testing of the hemaPEN microsampling device for the quantification of acetaminophen in human blood.

The hemaPEN is a liquid microsampling device for the reproducible collection and storage of blood samples as dried blood spots, for subsequent quantitative analysis. We examined the device's ability to collect accurate and precise blood volumes, at different hematocrit levels, via studies using acetaminophen in human blood. We also investigated the impact of different user training approaches on device performance.

Analysis of polar urinary metabolites for metabolic phenotyping using supercritical fluid chromatography and mass spectrometry.

Supercritical fluid chromatography (SFC) is frequently used for the analysis and separation of non-polar metabolites, but remains relatively underutilised for the study of polar molecules, even those which pose difficulties with established reversed-phase (RP) or hydrophilic interaction liquid chromatographic (HILIC) methodologies. Here, we present a fast SFC-MS method for the analysis of medium and high-polarity (-7≤cLogP≤2) compounds, designed for implementation in a high-throughput metabonomics setting. Sixty polar analytes were first screened to identify those most suitable for inclusion in chromatographic test mixtures; then, a multi-dimensional method development study was conducted to determine the optimal choice of stationary phase, modifier additive and temperature for the separation of such analytes using SFC.

Lipidomics comparing DCD and DBD liver allografts uncovers lysophospholipids elevated in recipients undergoing early allograft dysfunction.

Finding specific biomarkers of liver damage in clinical evaluations could increase the pool of available organs for transplantation. Lipids are key regulators in cell necrosis and hence this study hypothesised that lipid levels could be altered in organs suffering severe ischemia. Matched pre- and post-transplant biopsies from donation after circulatory death (DCD, n = 36, mean warm ischemia time = 2 min) and donation after brain death (DBD, n = 76, warm ischemia time = none) were collected.

Extension and limits of the network of coupled motions correlated to hydride transfer in dihydrofolate reductase.

Enzyme catalysis has been studied extensively, but the role of enzyme dynamics in the catalyzed chemical conversion is still an enigma. The enzyme dihydrofolate reductase (DHFR) is often used as a model system to assess a network of coupled motions across the protein that may affect the catalyzed chemical transformation. Molecular dynamics simulations, quantum mechanical/molecular mechanical studies, and bioinformatics studies have suggested the presence of a "global dynamic network" of residues in DHFR.

Evidence of altered phosphatidylcholine metabolism in Alzheimer's disease.

Abberant lipid metabolism is implicated in Alzheimer's disease (AD) pathophysiology, but the connections between AD and lipid metabolic pathways are not fully understood. To investigate plasma lipids in AD, a multiplatform screen (n = 35 by liquid chromatography-mass spectrometry and n = 35 by nuclear magnetic resonance) was developed, which enabled the comprehensive analysis of plasma from 3 groups (individuals with AD, individuals with mild cognitive impairment (MCI), and age-matched controls). This screen identified 3 phosphatidylcholine (PC) molecules that were significantly diminished in AD cases.

Metabolic phenotype of the healthy rodent model using in-vial extraction of dried serum, urine, and cerebrospinal fluid spots.

High-throughput multiplatform metabolomics experiments are becoming an integral part of clinical and systems biology research. Such methods call for the adoption of robust sample storage and transport formats for small volumes of biofluids. One such format is the dried biofluid spot, which combines small volume requirements with easy portability.

Synthesis of radiolabeled nicotinamide cofactors from labeled pyridines: versatile probes for enzyme kinetics.

(14)C-labeled nicotinamide cofactors are widely employed in biomedical investigations, for example, to delineate metabolic pathways, to elucidate enzymatic mechanisms, and as substrates in kinetic isotope effect (KIE) experiments. The (14)C label has generally been located remote from the reactive position, frequently at the adenine ring. Rising costs of commercial precursors and disruptions in the availability of enzymes required for established syntheses have recently made the preparation of labeled nicotinamides such as [Ad-(14)C]NADPH unviable.

A [2+2] cross-photodimerisation of photostable olefins via a three-component cocrystal solid solution.

A ditopic hydrogen-bond-donor template in the form of resorcinol facilitates a [2+2] cross-photodimerisation of 4-Cl-stilbazole and 4-Me-stilbazole in a rare cocrystal solid solution. A photoreaction does not proceed with the olefins individually or as a solid solution composed solely of the two olefins.

Self-assembled enzymatic monolayer directly bound to a gold surface: activity and molecular recognition force spectroscopy studies.

Escherichia coli dihydrofolate reductase (ecDHFR) has one surface cysteine, C152, located opposite and distal to the active site. Here, we show that the enzyme spontaneously assembles on an ultraflat gold surface as a homogeneous, covalently bound monolayer. Surprisingly, the activity of the gold-immobilized ecDHFR as measured by radiographic analysis was found to be similar to that of the free enzyme in solution.

Triple isotopic labeling and kinetic isotope effects: exposing H-transfer steps in enzymatic systems.

Kinetic isotope effect (KIE) studies can provide insight into the mechanism and kinetics of specific chemical steps in complex catalytic cascades. Recent results from hydrogen KIE measurements have examined correlations between enzyme dynamics and catalytic function, leading to a surge of studies in this area. Unfortunately, most enzymatic H-transfer reactions are not rate limiting, and the observed KIEs do not reliably reflect the intrinsic KIEs on the chemical step of interest.

Microscale synthesis and kinetic isotope effect analysis of ()-[Ad-C, 4-H] NADPH and ()-[Ad-H,4-H] NADPH.

We present a one-pot chemo-enzymatic microscale synthesis of NADPH with two different patterns of isotopic labels: ()-[Ad-C,4-H] NADPH and ()-[Ad-H,4-H] NADPH. These co-factors are required by an enormous range of enzymes, and isotopically labeled nicotinamides are consequently of significant interest to researchers. In the current procedure, [Ad-C] NAD and [Ad-H] NAD were phosphorylated by NAD kinase to produce [Ad-C] NADP and [Ad-H] NADP, respectively.