Correction to: Efficacy of Ronopterin (VAS203) in Patients with Moderate and Severe Traumatic Brain Injury (NOSTRA phase III trial): study protocol of a confirmatory, placebocontrolled, randomised, double blind, multi-centre study.

After publication of our article [1] the authors have notified us that one of the names has been incorrectly spelled.

Efficacy of Ronopterin (VAS203) in Patients with Moderate and Severe Traumatic Brain Injury (NOSTRA phase III trial): study protocol of a confirmatory, placebo-controlled, randomised, double blind, multi-centre study.

BACKGROUND: Traumatic brain injury is a leading cause of death and disability worldwide. The nitric oxide synthase inhibitor Ronopterin was shown to improve clinical outcome by enhancing neuroprotection in a phase IIa trial. METHODS/DESIGN: The NOSTRA phase III trial (Ronopterin in traumatic brain injury) is a multi-centre, prospective, randomised, double-blinded, placebo-controlled, phase III trial in Europe. It aims at determining whether the administration of Ronopterin compared to placebo improves neurological outcome in patients with moderate or severe traumatic brain injury at 6 months after injury. The trial is designed to recruit patients between 18 and 60 years of age with moderate or severe traumatic brain injury (Glasgow Coma Scale score ≥ 3) and requiring insertion of an intracranial pressure probe. Trial patients will receive a 48-h intravenous infusion of either Ronopterin or placebo starting at the earliest 6 h and at the latest 18 h after injury. The primary outcome will be the extended Glasgow Outcome Score (eGOS) at 6 months. Secondary outcomes will include the Quality of Life Index (QOLIBRI) at 6 months after the injury and the eGOS at 3 months after the injury. Additionally, effects on mortality, intracranial pressure and cerebral perfusion pressure are evaluated. DISCUSSION: The trial aims to provide evidence on the efficacy and safety of Ronopterin in patients with traumatic brain injury. TRIAL REGISTRATION: EudraCT, 2013-003368-29. Registered on 9 March 2016. ClinicalTrials.gov, NCT02794168. Registered on 8 June 2016. Protocol version 14.0 from 05 November 2018.

Effects of the nitric oxide synthase inhibitor ronopterin (VAS203) on renal function in healthy volunteers.

AIMS: Reduced nitric oxide (NO) availability may adversely affect renal perfusion and glomerular filtration. The aim of the present study was to characterize in detail the pharmacological effects of VAS203, an inhibitor of NO synthase, on renal haemodynamics in humans. METHODS: This double-blind, randomized, placebo-controlled, cross-over phase-I-study comprised 18 healthy men. Renal haemodynamics were assessed with constant-infusion input-clearance technique with p-aminohippurate and inulin for renal plasma flow (RPF) and glomerular filtration rate (GFR), respectively. After baseline measurement, a constant infusion of the tetrahydrobiopterin analogue ronopterin (VAS203, total 10 mg/kg body weight) or placebo was administered at random order for 6 hours additionally. After a wash-out phase of 28 days, the second course was applied. In parallel, markers of early kidney injury and renal function were assessed repeatedly up to 48 hours after starting VAS203/placebo-infusion. RESULTS: VAS203-infusion resulted in a significant decrease of RPF (P < .0001) and GFR (P < .001) compared to placebo, but magnitude was within the physiological range. RPF and GFR recovered partly 2 hours after end of VAS203-infusion and was normal at beginning of the second infusion period. Compared to placebo, preglomerular resistance (P < .0001), and to lesser extent postglomerular resistance (P < .0001) increased, resulting in a decrease of intraglomerular pressure (P < .01). No treatment related effect on markers of early kidney injury, and on renal function (P for all >.20) have been observed. CONCLUSIONS: Our phase-I-study in healthy humans indicates that VAS203 (10 mg/kg body weight) reduces renal perfusion and glomerular function within the physiological range mainly due to vasoconstriction at the preglomerular site.

Who is a 'healthy subject'?-consensus results on pivotal eligibility criteria for clinical trials.

INTRODUCTION/METHODS: A discussion forum was hosted by the German not-for-profit Association for Applied Human Pharmacology (AGAH e.V.) to critically review key eligibility criteria and stopping rules for clinical trials with healthy subjects, enrolling stakeholders from the pharmaceutical industry, contract research organisations, academia, ethics committees and competent authority. RESULTS: Pivotal eligibility criteria were defined for trials with new investigational medicinal products (IMPs) or with clinically established IMPs. In general, a pulse rate ranging between 50 and 90 beats/min is recommended for first-in-human (FIH) trials, while wider ranges seem acceptable for trials with clinically established IMPs, provided there are no indications of thyroid dysfunction. Hepatic laboratory parameters not to exceed the upper limit of normal (ULN) comprise ALT (alanine aminotransferase) and AST (aspartate aminotransferase) in FIH trials, whereas slight elevations (10% above ULN) seem acceptable in trials with clinically established IMPs without known hepatotoxicity. A normal renal function is required for any clinical trial in healthy subjects. A risk-adapted approach for stopping rules was adopted. Stopping rules for an individual subject are one adverse event of severe intensity or one serious adverse event. In case of a severe adverse event, some stakeholders demand a causal relationship with the IMP (i.e. an adverse reaction). Stopping rules for a cohort are one serious adverse reaction or ≥50% of subjects experiencing any adverse reaction of moderate or severe intensity. CONSEQUENCES: The application of this consensus resulted in a reduction in protocol deficiencies issued by the competent authority.

Endothelial nitric oxide synthase mediates arteriolar vasodilatation after traumatic brain injury in mice.

Brain edema and increased cerebral blood volume (CBV) contribute to intracranial hypertension and hence to unfavorable outcome after traumatic brain injury (TBI). The increased post-traumatic CBV may be caused in part by arterial vasodilatation. The aim of the current study was to uncover the largely unknown mechanisms of post-traumatic arteriolar vasodilatation. The diameter of pial arterioles and venules was monitored by intravital fluorescence microscopy before (baseline) and for 30 min after controlled cortical impact in C57BL/6 and endothelial nitric oxide synthase (eNOS)-/- mice (n=5-6/group) and in C57BL/6 mice (n=6/group) receiving vehicle (phosphate-buffered saline [PBS]) or 4-amino-tetrahydro-L-biopterine (VAS203), a NOS inhibitor previously shown to reduce post-traumatic intracranial hypertension. Temperature, end-tidal partial pressure of carbon dioxide (pCO2), and mean arterial blood pressure were kept within the physiological range throughout the experiments. Arteriolar diameters were stable during baseline monitoring but increased significantly in C57BL/6 mice after controlled cortical impact (136±7% of baseline; p<0.001 vs. baseline). This response was reduced by 78% in eNOS-/- mice (108±3% of baseline; p<0.005 vs. wild-type). Application of VAS203, a NOS inhibitor, or PBS did not affect vessels diameter before TBI. After trauma, however, administration of VAS203 reduced arteriolar diameter to 92±2% of baseline (p<0.05). The diameter of pial veins was not affected. Our results suggest that arteriolar vasodilatation after TBI is largely mediated by excess production of endothelial nitric oxide. Accordingly, our data may explain the beneficial effects of the NOS inhibitor VAS203 in the early phase after TBI and suggest that inhibition of excess endothelial nitric oxide production may represent a novel therapeutic strategy following TBI.

Nitric oxide synthase inhibition with the antipterin VAS203 improves outcome in moderate and severe traumatic brain injury: a placebo-controlled randomized Phase IIa trial (NOSTRA).

Traumatic brain injury (TBI) is an important cause of death and disability. Safety and pharmacodynamics of 4-amino-tetrahydrobiopterin (VAS203), a nitric oxide (NO)-synthase inhibitor, were assessed in TBI in an exploratory Phase IIa study (NOSynthase Inhibition in TRAumatic brain injury=NOSTRA). The study included 32 patients with TBI in six European centers. In a first open Cohort, eight patients received three 12-h intravenous infusions of VAS203 followed by a 12-h infusion-free interval over 3 days (total dose 15 mg/kg). Patients in Cohorts 2 and 3 (24) were randomized 2:1 to receive either VAS203 or placebo as an infusion for 48 or 72 h, respectively (total dose 20 and 30 mg/kg). Effects of VAS203 on intracranial pressure (ICP), cerebral perfusion pressure (CPP), brain metabolism using microdialysis, and the therapy intensity level (TIL) were end points. In addition, exploratory analysis of the extended Glasgow Outcome Score (eGOS) after 6 months was performed. Metabolites of VAS203 were detected in cerebral microdialysates. No significant differences between treatment and placebo groups were observed for ICP, CPP, and brain metabolism. TIL on day 6 was significantly decreased (p<0.04) in the VAS203 treated patients. The eGOS after 6 months was significantly higher in treated patients compared with placebo (p<0.01). VAS203 was not associated with hepatic, hematologic, or cardiac toxic effects. At the highest dose administered, four of eight patients receiving VAS203 showed transitory acute kidney injury (stage 2-3). In conclusion, the significant improvement in clinical outcome indicates VAS203-mediated neuroprotection after TBI. At the highest dose, VAS203 is associated with a risk of acute kidney injury.

Angiotensin II induces DNA damage via AT1 receptor and NADPH oxidase isoform Nox4.

Epidemiological studies revealed increased renal cancer incidences and higher cancer mortalities in hypertensive individuals. Activation of the renin-angiotensin-aldosterone system leads to the formation of reactive oxygen species (ROS). In vitro, in renal cells, and ex vivo, in the isolated perfused mouse kidney, we could show DNA-damaging potential of angiotensin II (Ang II). Here, the pathway involved in the genotoxicity of Ang II was investigated. In kidney cell lines with properties of proximal tubulus cells, an activation of NADPH oxidase and the production of ROS, resulting in the formation of DNA strand breaks and micronuclei induction, was observed. This DNA damage was mediated by the Ang II type 1 receptor (AT1R), together with the G protein G ( α-q/11 ) . Subsequently, phospholipase C (PLC) was activated and intracellular calcium increased. Both calcium stores of the endoplasmic reticulum and extracellular calcium were involved in the genotoxicity of Ang II. Downstream, a role for protein kinase C (PKC) could be detected, because its inhibition hindered Ang II from damaging the cells. Although PKC was activated, no involvement of its known target, the NADPH oxidase isoform containing the Nox2 subunit, could be found, as tested by small-interfering RNA down-regulation. Responsible for the DNA-damaging activity of Ang II was the NADPH oxidase isoform containing the Nox4 subunit. In summary, in kidney cells the DNA-damaging activity of Ang II depends on an AT1R-mediated activation of NADPH oxidase via PLC, PKC and calcium signalling, with the NADPH subunit Nox4 playing a crucial role.

Aldosterone increases kidney tubule cell oxidants through calcium-mediated activation of NADPH oxidase and nitric oxide synthase.

Chronic hyperaldosteronism has been associated with an increased cancer risk. We recently showed that aldosterone causes an increase in cell oxidants, DNA damage, and NF-κB activation. This study investigated the mechanisms underlying aldosterone-induced increase in cell oxidants in kidney tubule cells. Aldosterone caused an increase in both reactive oxygen and reactive nitrogen (RNS) species. The involvement of the activation of NADPH oxidase in the increase in cellular oxidants was demonstrated by the inhibitory action of the NADPH oxidase inhibitors DPI, apocynin, and VAS2870 and by the migration of the p47 subunit to the membrane. NADPH oxidase activation occurred as a consequence of an increase in cellular calcium levels and was mediated by protein kinase C. The prevention of RNS increase by BAPTA-AM, W-7, and L-NAME indicates a calcium-calmodulin activation of NOS. A similar pattern of effects of the NADPH oxidase and NOS inhibitors was observed for aldosterone-induced DNA damage and NF-κB activation, both central to the pathogenesis of chronic aldosteronism. In summary, this paper demonstrates that aldosterone, via the mineralocorticoid receptor, causes an increase in kidney cell oxidants, DNA damage, and NF-κB activation through a calcium-mediated activation of NADPH oxidase and NOS. Therapies targeting calcium, NOS, and NADPH oxidase could prevent the adverse effects of hyperaldosteronism on kidney function as well as its potential oncogenic action.

A protein phosphorylation-based assay for screening and monitoring of drugs modulating cyclic nucleotide pathways.

Cyclic nucleotide regulation is an important target for drug development, particularly for treatment and prophylaxis of cardiovascular diseases. Determination of cyclic nucleotide levels for screening and monitoring of cyclic nucleotide modulating drug action is necessary, yet the techniques available are cumbersome and not sufficiently accurate. Here we present an approach based on the detection of cyclic nucleotide-dependent protein phosphorylation. By use of a common substrate of cyclic nucleotide-dependent protein kinases, the protein vasodilator-stimulated phosphoprotein (VASP) featuring two phosphorylation sites specifically phosphorylated by these kinases, an assay was developed for the monitoring of intracellular cyclic nucleotide levels. The assay was tested with human platelets ex vivo treated with stimulants of nucleotide cyclases, kinases, and phosphodiesterase inhibitors. Phosphorylation of the protein VASP correlates with intracellular cyclic nucleotide concentration (R(2)>0.90 for cGMP and cAMP); however, VASP phosphorylation is more sensitive to elevated cyclic nucleotide levels and significantly more stable over time. Quantification of VASP phosphorylation offers a reliable and robust tool for fast and easy monitoring of cyclic nucleotide levels and is also applicable to unprocessed biological matrices. Owing to these properties, VASP is a promising biomarker for screening and monitoring of cyclic nucleotide modulating drugs.

The novel nitric oxide synthase inhibitor 4-amino-tetrahydro-L-biopterine prevents brain edema formation and intracranial hypertension following traumatic brain injury in mice.

Brain edema formation, resulting in increased intracranial pressure (ICP), is one of the most deleterious consequences of traumatic brain injury (TBI). Nitric oxide (NO) has previously been shown to be involved in the damage of the blood-brain barrier (BBB) and, thus, in the formation of post-traumatic brain edema; however, this knowledge never resulted in a clinically relevant therapeutic option because available NO synthase inhibitors have serious side effects in man. The aim of the current study was to investigate the therapeutic efficacy of VAS203, a novel tetrahydrobiopterine (BH3)-based NOS inhibitor, in experimental TBI. When added to isolated vessels rings obtained from rat basilar and middle cerebral arteries (n = 32-35) VAS203 showed the same vasoconstrictive effect as the classical NO synthase inhibitor L-(G)-nitro-arginine-methylester (L-NAME). VAS203 passed the BBB both in healthy and traumatized mouse brain (C57/BL6, n = 5 per group) and did not show any systemic side effects at therapeutic concentrations. When administered 30 min after experimental TBI (controlled cortical impact, 2.2 mg/kg/min i.v., n = 7 per group), VAS203 prevented any further increase in ICP or deterioration of cerebral blood flow. This effect was dose-dependent and long-lasting (i.e., 24 h after trauma, brain edema formation was still significantly reduced [-40%, p < 0.008; n = 7 per group] and functional improvements were present up to 7 days after TBI [p < 0.02 on post-trauma day 6; n = 8 per group]). Therefore, VAS203 may represent a promising candidate for the treatment of acute intracranial hypertension following TBI.

Advanced glycation end product accumulation in rho(0) cells without a functional respiratory chain.

Advanced glycation end products (AGEs) accumulate during ageing with reactive oxygen species from the mitochondrial respiratory chain discussed as a driving force. To determine the role of mitochondrial activity for AGE formation, a rho(0) derivative of the 143B.TK(-) osteosarcoma cell line lacking the respiratory chain, was analysed. These cells exhibit decreased superoxide formation but unchanged mitochondrial SOD expression as well as unchanged antioxidative free sulfhydryl (SH) levels. Whereas total protein content shows no differences in AGE levels, cell fractionation and Western blotting demonstrates some changes in the AGE pattern. Thus, the absence of functional respiration has only a negligible impact on AGE accumulation.

Effects of oral niacin on endothelial dysfunction in patients with coronary artery disease: results of the randomized, double-blind, placebo-controlled INEF study.

High-density-lipoproteins-cholesterol (HDL-C) is invertedly related to the incidence of cardiovascular events. Recent studies suggest that HDL-C directly improves endothelial function. Nicotinic acid (niacin) effectively raises serum HDL-C. We therefore hypothesized that treatment with niacin improves endothelial dysfunction in patients with coronary artery disease (CAD). One hundred seven patients with CAD were randomly assigned to double-blinded treatment for 12 weeks with extended-release (ER)-niacin 1000 mg/day (N) or placebo (C), respectively. Flow-mediated dilation (FMD) of the brachial artery, nitroglycerin-mediated endothelium-independent dilation (NMD) and serum lipid concentrations were measured before and after treatment. Triglycerides (P=0.013), low-density-lipoprotein-cholesterol (LDL-C) (P=0.013) and HDL-C (P<0.0001) were altered by N compared to C. Niacin treatment was without effect on FMD or NMD, respectively, compared to placebo. However, post-hoc subgroup analysis revealed an improvement in FMD in patients with low HDL-C at baseline (absolute change in FMD (mean+/-S.D.) N: +3.25+/-3.88%, C: +1.03+/-2.71% in low tertile HDL-C

A single loading dose of clopidogrel causes dose-dependent improvement of endothelial dysfunction in patients with stable coronary artery disease: results of a double-blind, randomized study.

Clinical studies have demonstrated beneficial effects for clopidogrel in patients with atherothrombotic disease. Recent in vitro studies identified stimulating effects of clopidogrel on endothelial cells, pointing towards mechanisms of action beyond the inhibition of platelet aggregation. We hypothesized that in vivo use of clopidogrel improves endothelial dysfunction in patients with coronary artery disease (CAD). Fifty-eight patients with CAD were randomly assigned to double-blinded oral administration of one single dose of clopidogrel 300 mg (C300) or 600 mg (C600), respectively. Endothelial function was assessed by measurement of flow-mediated dilation (FMD) of the brachial artery before and 2, 4 and 22 h after dose administration, respectively. Inhibition of the platelet ADP P2Y12 receptor by clopidogrel was monitored by the ex vivo analysis of ADP effects on prostaglandin-induced platelet VASP phosphorylation. C600 significantly improved FMD at 2, 4 and 22 h, while C300 significantly improved FMD at 4 and 22 h. Clopidogrel dose- and time-dependently inhibited the platelet ADP P2Y12 receptor without correlation with its stimulatory effects on FMD. Our study demonstrates for the first time in vivo that clopidogrel dose-dependently improves endothelial dysfunction. These results may indicate a new and potentially important aspect of the effect of clopidogrel treatment in patients with CAD.

Maillard reaction products enriched food extract reduce the expression of myofibroblast phenotype markers.

Advanced glycation end products (AGE) are associated with a wide range of degenerative diseases. The present investigation aimed at analysing the influence of AGE containing nutritional extracts on cardiac fibroblasts (CFs) as the major cell type responsible for cardiac fibrosis. Mice CFs were treated with bread crust extract (BCE) which contained significant amounts of a variety of AGE modifications. BCE treatment with up to 30 mg/mL did not impair cell viability. Furthermore, BCE induced a moderate elevation of reactive oxygen species (ROS) production and activation of redox sensitive pathways like the p42/44(MAPK), p38(MAPK) and NF-kappaB but did not alter Akt kinase phosphorylation. Expression of smooth muscle alpha-actin and tropomyosin-1, which represent markers for myofibroblast differentiation, was reduced after bread crust treatment. These data suggest a putative antifibrotic effect of melanoidin-rich food.

Genotoxicity of advanced glycation end products: involvement of oxidative stress and of angiotensin II type 1 receptors.

In patients with chronic renal failure, cancer incidence is increased. This may be related to an elevated level of genomic damage, which has been demonstrated by micronuclei formation as well as by comet assay analysis. Advanced glycation end products (AGEs) are markedly elevated in renal failure. In the comet assay, the model AGEs methylglyoxal- and carboxy(methyl)lysine-modified bovine serum albumin (BSA) induced significant DNA damage in colon, kidney, and liver cells. The addition of antioxidants prevented AGE-induced DNA damage, suggesting enhanced formation of reactive oxygen species (ROS). The coincubation with dimethylfumarate (DMF), an inhibitor of NF-kappaB translocation, reduced the genotoxic effect, thereby underscoring the key role of NF-kappaB in this process. One of the genes induced by NF-kappaB is angiotensinogen. The ensuing proteolytic activity yields angiotensin II, which evokes oxidative stress as well as proinflammatory responses. A modulator of the renin-angiotensin system (RAS), the angiotensin II (Ang II) receptor 1 antagonist, candesartan, yielded a reduction of the AGE-induced DNA damage, connecting the two signal pathways, RAS and AGE signaling. We were able to identify important participants in AGE-induced DNA damage: ROS, NF-kappaB, and Ang II, as well as modulators to prevent this DNA damage: antioxidants, DMF, and AT1 antagonists.

Increased protein glycation in cirrhosis and therapeutic strategies to prevent it.

Glycation of liver proteins by reactive aldehydes formed from the metabolism of ethanol and lipid peroxidation has been implicated in the development of both alcoholic and nonalcoholic liver cirrhosis. Modified proteins are targeted to the proteasome for proteolysis. Release of glycation-free adducts into the circulation may provide a diagnostic "signature" of hepatic protein damage. We quantitatively screened protein glycation, oxidation, and nitrosation adduct residues and free adducts in portal, hepatic, and peripheral venous blood plasma of cirrhotic patients; we also screened the hepatic and peripheral venous blood plasma of control subjects by liquid chromatography-mass spectrometry. There was a remarkable 14-16-fold increase of glyoxal-derived, hydroimidazolone-free adduct in portal and hepatic venous plasma of cirrhotic patients with respect to normal controls. There was only a twofold increase of glycation adduct residues in plasma proteins in cirrhotic patients, which was attributed mainly to decreased albumin turnover. Therapeutic strategies to decrease dicarbonyl compounds may be beneficial, such as dicarbonyl scavengers, glutathione repleting agents, and high-dose thiamine therapy.

Proteins of Thermus thermophilus are resistant to glycation-induced protein precipitation: an evolutionary adaptation to life at extreme temperatures?

In thermophilic bacteria, formation of Maillard products may occur at increased rates because this reaction is favored at higher temperatures. Therefore, specific protective mechanisms against glycation-induced protein precipitation are likely to exist in thermophilic bacteria. Indeed, Thermus thermophilus proteins remained soluble when a cell-free extract of T. thermophilus was incubated at 37 degrees C in the presence of glucose, fructose, or methylglyoxal; whereas E. coli proteins precipitated. In E. coli cell-free extracts, sugar-induced precipitation was accelerated by the addition of 5 microM Fe2+ and inhibited by metal chelators, suggesting that glycoxidation processes are involved in the formation of the precipitate. A low lysine content, endogenous small scavenger molecules, or enzymatic "antiglycation" mechanisms for the degradation of AGEs or their precursors could be excluded as possible causes for the resistance to protein precipitation in T. thermophilus. Therefore, the resistance to glycation-mediated protein precipitation is an endogenous property of thermophilic proteins that was acquired during evolution in environments with high glycation activity.

Monitoring of clopidogrel action: comparison of methods.

BACKGROUND: Clopidogrel is a potent drug for prevention of adverse effects during and after coronary intervention. Increasing experience indicates that a significant proportion of patients do not respond adequately to clopidogrel. Because failure of antiplatelet therapy can have severe consequences, there is need for a reliable assay to quantify the effectiveness of clopidogrel treatment. METHODS: Of 24 healthy volunteers admitted to the study, 18 were treated for 1 week with clopidogrel (300-mg loading dose and 75-mg maintenance dose), and 6 with placebo. Platelet function was monitored by 2 assays, based on flow cytometry and enzyme immunoassay, that measure the phosphorylation status of vasodilator-stimulated phosphoprotein (VASP) and by aggregometry, flow cytometry of P-selectin, and the platelet function analyzer at baseline, on days 1-5, and on day 9 of treatment. RESULTS: Aggregometry and VASP phosphorylation revealed a loss of platelet response to ADP within 12 h after clopidogrel intake. The phosphorylation status of VASP correlated with the inhibition of platelet aggregation. In contrast, neither P-selectin expression nor PFA-100 closure time was a clear indicator of clopidogrel effects on platelets. CONCLUSIONS: VASP phosphorylation assays are reliable for quantifying clopidogrel effects. Because the VASP assay directly measures the function of the clopidogrel target, the P2Y12 receptor, the assay is selective for clopidogrel effects rather than effects of other platelet inhibitors commonly in use.

Processing of protein glycation, oxidation and nitrosation adducts in the liver and the effect of cirrhosis.

BACKGROUND/AIMS: Plasma proteins are modified non-enzymatically in vivo by glycation, oxidation and nitrosation processes. Hepatic extraction of albumin glycated in vitro was reported but it is not clear if plasma proteins glycated in vivo also undergo hepatic extraction. We investigated the hepatic extraction of glycated, oxidised and nitrosated proteins in vivo. METHODS: Protein glycation, oxidation and nitrosation marker residues and free adducts were determined in portal, hepatic and peripheral venous blood plasma of cirrhotic patients and hepatic and peripheral venous blood plasma (as a surrogate of portal venous blood) of control subjects by liquid chromatography-mass spectrometry. RESULTS: There was no evidence for extraction of glycated, oxidised or nitrosated proteins or related free adducts by the liver in control subjects. There was limited extraction of methylglyoxal-modified proteins in cirrhotic patients and twofold increases in the concentrations of fructosyl-lysine and advanced glycation endproduct residues of plasma protein, with respect to controls. Remarkably, glyoxal-derived hydroimidazolone free adduct was increased 14-16-fold probably as a consequence of hepatic lipid peroxidation. CONCLUSIONS: We found no evidence for hepatic extraction of glycated, oxidised and nitrosated proteins or related free adducts in subjects with normal liver function and limited extraction of methylglyoxal-modified protein in cirrhotic subjects.

Advanced glycation endproducts induce changes in glucose consumption, lactate production, and ATP levels in SH-SY5Y neuroblastoma cells by a redox-sensitive mechanism.

Advanced glycation endproducts (AGEs) accumulate on long-lived proteins, including beta-amyloid plaques in Alzheimer's disease, and are suggested to contribute to neuronal dysfunction and cell death. We have investigated the effects of a model AGE upon glucose metabolism and energy production in a neuroblastoma cell line. AGEs decrease cellular ATP levels and increase glucose consumption and lactate production. All of the AGE-induced metabolic changes can be attenuated by antioxidants such as (R+)-alpha-lipoic acid and 17beta-estradiol. These antioxidants may become useful drugs against (AGE-mediated) effects in neurodegeneration through their positive effects on cellular energy metabolism.