Quantification of F(2)-isoprostanes is considered a reliable index of the oxidative stress status in vivo. Several immunoassays and chromatography/mass spectrometry-based assays are available for 15-F(2t)-isoprostane quantification. However, it remains unclear if results of immunoassays using different assays can be compared with those of liquid chromatography/mass spectrometry (LC/MS) assays. Previous studies comparing enzyme-linked immunosorbent assay (ELISA) and more specific gas chromatography/mass spectrometry assays have already indicated that ELISAs may overestimate 15-F(2t)-isoprostane concentrations in human plasma. Concentrations of 15-F(2t)-isoprostane in 25 human plasma and urine samples were measured by three commercially available ELISA assays (Assay Designs, Cayman Chemical and Oxford Biomedical Research) and compared with the concentrations measured with a validated, semi-automated high-throughput HPLC tandem mass spectrometry assay (LC/LC-MS/MS). All three ELISAs measured substantially higher 15-F(2t)-isoprostane concentrations (2.1-182.2-fold higher in plasma; 0.4-61.9-fold higher in urine) than LC/LC-MS/MS. Utilization of solid-phase extraction (SPE) columns, especially isoprostane affinity purification columns, brought ELISA isoprostane urine concentrations closer to the LC/LC-MS/MS results. However, SPE did not have much of an effect on ELISA plasma concentrations which remained significantly higher than corresponding LC/LC-MS/MS results. A poor correlation not only between LC/LC-MS/MS and immunoassay results, but also among the immunoassays was found. Especially in plasma, ELISAs grossly overestimate 15-F(2t)-isoprostane concentrations and are not comparable with each other or with LC/LC-MS/MS. It is most disturbing that a sample with relatively high concentrations measured with one ELISA may show low concentrations with another ELISA, and vice versa, potentially affecting the conclusions drawn from such data. The use of specific mass spectrometry-based assays seems advisable.

IntroductionStatins are cholesterol-lowering drugs with pleiotropic activities including inhibition of isoprenylation and reduction of signals driving cell proliferation and survival responses.MethodsIn this study we evaluated the effects of lovastatin acid and lactone on breast cancer MDAMB231 and MDAMB468 cells using a combination of proteomic and metabonomic profiling techniques.ResultsLovastatin inhibited proliferation of breast cancer cell lines. MDAMB231 cells were more sensitive to its effects, and in most cases lovastatin acid showed more potency towards the manipulation of protein expression than lovastatin lactone. Increased expression of Rho inhibitor GDI-2 stabilized the non-active Ras homolog gene family member A (RhoA) leading to a decreased expression of its active, membrane-bound form. Its downstream targets cofilin, CDC42 and G3BP1 are members of the GTPase family affected by lovastatin. Our data indicated that lovastatin modulated the E2F1-pathway through the regulation of expression of prohibitin and retinoblastoma (Rb). This subsequently leads to changes of E2F-downstream targets minichromosome maintenance protein 7 (MCM7) and MutS homolog 2 (MSH2). Lovastatin also regulated the AKT-signaling pathway. Increased phosphatase and tensin homolog (PTEN) and decreased DJ-1 expression lead to a down-regulation of the active pAkt. Lovastatin's involvement in the AKT-signaling pathway was confirmed by an upregulation of its downstream target, tumor progressor NDRG1. Metabolic consequences to lovastatin exposure included suppression of glycolytic and Krebs cycle activity, and lipid biosynthesis.ConclusionsThe combination of proteomics and metabonomics enabled us to identify several key targets essential to the antitumor activity of lovastatin. Our results imply that lovastatin has the potential to reduce the growth of breast cancer cells.

Mycophenolic acid (MPA) is used as an immunosuppressant after organ transplantation and for the treatment of immune diseases. There is increasing evidence that therapeutic drug monitoring and plasma concentration-guided dose adjustments are beneficial for patients to maintain immunosuppressive efficacy and to avoid toxicity. The major MPA metabolite that can be found in high concentrations in plasma is MPA glucuronide (MPAG). A metabolite usually present at lower concentrations, MPA acyl-glucuronide (AcMPAG), has been implicated in some of the adverse effects of MPA. We developed and validated an automated high-throughput ultra-high performance chromatography-tandem mass spectrometry (U-HPLC-MS/MS) assay using liquid-handling robotic extraction for the quantification of MPA, MPAG, and AcMPAG in human EDTA plasma and urine. The ranges of reliable response were 0.097 (lower limit of quantitation) to 200 μg/mL for MPA and MPAG and 0.156– 10 μg/mL for AcMPAG in human urine and plasma. The inter-day accuracies were 94.3–104.4%, 93.8–105.0% and 94.4–104.7% for MPA, MPAG and AcMPAG, respectively. Inter-day precisions were 0.7–7.8%, 0.9–6.9% and 1.6–8.6% for MPA, MPAG and AcMPAG. No matrix interferences, ion suppression/enhancement and carry-over were detected. The total assay run time was 2.3 min. The assay met all predefined acceptance criteria and the quantification of MPA was successfully cross-validated with an LC-MS/MS assay routinely used for clinical therapeutic drug monitoring. The assay has proven to be robust and reliable during the measurement of samples from several pharmacokinetics trials.

The bacterium Actinoplanes sp. ATCC 53771 is known to perform drug metabolism of several xenobiotics similarly to humans. We identified a cytochrome P450 enzyme from this strain, CYP107E4, and expressed it in Escherichia coli using the pET101 vector. The purified enzyme showed the characteristic reduced-CO difference spectra with a peak at 450 nm, indicating the protein is produced in the active form with proper heme incorporation. The CYP107E4 enzyme was found to bind the drug diclofenac. Using redox enzymes from spinach, the reconstituted system is able to produce hydroxylated metabolites of diclofenac. Production of the human 4'-hydroxydiclofenac metabolite by CYP107E4 was confirmed, and a second hydroxylated metabolite was also produced.

Enhancement of calcineurin inhibitor nephrotoxicity by sirolimus (SRL) is limiting the clinical use of this drug combination. We compared the dose-dependent effects of the structurally related everolimus (EVL) and sirolimus (SRL) alone, and in combination with cyclosporine (CsA), on the rat kidney. Lewis rats were treated by oral gavage for 28 days using a checkerboard dosing format (0, 3.0, 6.0 and 10.0 CsA and 0, 0.5, 1.5 and 3.0 mg/kg/day SRL or EVL, n = 4/dose combination). After 28 days, oxidative stress, energy charge, kidney histologies, glomerular filtration rates, and concentrations of the immunosuppressants were measured along with 1H-magnetic resonance spectroscopy (MRS) and gas chromatography- mass spectrometry profiles of cellular metabolites in urine. The combination of CsA with SRL led to higher urinary glucose concentrations and decreased levels of urinary Krebs cycle metabolites when compared to controls, suggesting that CsA+SRL negatively impacted proximal tubule metabolism. Unsupervised principal component analysis of MRS spectra distinguished unique urine metabolite patterns of rats treated with CsA+SRL from those treated with CsA+EVL and the controls. SRL, but not EVL blood concentrations were inversely correlated with urine Krebs cycle metabolite concentrations. Interestingly, the higher the EVL concentration, the closer urine metabolite patterns resembled those of controls, while in contrast, the combination of the highest doses of CsA+SRL showed the most significant differences in metabolite patterns. Surprisingly in this rat model, EVL and SRL in combination with CsA had different effects on kidney biochemistry, suggesting that further exploration of EVL in combination with low dose calcineurin inhibitors may be of potential benefit.