N-Acetylcysteine inhibits platelet-monocyte conjugation in patients with type 2 diabetes with depleted intraplatelet glutathione: a randomised controlled trial

AIMS/HYPOTHESIS: The aim of this study was to determine whether oral dosing with N-acetylcysteine (NAC) increases intraplatelet levels of the antioxidant, glutathione (GSH), and reduces platelet–monocyte conjugation in blood from patients with type 2 diabetes. METHODS: In this placebo-controlled randomised crossover study, the effect of oral NAC dosing on platelet–monocyte conjugation and intraplatelet GSH was investigated in patients with type 2 diabetes (eligibility criteria: men or post-menopausal women with well-controlled diabetes (HbA(1c) < 10%), not on aspirin or statins). Patients (n = 14; age range 43–79 years, HbA(1c) = 6.9 ± 0.9% [52.3 ± 10.3 mmol/mol]) visited the Highland Clinical Research Facility, Inverness, UK on day 0 and day 7 for each arm of the study. Blood was sampled before and 2 h after oral administration of placebo or NAC (1,200 mg) on day 0 and day 7. Patients received placebo or NAC capsules for once-daily dosing on the intervening days. The order of administration of NAC and placebo was allocated by a central office and all patients and research staff involved in the study were blinded to the allocation until after the study was complete and the data fully analysed. The primary outcome for the study was platelet–monocyte conjugation. RESULTS: Oral NAC reduced platelet–monocyte conjugation (from 53.1 ± 4.5% to 42.5 ± 3.9%) at 2 h after administration and the effect was maintained after 7 days of dosing. Intraplatelet GSH was raised in individuals with depleted GSH and there was a negative correlation between baseline intraplatelet GSH and platelet–monocyte conjugation. There were no adverse events. CONCLUSIONS/INTERPRETATION: The NAC-induced normalisation of intraplatelet GSH, coupled with a reduction in platelet–monocyte conjugation, suggests that NAC might help to reduce atherothrombotic risk in type 2 diabetes. FUNDING: Chief Scientist Office (CZB/4/622), Scottish Funding Council, Highlands & Islands Enterprise and European Regional Development Fund. TRIAL REGISTRATION: isrctn.org ISRCTN89304265 ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00125-012-2685-z) contains peer-reviewed but unedited supplementary material, which is available to authorised users

Biological variability dominates and influences analytical variance in HPLC-ECD studies of the human plasma metabolome

<p>Abstract</p> <p>Background</p> <p>Biomarker-based assessments of biological samples are widespread in clinical, pre-clinical, and epidemiological investigations. We previously developed serum metabolomic profiles assessed by HPLC-separations coupled with coulometric array detection that can accurately identify <it>ad libitum </it>fed and caloric-restricted rats. These profiles are being adapted for human epidemiology studies, given the importance of energy balance in human disease.</p> <p>Methods</p> <p>Human plasma samples were biochemically analyzed using HPLC separations coupled with coulometric electrode array detection.</p> <p>Results</p> <p>We identified these markers/metabolites in human plasma, and then used them to determine which human samples represent blinded duplicates with 100% accuracy (N = 30 of 30). At least 47 of 61 metabolites tested were sufficiently stable for use even after 48 hours of exposure to shipping conditions. Stability of some metabolites differed between individuals (N = 10 at 0, 24, and 48 hours), suggesting the influence of some biological factors on parameters normally considered as analytical.</p> <p>Conclusion</p> <p>Overall analytical precision (mean median CV, ~9%) and total between-person variation (median CV, ~50–70%) appear well suited to enable use of metabolomics markers in human clinical trials and epidemiological studies, including studies of the effect of caloric intake and balance on long-term cancer risk.</p

Molecular dynamics study of protein-oligosaccharide interaction mechanisms in chitinases engineered towards neolectins

Protein-carbohydrate interactions are essential in many biomolecular recognition events, such as inflammation, cell-cell recognition and adhesion, immunochemistry and human blood group type determination. A great deal of interest has thus arisen in isolation of medically important oligosaccharides. In this study our aim is to obtain atomic level information of the binding and to gain a deeper understanding of the factors determining the interactions between an oligosaccharide and a protein by using extended molecular dynamic (MD) simulations with explicit water. In addition to a conventional force field, a novel soft-core potential 1,2 originally developed for a priori modelling of surface loops of proteins without additional restraints was used here to the whole binding site of the modelled protein. The study concentrates on fungal 42kDa chitinase from Trichoderma harzianum 3, a naturally chitin degrading enzyme, containing an extended binding site providing a number of strong and specific interactions with up to 6-7 sugar units. Since these interactions come from a limited number of loops, the structure of chitinase (-barrel fold) provides an excellent platform for directed evolution studies. By mutating first the functional amino acid(s) and then altering the substrate specificity by locally directed saturation mutagenesis the functionality of a protein can be changed from a degrading enzyme to a specific binder. While experimentally determined three-dimensional structures were not available for the enzyme of interest, structural models were constructed based on the known structures of homologues. Experimentally determined sugar-protein complex structures of related chitinases were used in the initial simulations to evaluate the suitability of the force field parameters and simulation procedures. Classical MD (Gromacs) with a conventional force field and with a soft-core potential 1,2 is used to explore the conformational space of the chitinase loops and to study the functional behavior of the N-acetylglucosamine oligosaccharides and their derivates. Trajectories obtained from the simulations are used in analyzing the binding, especially the hydrogen bonding and hydrophobic interactions occurring via N/O-acetyl or O-methyl groups. The results from modeling are compared with the experimental data (mutagenesis, mass spectroscopy and nuclear magnetic resonance). Computational studies with the experimental work aim at development of neolectins, i.e. proteins selectively binding to given oligosaccharide structures, achieved by deactivating and engineering fungal chitinases towards the desired specificity and affinity. 1. Tappura, K.; Lahtela-Kakkonen, M; Teleman, O. J. Comput. Chem. 2000, 21(5), 388-97. 2. Tappura, K. Proteins 2001, 44(3), 167-79. 3. Boer, H.; Munck, N.; Natunen, J.; Wohlfahrt, G.; Söderlund, H.; Renkonen, O.; Koivula, A. (submitted 2004)