Rubus fruticosus L. (blackberry) and Rubus idaeus L. (raspberry). The anti-inflammatory activity was tested on gastric cell line AGS stimulated by TNF-α and IL-1ß for evaluating the effect on NF-kB driven transcription, nuclear translocation and IL-8 secretion. In vivo the protective effect of ellagitannins was evaluated in a rat model of ethanol-induced gastric lesions. Rats were treated orally for ten days with 20 mg/kg/day of ETs, and ethanol was given one hour before the sacrifice. Gastric mucosa was isolated and used for the determination of IL-8 release, NF-kB nuclear translocation, Trolox equivalents, superoxide dismutase and catalase activities. In vitro, ETs inhibited TNF-α induced NF-kB driven transcription (IC50: 0.67-1.73 µg/mL) and reduced TNF-α-induced NF-kB nuclear translocation (57%-67% at 2 µg/mL). ETs inhibited IL-8 secretion induced by TNF-α and IL-1ß at low concentrations (IC50 range of 0.7-4 µg/mL). Sanguiin H-6 and lambertianin C, the major ETs present in the extracts, were found to be responsible, at least in part, for the effect of the mixtures. ETs of blackberry and raspberry decreased Ulcer Index by 88% and 75% respectively and protected from the ethanol induced oxidative stress in rats. CINC-1 (the rat homologue of IL-8) secretion in the gastric mucosa was reduced in the animals receiving blackberry and raspberry ETs. The effect of ETs on CINC-1 was associated to a decrease of NF-κB nuclear translocation in ETs treated animals. The results of the present study report for the first time the preventing effect of ETs in gastric inflammation and support for their use in dietary regimens against peptic ulcer.

An amino acids mixture improves the hepatotoxicity induced by acetaminophen in mice.

Acetaminophen (APAP) is a widely used analgesic and antipyretic drug, but at high dose it leads to undesirable side effects, such as hepatotoxicity and nephrotoxicity. The aim of this study was to evaluate the protective role of DDM-GSH, a mixture of L-cysteine, L-methionine, and L-serine in a weight ratio of 2 : 1 : 1, in comparison to N-acetylcysteine (NAC), against acetaminophen- (APAP-) induced hepatotoxicity in mice. Toxicity was induced in mice by the intraperitoneal (ip) administration of low dose (2 mmol/kg) or high dose (8 mmol/kg) of APAP. DDM-GSH (0.4 to 1.6 mmol/kg) was given ip to mice 1 h before the APAP administration. The same was done with NAC (0.9 to 3.6 mmol/kg), the standard antidote of APAP toxicity. Mice were sacrificed 8 h after the APAP injection to determine liver weight, serum alanine aminotransferase (ALT), and total glutathione (GSH) depletion and malondialdehyde (MDA) accumulation in liver tissues. DDM-GSH improved mouse survival rates better than NAC against a high dose of APAP. Moreover, DDM-GSH significantly reduced in a dose-dependent manner not only APAP-induced increases of ALT but also APAP-induced hepatic GSH depletion and MDA accumulation. Our results suggest that DDM-GSH may be more potent than NAC in protecting the liver from APAP-induced liver injury.

Factors involved in sudden coagulation observed in patients with acute myocardial infarction.

Coronary artery diseases (CAD) evolving into acute myocardial infarction (AMI) is associated with coagulation and thrombotic occlusion of coronary vessels in the presence of unstable atheroma. The atheromatous plaque becomes unstable when it is infiltrated by monocytes, macrophages and neutrophils capable of secreting proteases that induce plaque erosion, rupture and initialize the coagulation process. The aim of this study was (a) to analyse the plasma of patients with AMI for the presence of proteases that may activate rapid coagulation, (b) to evaluate coagulation markers as prothrombin fragment (F1+2) and antithrombin III and (c) to find an interrelation between proteases and coagulation markers. The examined plasma showed high values of prothrombin fragment (F1+2) and low levels of antithrombin III. These markers showed a highly significant negative-correlation. The plasma also exhibited increased levels of matrix metalloproteinase-9 (MMP-9) which were positively-correlated with the prothrombin fragment (F1+2). MMP-9 seems to cause the coagulation activity by increasing the level of prothrombin fragment (F1+2) and the consumption of antithrombin III. The examined plasma also exhibited high levels of neutrophil gelatinase-associated lipocalin (NGAL), which is known to modulate MMP-9 activity. The high plasma levels of MMP-9 and NGAL can be attributed to plaque instability and appear to activate sudden coagulation. MMP-9 and NGAL, in the presence of altered values of prothrombin fragment (F1+2) and antithrombin III in AMI patients, seem to be suitable markers to be studied in unstable plaque patients, for the prediction and prevention of acute coronary syndrome.

The influence of the polar head and the hydrophobic chain on the skin penetration enhancement effect of poly(ethylene glycol) derivatives.

The effect of a homologue series of nonionic surfactants, namely poly(ethylene glycol) (PEG) fatty acid esters, differing in oxyethylene (PEG 8, PEG 12, and PEG 40) and fatty acid (stearate, mono and di-laurate, and mono and di-oleate) chain lengths, on in vitro skin permeability of ketoprofen (KTP) vehicled in plasters was investigated. The drug diffusion through hairless mouse skin as well as the effect of the surfactant type and strength was studied by Franz diffusion cells and ATR-FTIR spectroscopy. The use of PEG stearate series revealed that the surfactant with the largest polar head, namely PEG 40, was ineffective in enhancing the skin permeation of KTP, independently of the plaster concentrations. The effect of the hydrophobic chain was investigated only by using the shortest oxyethylene chains. The experimental results revealed that the oxyethylene chain length of surfactants appeared to be more influent than the alkyl chain. The prediction of the absorption enhancing capability of these PEG derivatives appeared related to the vehicle other than the proper combination of the number of ethylene oxide groups and alkyl groups.

Methylprednisolone-loaded PLGA microspheres: a new formulation for sustained release via intra-articular administration. A comparison study with methylprednisolone acetate in rats.

Methylprednisolone (MP) released by poly(d,l-lactide-co-glycolide) microspheres (PLGA MS) was monitored in plasma after intra-articular (i.a.) administration into rat joint. A validated LC-ESI-MS/MS method was used to quantify the plasmatic concentrations of MP. The calculated pharmacokinetic parameters were compared to those obtained after the i.a. administration of a commercially available suspension of MP acetate (MPA). Different pharmacokinetic profiles were observed in the two formulations, and a lower peak level (C(max) = 13.7 ± 4.3 ng · mL(-1)) and AUC(0-72 h) (198 ± 45 ng · mL(-1) · h) were observed for MP-PLGA MS than MPA (C(max) = 18.4 ± 2.7 ng · mL(-1)) and AUC(0-72 h) (943 ± 249 ng · mL(-1) · h). The administration of MP-PLGA MS resulted in a rapid increase in the MP concentration at 30 min, with a t(max) at 0.8 ± 0.3 h. Instead, for the MPA suspension the t(max) was 32.0 ± 13.9 h. These differences were indirectly confirmed by the evaluation of the extra-articular effects, namely, carrageenan-induced paw edema, since MP-PLGA MS showed a lower anti-inflammatory activity than MPA.

Design, synthesis, ADME properties, and pharmacological activities of ß-alanyl-D-histidine (D-carnosine) prodrugs with improved bioavailability.

ß-Alanyl-D-histidine (D-CAR, the enantiomer of the natural dipeptide carnosine) is a selective and potent sequestering agent of reactive carbonyl species (RCS) that is stable against carnosinase, but is poorly absorbed in the gastrointestinal tract. Herein we report a drug discovery approach aimed at increasing the oral bioavailability of D-CAR. In our study we designed, synthesized, and evaluated a series of novel lipophilic D-CAR prodrugs. The considered prodrugs can be divided into two categories: 1) derivatives with both terminal groups modified, in which the carboxyl terminus is always esterified while the amino terminus is protected by an amidic (N-acetyl derivatives) or a carbamate (ethyloxy or benzyloxy derivatives) function; 2) derivatives with only one terminus modified, which can be alkyl esters as well as amidic or carbamate derivatives. The prodrugs were designed considering their expected lipophilicity and their hydrolysis predicted by docking simulations on the most important human carboxylesterase (hCES1). The stability and metabolic profile of the prodrugs were studied by incubating them with rat and human serum and liver fractions. The octyl ester of D-CAR (compound 13) was chosen as a candidate for further pharmacological studies due to its rapid hydrolysis to the bioactive metabolite in vitro. Pharmacokinetic studies in rats confirmed the in vitro data and demonstrated that the oral bioavailability of D-CAR is increased 2.6-fold if given as an octyl ester relative to D-CAR. Compound 13 was then found to dose-dependently (at daily doses of 3 and 30 mg kg(-1) equivalent of D-CAR) decrease the development of hypertension and dyslipidemia, to restore renal functions of Zucker fa/fa obese rats, and to inhibit the carbonylation process (AGEs and pentosidine) as well as oxidative stress (urinary 8-epi-prostaglandin F2α and nitrotyrosine). A plausible mechanism underlying the protective effects of 13 is RCS sequestration, as evidenced by the significant increase in the level of adduct between CAR and 4-hydroxy-trans-2-nonenal (HNE, the main RCS generated by lipid oxidation) in the urine of treated animals.

The carbonyl scavenger carnosine ameliorates dyslipidaemia and renal function in Zucker obese rats.

The metabolic syndrome is a risk factor that increases the risk for development of renal and vascular complications. This study addresses the effects of chronic administration of the endogenous dipeptide carnosine (ß-alanyl-L-histidine, L-CAR) and of its enantiomer (ß-alanyl-D-histidine, D-CAR) on hyperlipidaemia, hypertension, advanced glycation end products, advanced lipoxidation end products formation and development of nephropathy in the non-diabetic, Zucker obese rat. The Zucker rats received a daily dose of L-CAR or D-CAR (30 mg/kg in drinking water) for 24 weeks. Systolic blood pressure was recorded monthly. At the end of the treatment, plasma levels of triglycerides, total cholesterol, glucose, insulin, creatinine and urinary levels of total protein, albumin and creatinine were measured. Several indices of oxidative/carbonyl stress were also measured in plasma, urine and renal tissue. We found that both L- and D-CAR greatly reduced obese-related diseases in obese Zucker rat, by significantly restraining the development of dyslipidaemia, hypertension and renal injury, as demonstrated by both urinary parameters and electron microscopy examinations of renal tissue. Because the protective effect elicited by L- and D-CAR was almost superimposable, we conclude that the pharmacological action of L-CAR is not due to a pro-histaminic effect (D-CAR is not a precursor of histidine, since it is stable to peptidic hydrolysis), and prompted us to propose that some of the biological effects can be mediated by a direct carbonyl quenching mechanism.

Activity of a new hydrogen sulfide-releasing aspirin (ACS14) on pathological cardiovascular alterations induced by glutathione depletion in rats.

We investigated the effects of the hydrogen sulfide (H2S)-releasing derivatives of aspirin (ACS14) and salicylic acid (ACS21) in a rat model of metabolic syndrome induced by glutathione (GSH) depletion, causing hypertension and other pathological cardiovascular alterations. GSH depletion was induced in normal rats by the GSH-synthase inhibitor buthionine sulfoximine (BSO, 30 mmol/L day for seven days in the drinking water). Systolic blood pressure and heart rate were measured daily by the tail-cuff method, and plasma thromboxane B2, 6-keto-prostaglandin F(2α), 8-isoprostane, GSH, insulin and glucose were determined at the end of the seven-day BSO schedule. In addition, ischemia/reperfusion-induced myocardial dysfunction and endothelial dysfunction were assayed on isolated heart and aortic rings, respectively. Unlike aspirin and salicylic acid, ACS14 and ACS21 reduced BSO-induced hypertension, also lowering plasma levels of thromboxane B2, 8-isoprostane and insulin, while GSH remained in the control range. Neither ACS14 nor ACS21 caused gastric lesions. Both restored the endothelial dysfunction observed in aortic rings from BSO-treated rats, and in ischemia/reperfusion experiments they lowered left ventricular end-diastolic pressure, consequently improving the developed pressure and the maximum rise and fall of left ventricular pressure. Together with this improvement of heart mechanics there were reductions in the activity of creatine kinase and lactate dehydrogenase in the cardiac perfusate. This implies that H2S released by both ACS14 and ACS21 was involved in protecting the heart from ischemia/reperfusion, and significantly limited vascular endothelial dysfunction in aortic tissue and the related hypertension.

Pretreatment with tetrandrine has protective effects against isoproterenol-induced myocardial infarction in rabbits.

Tetrandrine, the active principle of Stephania tetrandra radix extracts, has broad pharmacological activity, including effects on the cardiovascular system: it has been shown to reduce the size of acute myocardial infarction in rats undergoing coronary vessel ligation and to improve heart lesions in the constriction/reperfusion model by means of mechanisms involving peroxidation, calcium antagonism and coagulation. The aim of this study was to investigate whether tetrandrine has anti-infarction, antioxidant and anticoagulant effects in rabbits treated with isoproterenol, a drug capable of causing peroxide generation, calcium overload and coagulation alterations, and inducing myocardial infarction. The results showed that pretreatment with tetrandrine protects against the myocardial injuries caused by isoproterenol. It counteracted the appearance of myocardial necrotic lesions and ischemic electrocardiographic modifications, such as ST segment alterations, prevented the appearance of the plasma cardiac necrosis markers c-troponin I and myoglobin, lowered malondialdehyde levels, and prolonged partial thromboplastin time. The protective effects of tetrandrine can be attributed to its antioxidant action in lowering peroxide levels and its ability to counteract coagulating activity. Tetrandrine seems to offer full protection against myocardial infarction experimentally induced by the non-invasive treatment of rabbits with isoprotenerol.

A sensitive and specific precursor ion scanning approach in liquid chromatography/electrospray ionization tandem mass spectrometry to detect methylprednisolone acetate and its metabolites in rat urine.

A new, simple, sensitive and specific liquid chromatography/electrospray ionization tandem mass spectrometric (LC/ESI-MS/MS) method in precursor ion scanning (PIS) mode has been developed for the rapid detection of methylprednisolone acetate (MPA) and its metabolites in rat urine. A suitable product ion specific for methylprednisolone (MP) and MPA was selected after a fragmentation study on 20 (cortico)steroids at different collision energies (5-40 eV). Urine samples were simply treated with acetonitrile then dried in a SpeedVac system. The method was validated and compared with other PIS methods for detecting corticosteroids in human urine. It was more sensitive, with limit of detection (LOD) and lower limit of quantitation (LLOQ), respectively, of 5 and 10 ng mL(-1). The method was applied for the analysis of rat urine collected before and after (24, 48, 72 h) intra-articular (IA) injection of a marketed formulation of MPA (Depo-Medrol(R)). MS/MS acquisitions were taken at different collision energies for the precursor ions of interest, detected in PIS mode, to verify the MP-related structure. Six different metabolites were detected in rat urine, and their chemical structures were assigned with a computational study.

Synthesis of human renalase1 in Escherichia coli and its purification as a FAD-containing holoprotein.

Renalase is a protein ubiquitous in vertebrates, which has been proposed to modulate blood pressure and heart rate, and whose downregulation might result in hypertension. Despite its potential relevance for human health, the biochemical characterization of renalase is still lacking, possibly due to difficulties in obtaining it in recombinant form. By expressing two different gene constructs, we found that the major isoform of human renalase, renalase1, is mainly produced in Escherichia coli in inclusion bodies. However, by optimizing the expression conditions, significant amounts of soluble products were obtained. Both soluble renalase forms have been purified to homogeneity exploiting their N-terminal His-tag. Linking of the protein of interest to the SUMO protein did not improve solubility, but yielded untagged renalase1 after proteolytic processing of the fusion product. The two recombinant renalase forms displayed the same molecular properties. They bind equimolar amounts of FAD and appear to be correctly folded by various criteria. The procedures for the production and isolation of recombinant renalase1 here reported are expected to boost the much awaited biochemical studies on this remarkable protein.

Selective estrogen receptor-alpha agonist provides widespread heart and vascular protection with enhanced endothelial progenitor cell mobilization in the absence of uterotrophic action.

The beneficial effects of estrogens on the cardiovascular system are associated with adverse effects on reproductive tissues. On the basis of previous work indicating a major role for estrogen receptor (ER)-alpha in maintaining cardiovascular health, we evaluated the tissue selectivity of the ER alpha-selective agonist propyl pyrazole triol (PPT) compared with 17beta-estradiol (E2) in vivo. Four weeks postovariectomy, equimolar doses of PPT and E2 were administered to rats in subcutaneous implants for 5 d. Both treatments restored rapid vasorelaxation of aortic tissue to estrogenic agents and prevented coronary hyperresponsiveness to angiotensin II in isolated heart preparations. Accordingly, multiple endpoints of myocardial ischemia-reperfusion injury exacerbated by ovariectomy returned to baseline following treatment. These protective effects were linked to increased in vivo levels of endothelial progenitor cells (EPCs). Human EPC function was enhanced in vitro after PPT treatment. In sharp contrast to E2, PPT treatment had no effect on uterine weight and histomorphology except for vessel density, and failed to up-regulate classic estrogen target genes. Dissection of the effects on vascular reactivity and uterine morphology was also observed following increased exposure to PPT at a higher dose for longer time. These data provide the first in vivo evidence for tissue-specific ER alpha activation. By conferring cardiovascular protection dissected from unwanted uterotrophic effects, ER alpha-selective agonists may represent a potential safer alternative to natural hormones.

Interaction between ticlopidine or warfarin or cardioaspirin with a highly standardized deterpened Ginkgo biloba extract (VR456) in rat and human.

Ginkgo biloba is available in Europe as an over-the-counter drug and it is reported to cause hemorrhage when co-administered with other anti-platelet agents. We set out to study the interactions of ticlopidine with Ginkgo biloba extract or VR456, a new highly standardized deterpened extract from Ginkgo biloba leaves. Male Wistar rats were used to study the effects of ticlopidine (50-100 mg/kg/day), given alone and in combination for 5 days with Ginkgo biloba extract (50 mg/kg/day) or VR456 (50 mg/kg/day), on bleeding time and ex vivo ADP-induced platelet aggregation measurements. In addition, human studies were performed with the compounds under investigation. Combined treatment of ticlopidine and undeterpened Ginkgo biloba extract increased anti-platelet effect and prolonged the bleeding time in the rat. On the contrary, the combination treatment of ticlopidine and VR456 increased anti-platelet effect but did not prolong bleeding time. Moreover, daily administration of 360 mg of VR456 for 14 days to ticlopidine-treated humans did not highlight any unwanted effect and did not alter PT/INR and PTT parameters. Same results have been also obtained in warfarin or in cardioaspirin-treated patients. These data point out the clear role played by the terpenoid, PAF-antagonist fraction of Ginkgo biloba extract in affecting bleeding risk in anticoagulant-treated subjects and suggest VR456 as a possible option treatment in geriatric people subjected to anticoagulant treatment where the use of standard Ginkgo biloba extracts are discouraged.

Changes in sympathetic activity in prion neuroinvasion.

Prion diseases are neurodegenerative diseases affecting humans and animals in which the infectious agent or prion is PrP(res), a protease-resistant conformer of the cell protein PrP. The natural transmission route of prion diseases is peripheral infection, with the lymphoreticular system (LRS) and peripheral nerves being involved in animal models of scrapie neuroinvasion and human prion diseases. To study the effects of PrP neuroinvasion on sympathetic nerve function, we measured plasma catecholamine levels, blood pressure, heart rate, and PrP tissue levels in intraperitoneally or intracerebrally infected mice. The results indicate a specific alteration in sympathetic nerve function because the levels of noradrenaline (but not adrenaline) were increased in the animals infected peripherally (but not in those infected intracerebrally) and correlated with increased blood pressure. These findings confirm that prion neuroinvasion uses the sympathetic nervous system to spread from the periphery to the central nervous system after invading the LRS.

Plasma cardiac necrosis markers C-troponin I and creatine kinase, associated with increased malondialdehyde levels, induced in rabbits by means of 5-aminolevulinic acid injection.

A number of papers have described high levels of 5-aminolevulinic acid in cases of heart damage due to acute myocardial infarction, acute intermittent porphyria or chronic kidney failure, but it is not known whether the heart damage is directly associated with 5-aminolevulinic acid. The aim of this study was to verify whether such an association exists by injecting rabbits with 5-aminolevulinic acid and searching for the appearance of cardiac necrosis markers and histological heart alterations, and investigate whether the cardiotoxic activity of 5-aminolevulinic acid may involve peroxidation by seeking the presence of the peroxide marker malondialdehyde. The administration of 5-aminolevulinic acid led to the appearance of c-troponin I and creatine kinase, induced histological heart alterations and increased the malondialdehyde levels. The plasma levels of malondialdehyde and cardiac necrosis markers were also measured after the injection of 5-aminolevulinic acid in combination with the daunorubicin agent inducing peroxidation. The combined administration very significantly increased the plasma levels of 5-aminolevulinic acid, malondialdehyde, and the cardiac necrosis markers c-troponin I and creatine kinase. It therefore seems that there is a close relationship between altered 5-aminolevulinic acid levels, malondialdehyde and cardiac necrosis markers, which is attributable to the capacity of 5-aminolevulinic acid to generate toxic oxygen species that damage the heart. High plasma 5-aminolevulinic acid levels should be considered a factor contributing to cardiotoxicity and to the appearance of cardiac necrosis markers.

Anti-ischemic activity and endothelium-dependent vasorelaxant effect of hydrolysable tannins from the leaves of Rhus coriaria (Sumac) in isolated rabbit heart and thoracic aorta.

The aim of this work was to investigate the cardioprotective activity of hydrolysable gallotannins from Rhus coriaria L. leaves extract (RCLE) in isolated rabbit heart preparations, submitted to low-flow ischemia/reperfusion damage. RCLE induces a dose-dependent normalization of coronary perfusion pressure (CPP), reducing left ventricular contracture during ischemia, and improving left ventricular developed pressure and the maximum rate of rise and fall of left ventricular pressure at reperfusion. Creatinine kinase (CK) and lactate dehydrogenase (LDH) outflow were significantly reduced during reperfusion. In parallel there was a rise in the release of the cytoprotective 6-ketoprostaglandin F (1alpha) (6-keto-PGF (1alpha)) and a decrease of tumor necrosis factor-alpha (TNF-alpha), both significant only at the highest RCLE concentrations (150-500 microg/mL). The vasorelaxant activity of RCLE was studied in isolated rabbit aorta rings precontracted with norepinephrine (NE) with and without endothelium. The vasorelaxation induced by RCLE was predominantly endothelium-dependent as demonstrated by the loss of RCLE vasorelaxant ability in i) de-endothelized rings and ii) in intact aortic rings after pretreatment with NG-monomethyl- L-arginine (L-NMMA) and 1 H-[1.2.4]oxadiazolo[4.3- A]quinoxalin-1-one (ODQ). The inhibition of vasorelaxation in intact rings by indomethacin (INDO) demonstrates the ability of RCLE to modulate the coronary endothelium cyclooxygenase (COX) pathway. The K-ATP channel antagonist glibenclamide (GLIB) was ineffective. The antioxidant activity of RCLE, investigated in the 1,1-diphenyl-2-picrylhydrazyl (DPPH) model and in living cell systems (rat erythrocytes), was stronger than that of gallic acid, ascorbic acid and trolox. The structure of its main bioactive constituents, profiled by HPLC-ESI-HR-S, comprised a mixture of polygalloylated D-glucopyranose with different degrees of galloylation and 3- O-methylgallic acid. The cardiovascular protective effect of RCLE seems to be due to an interplay of different factors: COX pathway activation, TNF-alpha inhibition, endothelial nitric oxide synthase (eNOS) activation, and free radical and ROS scavenging.

Liquid chromatography/atmospheric pressure chemical ionization ion trap mass spectrometry of bilobalide in plasma and brain of rats after oral administration of its phospholipidic complex.

Standardized extracts of Ginkgo biloba L. leaves are widely used in clinical practice for the symptomatic treatment of mild to moderate dementia syndromes, cerebral insufficiency and for the enhancement of cognitive function. The main active components present in G. biloba extracts are flavonol-glycosides and terpene-lactones. In recent investigations, the sesquiterpene trilactone bilobalide has been described to exert an interesting neuroprotective effect when administered systemically to experimental animals. Oral administration of terpene-lactones either as standardized extracts or purified products is characterized by a low bioavailability. While preparing phospholipidic complex of G. biloba extracts or bilobalide, plasma levels of terpenes and sesquiterpene increase. In the present study, phospholipidic complex of bilobalide (IDN 5604) has been administered orally to rats and bilobalide levels have been determined in plasma and brain by means of a validated method based on liquid chromatography coupled to atmospheric pressure chemical ionization ion trap mass spectrometry (LC/APCI-ITMS). Due to its sensitivity (about 3pmol/ml) and specificity, LC/APCI-ITMS method proved to be a very powerful tool for pharmacokinetic studies of Ginkgo terpene-lactones. The results of the present study clearly confirm the improvement of oral bioavailability of bilobalide administered as phospholipidic complex and, for the first time, demonstrate the detection of significative amounts of bilobalide in brain. This last finding agrees with the neuroprotective activity observed for bilobalide.