Touraj Shokati

Quantification of 15-F2t-isoprostane in human plasma and urine: results from enzyme-linked immunoassay and liquid chromatography/tandem mass spectrometry cannot be compared

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.

Effects of lovastatin on breast cancer cells: a proteo-metabonomic study

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. Lovastatins 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.

A high-throughput U-HPLC-MS/MS assay for the quantification of mycophenolic acid and its major metabolites mycophenolic acid glucuronide and mycophenolic acid acyl-glucuronide in human plasma and urine.

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.

Everolimus and Sirolimus in Combination with Cyclosporine Have Different Effects on Renal Metabolism in the Rat

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.

Quantification of the Immunosuppressant Tacrolimus on Dried Blood Spots Using LC-MS/MS.

The calcineurin inhibitor tacrolimus is the cornerstone of most immunosuppressive treatment protocols after solid organ transplantation in the United States. Tacrolimus is a narrow therapeutic index drug and as such requires therapeutic drug monitoring and dose adjustment based on its whole blood trough concentrations. To facilitate home therapeutic drug and adherence monitoring, the collection of dried blood spots is an attractive concept. After a finger stick, the patient collects a blood drop on filter paper at home. After the blood is dried, it is mailed to the analytical laboratory where tacrolimus is quantified using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) in combination with a simple manual protein precipitation step and online column extraction. For tacrolimus analysis, a 6-mm disc is punched from the saturated center of the blood spot. The blood spot is homogenized using a bullet blender and then proteins are precipitated with methanol/0.2 M ZnSO4 containing the internal standard D2,(13)C-tacrolimus. After vortexing and centrifugation, 100 µl of supernatant is injected into an online extraction column and washed with 5 ml/min of 0.1 formic acid/acetonitrile (7:3, v:v) for 1 min. Hereafter, the switching valve is activated and the analytes are back-flushed onto the analytical column (and separated using a 0.1% formic acid/acetonitrile gradient). Tacrolimus is quantified in the positive multi reaction mode (MRM) using a tandem mass spectrometer. The assay is linear from 1 to 50 ng/ml. Inter-assay variability (3.6%-6.1%) and accuracy (91.7%-101.6%) as assessed over 20 days meet acceptance criteria. Average extraction recovery is 95.5%. There are no relevant carry-over, matrix interferences and matrix effects. Tacrolimus is stable in dried blood spots at RT and at +4 °C for 1 week. Extracted samples in the autosampler are stable at +4 °C for at least 72 hr.

Mycophenolate Mofetil Enhances the Negative Effects of Sirolimus and Tacrolimus on Rat Kidney Cell Metabolism

Background and Purpose Mycophenolate mofetil (MMF) per se is not known to have negative effects on the kidney. MMF alone or in combination with sirolimus, can be the basis of calcineurin inhibitor (CNI)-free, kidney sparing drug protocols. However, long-term outcomes in patients on MMF/SRL seem to be inferior to those treated with regimens that include the CNI tacrolimus (TAC) due to an increased risk of allo-immune reactions. Interestingly, potential enhancement of the negative effects of SRL and TAC on the kidney by MMF has never been considered. Experimental Approach It was our aim to study the effects of TAC, SRL and MMF alone and evaluate their interactions when combined on the rat kidney. For this purpose we used a comprehensive molecular marker approach including measurements of urinary 8-isoprostane concentrations (oxidative stress marker) and changes of urinary metabolite patterns (1H-NMR spectroscopy) and comparing these markers to renal function (glomerular filtration rate (GFR)) and morphologic alterations (histology). Key Results While MMF alone did not impact GFR, its interaction with SRL and TAC led to a significant decrease of rats’ renal function. The decline went in parallel with a significant increase in urinary isoprostane concentrations and an enhancement of negative effects on urinary metabolite patterns. Conclusions In broad summary, the present study showed that MMF may enhance the negative effects of TAC on kidney function and may even display nephrotoxic properties when combined with SRL.

Genetic and Structure-Function Studies of Missense Mutations in Human Endothelial Lipase

Endothelial lipase (EL) plays a pivotal role in HDL metabolism. We sought to characterize EL and its interaction with HDL as well as its natural variants genetically, functionally and structurally. We screened our biethnic population sample (n = 802) for selected missense mutations (n = 5) and identified T111I as the only common variant. Multiple linear regression analyses in Hispanic subjects revealed an unexpected association between T111I and elevated LDL-C (p-value = 0.012) and total cholesterol (p-value = 0.004). We examined lipase activity of selected missense mutants (n = 10) and found different impacts on EL function, ranging from normal to complete loss of activity. EL-HDL lipidomic analyses indicated that EL has a defined remodeling of HDL without exhaustion of the substrate and a distinct and preference for several fatty acids that are lipid mediators and known for their potent pro- and anti-inflammatory properties. Structural studies using homology modeling revealed a novel α/β motif in the C-domain, unique to EL. The EL dimer was found to have the flexibility to expand and to bind various sizes of HDL particles. The likely impact of the all known missense mutations (n = 18) on the structure of EL was examined using molecular modeling and the impact they may have on EL lipase activity using a novel structure-function slope based on their structural free energy differences. The results of this multidisciplinary approach delineated the impact of EL and its variants on HDL. Moreover, the results suggested EL to have the capacity to modulate vascular health through its role in fatty acid-based signaling pathways.

Structural identification of SAR-943 metabolites generated by human liver microsomes in vitro using mass spectrometry in combination with analysis of fragmentation patterns.

SAR-943 (32-deoxo rapamycin) is a proliferation signal inhibitor via interaction with the mammalian target of rapamycin (mTOR). Most importantly, SAR-943 has improved chemical stability compared to rapamycin (sirolimus) and is currently under investigation as a drug coated on coronary stents. It was the goal of this study to identify the SAR-943 metabolites generated after incubation with human liver microsomes using high-resolution mass spectrometry (MS) and MS/iontrap (MS(n)) and comparison of fragmentation patterns of the metabolites with those of SAR-943 and other known rapamycin derivatives. Our study showed that SAR-943 is mainly hydroxylated and/or demethylated by human liver microsomes. The structures of the following metabolites were identified: O-demethylated metabolites: 39-O-desmethyl, 16-O-desmethyl and 27-O-desmethyl SAR-943; hydroxylated metabolites: hydroxy piperidine SAR-943, 11-hydroxy, 12-hydroxy, 14-hydroxy, 23-hydroxy, 24-hydroxy, 25-hydroxy, 46-hydroxy and 49-hydroxy SAR-943; didemethylated metabolites: 16,39-O-didesmethyl and 27,39-O-didesmethyl SAR-943; demethylated-hydroxylated metabolites: 39-O-desmethyl, 23- or 24-hydroxy and 39-O-desmethyl, hydroxy piperidine SAR-943 and dihydroxylated metabolites: 12-,23- or 24-dihydroxy SAR-943. In addition, several other demethylated-hydroxylated and dihydroxylated metabolites were detected. However, their exact structures could not be identified.