No abstract

Quantitative metabolite profiling in biological samples has the potential to reflect physiological status and to identify disease associated disturbances in metabolic networks. However, this approach is hampered by a wide range of preanalytical variables. Hence, the aim of our study was to develop a standardized preanalytical protocol for metabolite profiling of amino acids and acylcarnitines in human blood. Amino acids and acylcarnitines were simultaneous analyzed after butylation of 3 lL dried blood or 10 lL whole blood, serum and anticoagulated plasma using electrospray tandem-mass spectrometry. The influence of exogenous and endogenous preanalytical variables was investigated in healthy volunteers. Different sampling materials and anticoagulants for blood taking were investigated. Concentrations of long-chain acylcarnitines were 5-fold higher in EDTA-whole blood or dried whole blood compared to serum and anticoagulated plasma. Significant differences in amino acid concentrations were found for capillary versus venous blood taking. Fasting for 8 h before specimen collection minimized the nutritional influence. Physical activity significantly alters amino acid and short chain acylcarnitine concentrations. As a result of our preanalytical investigation we developed a pre-treatment protocol based on EDTA whole blood dried on filter paper to reduce the preanalytical variability and facilitate reproducible quantitative metabolite profiling in clinical trials.

The signaling pathway(s) and molecular target(s) for 5,6-dimethylxanthenone-4-acetic acid (DMXAA), a tumor vascular disrupting agent in late stages of clinical development, are still undefined. As an approach toward identifying potential targets for DMXAA, a tritiated azido-analog of DMXAA was used to probe for cellular binding proteins. More than 20 cytosolic proteins from murine splenocytes, RAW 264.7 cells, and the HECPP immortalized endothelial cells were photoaffinity-labeled. Although no protein domain, fold, or binding site for a specific ligand was found to be shared by all the candidate proteins, essentially all were noted to be oxidizable proteins, implicating a role for redox signaling in the action of DMXAA. Consistent with this hypothesis, DMXAA caused an increase in concentrations of reactive oxygen species (ROS) in RAW264.7 cells during the first 2 hours. This increase in ROS was suppressed in the presence of the antioxidant, N-acetyl-L-cysteine, which also suppressed DMXAA-induced cytokine production in the RAW 264.7 cells with no effects on cell viability. Short interfering RNA (siRNA)-mediated knockdown of one of the photoaffinity-labeled proteins, superoxide dismutase 1, an ROS scavenger, resulted in an increase in tumor necrosis factor-alpha production by RAW 264.7 cells in response to DMXAA compared with negative or positive controls transfected with nontargeting or lamin A/C-targeting siRNA molecules, respectively. The results from these lines of study all suggest that redox signaling plays a central role in cytokine induction by DMXAA.

DMXAA (5,6-dimethylxanthenone-4-acetic acid) is in late stages of clinical development as a tumor vascular disrupting agent. The signalling pathway(s) and molecular target(s) for this drug are still undefined. As an approach towards identifying potential targets for DMXAA, a tritiated azido-analog of DMXAA is used in the studies here to probe for cellular binding proteins. Unexpectedly, over 20 proteins were photo-affinity labelled from the cytosolic protein extracts from murine spleen cells and the RAW 264.7 murine macrophage-like cell line. While no protein domain, fold or binding site for a specific ligand was found to be shared by all the candidate proteins, almost all were noted to be oxidisable proteins, implicating a role for redox cycling in the action of DMXAA. Consistent with this hypothesis, we showed that DMXAA caused an accumulative increase in concentrations of reactive oxygen species (ROS) in RAW 264.7 cells over the first two hours. This increase in ROS was suppressed in the presence of the anti-oxidant, N-acetyl-L-cysteine, which also suppressed DMXAA-induced cytokine production in the RAW 264.7 cells with no effects on cell viability. SiRNA knock-down in RAW 264.7 cells of one of the photolabelled proteins, superoxide dismutase 1, an ROS scavenger resulted in an increase in tumour necrosis factor-alpha production in response to DMXAA compared with controls treated with transfection reagent alone, or with unrelated siRNA molecules. Knock-down of two other photoaffinity labelled proteins which do not have direct ROS scavenging properties, thioredoxin and protein disulfide isomerase 6, had no significant effect on tumour necrosis factor-alpha production. The results from these three lines of study have led us to speculate on a role of modulation of redox signalling in cytokine induction by DMXAA. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 24.