The expression of anaerobic metabolites in sweat and sebum from human skin subjected to intermittent and continuous mechanical loading.

Pressure ulcers (PUs) represent a substantial burden to both patients and healthcare providers. Accordingly, effective prevention strategies should follow early detection of PUs. Anaerobic metabolites, such as lactate and pyruvate, are promising noninvasive biomarkers indicative of tissue ischaemia, one of the major mechanisms leading to PU development. The aim of this study was to investigate if the temporal release profile of these metabolites in sweat and sebum is sensitive to detect local tissue changes resulting from prolonged mechanical loads. The sacrum of healthy volunteers was subjected to two different loading protocols. After a baseline measurement, the left and right side of the sacrum were subjected to continuous and intermittent loading regimen, respectively, at a pressure of 100 mmHg. Biomarker samples were collected every 20 min, with a total experimental time of 140 min. Sweat was collected at 37 ∘C and 80% relative humidity, and sebum at ambient conditions, from 11 to 13 volunteers, respectively. Both samples were analysed for lactate and pyruvate concentrations using ultra-high performance supercritical fluid chromatography mass spectrometry. Prior to analysis metabolite concentrations were normalized to individual baseline levels and, in the case of sweat, additional normalization was performed to an unloaded control site to account for fatigue of sweat glands. Although substantial variability was present, the temporal release profiles of both sweat and sebum metabolites reflected the applied loading regimen with increased levels upon load application, and recovery to baseline levels following load removal. Highest relative increases were 20% and 30% for sweat lactate and pyruvate, respectively, and 41% for sebum lactate. Sebum pyruvate was not present in quantifiable amounts. There was a linear correlation between the individual responses to intermittent and continuous loading. The present study revealed that metabolite biomarkers in both sweat and sebum were sensitive to the application of mechanical loads, indicative of local ischaemia within skin and soft tissues. Similar trends in metabolic biomarkers were observed in response to intermittent and continuous loading regimens in both sweat and sebum. Metabolites represent a potential means to monitor the health of loaded skin and soft tissues informing timely interventions of PU prevention.

Polyhydroxyalkanoate (PHA) biosynthesis from structurally unrelated carbon sources by a newly characterized Bacillus spp.

A newly acquired polyhydroxyalkanoate (PHA) producing Bacillus spp. was identified to be a strain of Bacillus cereus using a range of microbiological and molecular techniques. This strain, named B. cereus SPV, was found to be capable of using a wide range of carbon sources including glucose, fructose, sucrose, various fatty acids and gluconate for the production of PHAs, an advantage for the commercial production of the polymers. The media used for the polymer production was novel in the context of the genus Bacillus. The PHA, once produced, was found to remain at a constant maximal concentration, without any degradation, a great advantage for the commercial production of the PHAs. This particular strain of Bacillus spp. was able to synthesize various PHAs with 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV) and 4-hydroxybutyrate (4HB)-like monomer units from structurally unrelated carbon sources such as fructose, sucrose and gluconate. This is the first report of the incorporation of a 4HB related monomer containing PHA by the genus Bacillus and from structurally unrelated carbon sources. The PHAs isolated had molecular weights ranging between (0.4 and 0.8) x 10(6) and low polydispersity index values (M(W)/M(N)) ranging from 2.6 to 3.4.

Pyridoxal phosphate de-activation by pyrroline-5-carboxylic acid. Increased risk of vitamin B6 deficiency and seizures in hyperprolinemia type II.

We previously identified vitamin B6 deficiency in a child presenting with seizures whose primary diagnosis was the inherited disorder hyperprolinemia type II. This is an unrecognized association, which was not explained by diet or medication. We hypothesized that pyridoxal phosphate (vitamin B6 coenzyme) was de-activated by L-Delta(1)-pyrroline-5-carboxylic acid, the major intermediate that accumulates endogenously in hyperprolinemia type II. The proposed interaction has now been investigated in vitro with high resolution 1H nuclear magnetic resonance spectroscopy and mass spectrometry at a pH of 7.4 and temperature of 310 K. Three novel adducts were identified. These were the result of a Claisen condensation (or Knoevenagel type of reaction) of the activated C-4 carbon of the pyrroline ring with the aldehyde carbon of pyridoxal phosphate. The structures of the adducts were confirmed by a combination of high performance liquid chromatography, nuclear magnetic resonance, and mass spectrometry. This interaction has not been reported before. From preliminary observations, pyrroline-5-carboxylic acid also condenses with other aromatic and aliphatic aldehydes and ketones, and this may be a previously unsuspected generic addition reaction. Pyrroline-5-carboxylic acid is thus found to be a unique endogenous vitamin antagonist. Vitamin B6 de-activation may contribute to seizures in hyperprolinemia type II, which are so far unexplained, but they may be preventable with long term vitamin B6 supplementation.

Catalytic mechanism of a C-C hydrolase enzyme: evidence for a gem-diol intermediate, not an acyl enzyme.

2-Hydroxy-6-keto-nona-2,4-diene 1,9-dioic acid 5,6-hydrolase (MhpC) from Escherichia coli catalyses the hydrolytic cleavage of the extradiol ring fission product on the phenylpropionate catabolic pathway and is a member of the alpha/beta hydrolase family. The catalytic mechanism of this enzyme has previously been shown to proceed via initial ketonization of the dienol substrate (Henderson, I. M. J., and Bugg, T. D. H. (1997) Biochemistry 36, 12252-12258), followed by stereospecific fragmentation. Despite the implication of an active site serine residue in the alpha/beta hydrolase family, attempts to verify a putative acyl enzyme intermediate by radiochemical trapping methods using a (14)C-labeled substrate yielded a stoichiometry of <1% covalent intermediate, which could be accounted for by nonenzymatic processes. In contrast, incorporation of 5-6% of two atoms of (18)O from H(2)(18)O into succinic acid was observed using the natural substrate, consistent with the reversible formation of a gem-diol intermediate. Furthermore, time-dependent incorporation of (18)O from H(2)(18)O into the carbonyl group of a nonhydrolysable analogue 4-keto-nona-1,9-dioic acid was observed in the presence of MhpC, consistent with enzyme-catalyzed attack of water at the ketone carbonyl. These results favor a catalytic mechanism involving base-catalyzed attack of water, rather than nucleophilic attack of an active site serine. The implication of this work is that the putative active site serine in this enzyme may have an alternative function, for example, as a base.

Simplified sample preparation for the analysis of oligonucleotides by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry.

Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) for the analyses of oligonucleotides has generally been carried out using negative ionisation conditions, usually following ammonium ion-exchange chromatography and the addition of ammonium buffers to the MALDI matrix. The molecular ion region is complex, due to the varying degrees of ammoniation of the phosphate backbone of the oligonucleotide. This gives rise to an overall decrease in sensitivity compared with similar size peptides and can cause ambiguity of assignment of the relative molecular mass of the sample. This study describes the use of H(+) ion exchange resin in situ as the means of removing alkali metal ions from the phosphate backbone of the oligonucleotide. An increase in resolution, sensitivity and identification of the molecular species is reported, with little or no difference in sensitivity observed between positive or negative ionisation spectra. This method is now used for routine screening of synthetic oligonucleotides with a gain in sensitivity of 1-2 orders of magnitude compared with previous methods, and mass assignment errors of +/-0.1% are routinely recorded for externally calibrated data.

Mass spectrometric analysis in combinatorial chemistry.

The continuing success of the combinatorial approach is heavily reliant on analytical methodologies, which allow for the rapid and accurate characterisation of medicinally relevant molecules from compound libraries. Mass spectrometry has recently been touted as the most suitable tool for a range of combinatorial applications such as structural elucidation and screening. The refinement of conventional methods, developments of techniques such as Fourier transform ion cyclotron resonance and new screening methodologies have allowed the medicinal chemist to tackle the growing analytical challenges posed by combinatorial chemistry.

Determination of hydrazine in biofluids by capillary gas chromatography with nitrogen-sensitive or mass spectrometric detection.

Plasma and liver levels of hydrazine were determined at 10, 30, 90 and 270 min in rats given 0.09, 0.27, 0.84 and 2.53 mmol of hydrazine per kg body weight orally by capillary gas chromatography-mass spectrometry of its pentafluorobenzaldehyde adduct (DFBA, m/z 388) using selected ion monitoring with 15N2-labelled hydrazine as the internal standard (adduct, m/z 390). The mean half-life for hydrazine in the plasma was approximately 2 h but varied with dose. Urinary excretion (0-24 h) of hydrazine and its metabolite acetylhydrazine were determined employing nitrogen-phosphorus detection of the adducts utilising a novel internal standard, pentafluorophenylhydrazine, the adduct of which structurally resembles DFBA. The fraction of the original dose excreted as hydrazine (and acetylhydrazine) declined with increasing dose.