Metabolic profiling reveals differences in concentrations of oxylipins and fatty acids secreted by the infrapatellar fat pad of donors with end-stage osteoarthritis and normal donors.

OBJECTIVE: The infrapatellar fat pad (IPFP) in the knee joint is hypothesized to contribute to osteoarthritis (OA) development by the IFPF possibly by influencing inflammatory processes. Oxylipins are essential mediators in the inflammatory process. We undertook this study to investigate secretion by the IFPF of fatty acids and oxylipins derived from those fatty acids. METHODS: IPFP explants from 13 OA donors undergoing joint replacement surgery and from 10 normal donors postmortem were cultured for 24 hours, and supernatants (fat-conditioned medium [FCM]) were collected. Liquid chromatography tandem mass spectrometry detected fatty acids and oxylipins in FCM samples. Univariate and multivariate (partial least-squares discriminant analysis [PLS-DA]) analyses were performed, followed by pathway analysis. To validate these outcomes, a second set of OA FCM samples was measured (n=23). RESULTS: Twenty-nine oxylipins and fatty acids could be detected in FCM. Univariate analysis showed no differences between normal donor and OA donor FCM; however, PLS-DA revealed an oxylipin/fatty acid profile consisting of 14 mediators associated with OA (accuracy rate 72%). The most important contributors to the model were lipoxin A4 (decreased), thromboxane B2 (increased), and arachidonic acid (increased). The statistical model predicted 64% of the second set of OA FCM samples correctly. Pathway analysis indicated differences in individual mediators rather than in complete pathways. CONCLUSION: The IPFP secretes multiple and different oxylipins, and a subset of these oxylipins provides a distinctive profile for OA donors. It is likely that the observed changes are regulated by the OA process rather than being a consequence of basal metabolism changes, as an increase in fatty acid levels was not necessarily associated with an increase in oxylipins derived from that fatty acid.

The acute effects of olanzapine on ghrelin secretion, CCK sensitivity, meal size, locomotor activity and body temperature.

OBJECTIVE: Significant weight gain is a problematic side effect of treatment with the antipsychotic drug olanzapine (OLA). Previous studies in rats suggest that one of the contributing factors is an impairment in satiation that results in increased food intake. However, the mechanisms underlying this impairment in satiation remain largely unclear. METHODS AND RESULTS: In this study, we determined the effect of OLA on levels of leptin, insulin, ghrelin, cholecystokinin (CCK), glucagon-like peptide-1, peptide YY and amylin in male rats that had received a fixed amount of food. OLA did not affect the secretion of any of these hormones, except for ghrelin levels, which were increased compared with controls. Furthermore, when ghrelin levels were determined in rats just before they received their meal, OLA caused a significant increase in ghrelin levels compared with controls, whereas OLA failed to affect baseline ghrelin levels. Next, we investigated the effect of OLA on the efficacy of CCK to reduce meal size. With coadministration, OLA pretreatment counteracted the reduction in meal size by CCK, although there was no significant interaction between the treatments. Finally, telemetry measurements revealed that acute OLA treatment causes a temporary decrease in both locomotor activity and body core temperature. CONCLUSION: Taken together, this study shows that acute injection of OLA selectively increases meal-related ghrelin secretion and this may partially underlie the impairment in satiation by OLA.

The art and practice of systems biology in medicine: mapping patterns of relationships.

Systems biology has developed in recent years from a technology-driven enterprise to a new strategic tool in Life Sciences, particularly for innovative drug discovery and drug development. Combining the ultimate in systems phenotyping with in-depth investigations of biomolecular mechanisms will enable a revolution in our understanding of disease pathology and will advance translational medicine, combination therapies, integrative medicine, and personalized medicine. A prerequisite for deriving the benefits of such a systems approach is a reliable and well-validated bioanalytical platform across complementary measurement modalities, especially transcriptomics, proteomics, and metabolomics, that operates in concert with a megavariate integrative biostatistical/bioinformatics platform. The applicable bioanalytical methodologies must undergo an intense development trajectory to reach an optimal level of reliable performance and quantitative reproducibility in daily practice. Moreover, to generate such enabling systems information, it is essential to design experiments based on an understanding of the complexity and statistical characteristics of the large data sets created. Novel insights into biology and system science can be obtained by evaluating the molecular connectivity within a system through correlation networks, by monitoring the dynamics of a system, or by measuring the system responses to perturbations such as drug administration or challenge tests. In addition, cross-compartment communication and control/feed-back mechanisms can be studied via correlation network analyses. All these data analyses depend critically upon the generation of high-quality bioanalytical platform data sets. The emphasis of this paper is on the characteristics of a bioanalytical platform that we have developed to generate such data sets. The broad applicability of Systems Biology in pharmaceutical research and development is discussed with examples in disease biomarker research, in pharmacology using system response monitoring, and in cross-compartment system toxicology assessment.

Capillary electrochromatography/nanoelectrospray mass spectrometry for attomole characterization of peptides.

The successful coupling of capillary electrochromatography (CEC) to an ion trap mass spectrometer via a nanoelectrospray interface (nESI) is described. Using a conductively coated tip butted to the end of a CEC column, it was possible to obtain a stable spray without any sheath liquid being employed. Selected small peptides were separated with CEC columns (100 microm i.d./25 cm long) packed with 3 microm Hypersil C8 or C18 bonded silica particles with an eluent composed of ammonium acetate/acetonitrile. Peptide mixtures of desmopressin, peptide A, oxytocin, carbetocin and [Met(5)]-enkephalin were detected in the mid-attomole range, which is the lowest amount analyzed using CEC combined with MS detection. It was also observed that sensitivity can be compromised at higher separation voltages. We demonstrate that CEC/nESI-MS, at the current stage of development, represents one of the most sensitive systems for peptide analysis.

Quantification of perifosine, an alkylphosphocholine anti-tumour agent, in plasma by pneumatically assisted electrospray tandem mass spectrometry coupled with high-performance liquid chromatography.

An HPLC assay with tandem mass spectrometric detection in the positive-ion Turbo-Ion-Spray (TISP) mode for the fast and sensitive determination of perifosine ((I), D-21266) in human plasma was developed, utilising the structural analogue, miltefosine ((II), D-18506), as internal standard. Automated solid-phase extraction of diluted plasma samples, based on 250-microl plasma aliquots, at pH 6.5, allowed a reliable quantification of perifosine down to 4 ng/ml. Injection of 200 microl of plasma extracts onto a 100x3 mm normal-phase analytical column at a flow-rate of 0.5 ml/min provided retention-times of 2.4 and 2.1 min for perifosine (I) and the internal standard (II), respectively. The standard curves were linear from 4 to 2000 ng/ml using weighted linear regression analysis (1/Y2). The inter-assay and intra-assay accuracies for the calibration standards were within +0.9% and -0.2%, exhibiting precisions (C.V.) of +/-6.5 and +/-7.3%, respectively. Up to 100 unknowns may be analysed each 24 h per analyst.

Screening and confirmatory determination of ractopamine residues in calves treated with growth promoting doses of the beta-agonist.

Ractopamine (RCT) is a phenethanolamine member of the family of beta-adrenergic agonists (beta-agonists). This class of compounds have become notable for their properties of enhancing the growth rates of farm animal species but are not licensed for use in Europe. An ELISA procedure employing a polyclonal antibody raised in a goat was developed to detect RCT residues in bovine urine samples. The assay had a high sensitivity (calibration curve mid-point of 22 pg per well), allowing the analysis of urine samples without the need for sample clean-up. In addition, an LC-MS-MS confirmatory procedure was developed which was able to act as a confirmatory procedure for the ELSA results. Four calves were orally treated with RCT (0.1 mg kg-1 body mass for 17 d) and urine samples collected were assayed by both analytical procedures. It was observed that RCT residues were excreted mainly in the form of glucuronides and deconjugation could be achieved using two different sources of the enzyme beta-glucuronidase (Helix pomatia and Escherichia coli). High concentrations of RCT residues were found throughout the medication period (44-473 ng ml-1; LC-MS-MS data) and remained present for several days following removal of the drug from the diet. RCT residues were no longer detectable 2 weeks after withdrawal. Good agreement (r2 = 0.73) was achieved between the ELISA and LC-MS-MS results, especially when sample deconjugation was applied to the urine samples for sets of analyses. The results show that an effective screening and confirmatory system was devised to detect RCT residues in urine samples taken during treatment and close to withdrawal. However, alternative matrices may have to be selected to allow the illegal use of the substance to be detected following prolonged withdrawal times.

Characterization of sulfur mustard induced structural modifications in human hemoglobin by liquid chromatography--tandem mass spectrometry.

In this paper we describe the use of tandem mass spectrometry to identify modified sites in human hemoglobin after in vitro exposure to bis(2-chloroethyl) sulfide (sulfur mustard). Globin isolated from human whole blood which had been exposed to sulfur mustard was degraded with trypsin, and the digests were analyzed by micro LC/MS. Alkylated tryptic fragments (alpha-T1, alpha-T4, alpha-T6, alpha-T9, beta-T1, beta-T9, beta-T10, beta-T11, and beta-T10-S-S-beta-T12) could be tentatively assigned upon comparison with a digest from nonexposed globin. Subsequent tandem mass spectrometry of these peptides allowed unambiguous assignment of 5 specific modified residues: alpha-Val-1, alpha-His-20, beta-Val-1, beta-His-77, and beta-His-97. The results demonstrate the usefulness of microbore LC in combination with tandem mass spectrometry for the structural determination of chemically modified peptides and proteins.

Combined supercritical-fluid chromatography/mass spectrometry in the analysis of diuron in plasma using on-line phase-system switching.

On-line sample pretreatment by means of the phase-system switching approach is an interesting technique for the analysis of aqueous samples, e.g., plasma, by means of supercritical-fluid chromatography. In order to analyse plasma samples the following analytical procedure is used. The plasma sample is injected on to a short precolumn, which is washed with water and subsequently dried with nitrogen. Next, the solutes are desorbed with the supercritical mobile phase, analysed with packed-column supercritical-fluid chromatography and detected with either a UV detector or a mass spectrometer, equipped with a moving-belt interface. The herbicide diuron is selected as a test compound to study the feasibility of this approach. Using a selective detector the procedure is sufficiently sensitive to detect diuron in plasma, but not appropriate to detect the diuron metabolites in a post-mortem plasma sample. These have been identified with liquid chromatography/mass spectrometry. The detection limit of diuron in plasma using the procedure described is about 30 ng/mL.

Identification of diuron and four of its metabolites in human postmortem plasma and urine by LC/MS with a moving-belt interface.

Unknown compounds that were not amenable to GC/MS were found during routine benzodiazepine HPLC screening in a postmortem case. The apparent thermolability made the application of liquid chromatography with mass spectrometry mandatory. The moving-belt interface was used because of its value for identification based on the use of both electron impact and chemical ionization, which provided information on both structure and molecular weight. The herbicide diuron and four of its metabolites were identified in plasma and urine and had a total concentration as high as 100 mg/L. Metabolism via demethylation and hydroxylation appeared to be the major routes.

Induction of triterpene biosynthesis by elicitors in suspension cultures of Tabernaemontana species.

Treatment of suspension cultures of some Tabernaemontana species (Apocynaceae) with elicitors (e.g. cellulase, Candida albicans) result in a rapid de novo production of antimicrobial active triterpenes. The triterpenes are identified as ursene carboxylic acid derivatives. These triterpenes are not produced by an elicited cell suspension culture of Catharanthus roseus, another Apocynaceae.