E D Kharasch

Washington University in St. Louis, San Luis, Missouri, United States

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Publications (222)1026.71 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Interindividual variability and drug interaction studies suggest that blood-brain barrier drug transporters mediate human methadone brain biodistribution. In vitro and animal studies suggest that methadone is a substrate for the efflux transporter P-glycoprotein, and that P-glycoprotein-mediated transport influences brain access and pharmacologic effect. This investigation tested whether methadone is a transporter substrate in humans.
    Anesthesiology 07/2014; · 5.16 Impact Factor
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    ABSTRACT: Ritonavir, an HIV protease inhibitor, is successfully used for the prevention and treatment of HIV infections. Ritonavir pharmacokinetics are complicated by inhibition, induction and pharmacogenetics of cytochrome P450 (CYP) enzymes mediating its clearance. This investigation revealed that CYP2J2, along with CYP3A4/5 and CYP2D6, efficiently metabolizes ritonavir, and to a CYP2J2-specific (minor) metabolite. Chemical inhibition of ritonavir metabolism, clearance, KI/kinact and abundance of CYP2J2 in liver microsomes were evaluated and then applied to an in vitro-in vivo static scaling model to estimate the contribution of each isozyme, as a function of CYP abundance, activity, and genotype. Disposition of the CYP2J2- specific metabolite was also evaluated in vivo. In plasma, metabolite abundance was well above previously reported levels with circulating concentrations measured at 2μM for the main hydroxylisopropyl metabolite. Ritonavir and metabolite plasma profiles were simulated using Simcyp®. A modest (2-6%) contribution of CYP2J2 to ritonavir clearance is predicted which increases to more than 20% in subjects carrying CYP2D6 poor metabolizer polymorphisms and CYP3A4 irreversible inhibition. These results indicate that minor drug metabolizing enzymes could become quantitatively important in RTV clearance if main metabolic pathways are impeded.
    Biochemical pharmacology. 06/2014;
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    ABSTRACT: Printable multi-marker biochips that enable simultaneous quantitative detection of multiple target biomarkers in point-of-care and resource-limited settings are a holy grail in the field of biodiagnostics. However, preserving the functionality of biomolecules, which are routinely employed as recognition elements, during conventional printing approaches remains challenging. In this article, we introduce a simple yet powerful approach, namely plasmonic calligraphy, for realizing multiplexed label-free bioassays. Plasmonic calligraphy involves a regular ballpoint pen filled with biofunctionalized gold nanorods as plasmonic ink for creating isolated test domains on paper substrates. Biofriendly plasmonic calligraphy approach serves as a facile method to miniaturize the test domain size to few mm(2), which significantly improves the sensitivity of the plasmonic biosensor compared to bioplasmonic paper fabricated using immersion approach. Furthermore, plasmonic calligraphy also serves as a simple and efficient means to isolate multiple test domains on a single test strip, which facilitates multiplexed biodetection and multi-marker biochips. Plasmonic calligraphy, which can be potentially automated by implementing with a robotic arm, serves as an alternate path forward to overcome the limitations of conventional ink-jet printing.
    Biosensors & bioelectronics 03/2014; 59C:208-215. · 5.43 Impact Factor
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    ABSTRACT: Long-acting opioid agonists methadone and l-α-acetylmethadol (LAAM) prevent withdrawal in opioid-dependent persons. Attempts to synthesize [11C]-methadone for PET evaluation of brain disposition were unsuccessful. Owing, however, to structural and pharmacologic similarities, we aimed to develop [11C]LAAM as a PET ligand to probe the brain exposure of long-lasting opioids in humans. This manuscript describes [11C]LAAM synthesis and its biodistribution in mice. The radiochemical synthetic strategy afforded high radiochemical yield, purity and specific activity, thereby making the synthesis adaptable to automated modules.
    Applied Radiation and Isotopes. 01/2014; 91:135–140.
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    ABSTRACT: Printable multi-marker biochips that enable simultaneous quantitative detection of multiple target biomarkers in point-of-care and resource-limited settings are a holy grail in the field of biodiagnostics. However, preserving the functionality of biomolecules, which are routinely employed as recognition elements, during conventional printing approaches remains challenging. In this article, we introduce a simple yet powerful approach, namely plasmonic calligraphy, for realizing multiplexed label-free bioassays. Plasmonic calligraphy involves a regular ballpoint pen filled with biofunctionalized gold nanorods as plasmonic ink for creating isolated test domains on paper substrates. Biofriendly plasmonic calligraphy approach serves as a facile method to miniaturize the test domain size to few mm2, which significantly improves the sensitivity of the plasmonic biosensor compared to bioplasmonic paper fabricated using immersion approach. Furthermore, plasmonic calligraphy also serves as a simple and efficient means to isolate multiple test domains on a single test strip, which facilitates multiplexed biodetection and multi-marker biochips. Plasmonic calligraphy, which can be potentially automated by implementing with a robotic arm, serves as an alternate path forward to overcome the limitations of conventional ink-jet printing.
    Biosensors and Bioelectronics. 01/2014; 59:208–215.
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    ABSTRACT: We demonstrate that gold nanocages (AuNCs) with built-in artificial antibodies enable the detection of kidney injury biomarker from synthetic urine down to a concentration of 25 ng/ml. Molecularly imprinted AuNCs exhibit excellent selectivity against numerous interfering urinary proteins and remarkable stability over a wide range of pH and specific gravity.
    Journal of Materials Chemistry 11/2013; · 5.97 Impact Factor
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    ABSTRACT: To evaluate the trends in urine aquaporin-1 (AQP1) and perilipin 2 (PLIN2) concentrations in patients with clear cell and papillary renal cell carcinoma (RCC), we determined the relationship between the urine concentration of these biomarkers and tumor size, grade, and stage. The biomarker concentrations were determined by sensitive and specific Western blot procedures normalized to the urine creatinine excretion. The analysis included 61 patients undergoing partial or radical nephrectomy for clear cell or papillary RCC and 43 age- and sex-matched control patients. Relationships between urine biomarker concentrations and tumor size, stage, and grade were assessed. Patients with RCC had 35-fold and 9-fold higher median urinary AQP1 and PLIN2 concentrations, respectively, compared with controls. Both tumor markers decreased after tumor resection to concentrations equivalent to those of controls. The sensitivity and specificity were both 100% for AQP1 and 92% and 100%, respectively, for PLIN2. A significant linear correlation was found between the tumor size and the prenephrectomy AQP1 (Spearman coefficient 0.78, P <.001) and PLIN2 (Spearman coefficient 0.69, P <.001) concentrations. A correlation was found for both markers with tumor stage (overall P = .030), when the stage was dependent primarily on the tumor size (stages T1 and T2), but not with stage T3, which reflected extrarenal spread. Neither marker showed a significant correlation with tumor grade. AQP1 and PLIN2 were significantly increased in patients with clear cell and papillary RCC compared with controls. The preoperative urinary concentrations of these markers reflected the tumor size and stage.
    Urology 11/2013; · 2.42 Impact Factor
  • Evan D Kharasch, Kristi Stubbert
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    ABSTRACT: Plasma concentrations of orally administered methadone are reduced by the HIV protease inhibitor combination ritonavir/lopinavir, but the mechanism is unknown. Methadone metabolism, clearance and drug interactions have been attributed to CYP3A4, but this remains controversial. This investigation assessed effects of acute (2d) and steady-state (2 weeks) ritonavir/lopinavir on intravenous and oral methadone metabolism and clearance, hepatic and intestinal CYP3A4/5 activity (using the probe substrate intravenous and oral alfentanil), and intestinal transporter activity (using oral fexofenadine), in healthy volunteers. Plasma and urine concentrations of methadone and metabolite enantiomers, and other analytes, were determined by mass spectrometry. Acute and chronic ritonavir/lopinavir reduced plasma methadone enantiomer concentrations in half, with an average 2.6- and 1.5-fold induction of systemic and apparent oral methadone clearances. Induction was attributable to stereoselectively increased hepatic methadone N-demethylation, hepatic extraction, and hepatic clearance, and there was a strong correlation between methadone N-demethylation and clearance. Methadone renal clearance was unchanged. Alfentanil systemic clearance and hepatic extraction, apparent oral clearance, and intestinal extraction were reduced to 25%, 16% and 35% of control, indicating strong inhibition of hepatic and intestinal CYP3A activities. Ritonavir/lopinavir (acute>chronic) increased fexofenadine exposure, suggesting intestinal P-glycoprotein inhibition. There was no correlation between methadone clearance and CYP3A activity. Acute and steady-state ritonavir/lopinavir stereoselectively induced methadone N-demethylation and clearance, despite significant inhibition of hepatic and intestinal CYP3A activity. Ritonavir/lopinavir inhibited intestinal transporters activity, but had no effect on methadone bioavailability. These results do not support a significant role for CYP3A or ritonavir/lopinavir-inhibitable intestinal transporters in single-dose methadone disposition.
    Drug metabolism and disposition: the biological fate of chemicals 09/2013; · 3.74 Impact Factor
  • Karen J Regina, Evan D Kharasch
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    ABSTRACT: A new method using ultra-fast liquid chromatography and tandem mass spectrometry (UFLC-MS/MS) was developed for the simultaneous determination of buprenorphine and the metabolites norbuprenorphine, buprenorphine-3β-glucuronide, and norbuprenorphine-3β-glucuronide in plasma and urine. Sample handling, sample preparation and solid-phase extraction procedures were optimized for maximum analyte recovery. All four analytes of interest were quantified by positive ion electrospray ionization tandem mass spectrometry after solid-phase microextraction. The lower limits of quantification in plasma were 1pg/mL for buprenorphine and buprenorphine glucuronide, and 10pg/mL for norbuprenorphine and norbuprenorphine glucuronide. The lower limits of quantitation in urine were 10pg/mL for buprenorphine, norbuprenorphine and their glucuronides. Overall extraction recoveries ranged from 68-100% in both matrices. Interassay precision and accuracy was within 10% for all four analytes in plasma and within 15% in urine. The method was applicable to pharmacokinetic studies of low-dose buprenorphine.
    Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 09/2013; 939C:23-31. · 2.78 Impact Factor
  • Evan D Kharasch, Carl E Rosow
    Anesthesiology 09/2013; 119(3):504-6. · 5.16 Impact Factor
  • Scott D Campbell, Karen J Regina, Evan D Kharasch
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    ABSTRACT: Endothelial cells forming the blood-brain barrier limit drug access into the brain, due to tight junctions, membrane drug transporters, and unique lipid composition. Passive permeability, thought to mediate drug access, is typically tested using porcine whole-brain lipid. However, human endothelial cell lipid composition differs. This investigation evaluated the influence of lipid composition on passive permeability across artificial membranes. Permeability of CNS-active drugs across an immobilized lipid membrane was determined using three lipid models: crude extract from whole pig brain, human brain microvessel lipid, and microvessel lipid plus cholesterol. Lipids were immobilized on polyvinylidene difluoride, forming donor and receiver chambers, in which drug concentrations were measured after 2 h. The log of effective permeability was then calculated using the measured concentrations. Permeability of small, neutral compounds was unaffected by lipid composition. Several structurally diverse drugs were highly permeable in porcine whole-brain lipid but one to two orders of magnitude less permeable across human brain endothelial cell lipid. Inclusion of cholesterol had the greatest influence on bulky amphipathic compounds such as glucuronide conjugates. Lipid composition markedly influences passive permeability. This was most apparent for charged or bulky compounds. These results demonstrate the importance of using species-specific lipid models in passive permeability assays.
    Journal of Biomolecular Screening 08/2013; · 2.21 Impact Factor
  • Evan D Kharasch, Craig M Coopersmith
    Anesthesiology 07/2013; · 5.16 Impact Factor
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    ABSTRACT: The blood-brain barrier is richly populated by active influx and efflux transporters influencing brain drug concentrations. Morphine, a drug with delayed clinical onset, is a substrate for the efflux transporter P-glycoprotein in vitro and in animals. This investigation tested whether morphine is a transporter substrate in humans. Fourteen healthy volunteers received morphine (0.1 mg/kg, 1-h IV infusion) in a crossover study without (control) or with the infusion of validated P-glycoprotein inhibitor cyclosporine (5 mg/kg, 2-h infusion). Plasma and urine morphine and morphine glucuronide metabolite concentrations were measured by mass spectrometry. Morphine effects were measured by miosis and analgesia. Cyclosporine minimally altered morphine disposition, increasing the area under the plasma morphine concentration versus time curve to 100 ± 21 versus 85 ± 24 ng/ml·h (P < 0.05) without changing maximum plasma concentration. Cyclosporine enhanced (3.2 ± 0.9 vs. 2.5± 1.0 mm peak) and prolonged miosis, and increased the area under the miosis-time curve (18 ± 9 vs. 11 ± 5 mm·h), plasma effect-site transfer rate constant (ke, median 0.27 vs. 0.17 h), and maximum calculated effect-site morphine concentration (11.5 ± 3.7 vs. 7.6 ± 2.9 ng/ml; all P < 0.05). Analgesia testing was confounded by cyclosporine-related pain. Morphine is a transporter substrate at the human blood-brain barrier. Results suggest a role for P-glycoprotein or other efflux transporters in brain morphine access, although the magnitude of the effect is small, and unlikely to be a major determinant of morphine clinical effects. Efflux may explain some variability in clinical morphine effects.
    Anesthesiology 07/2013; · 5.16 Impact Factor
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    ABSTRACT: BACKGROUND:There is considerable interindividual and intraindividual variability in methadone metabolism and clearance. Methadone dosing is particularly challenging during initiation of therapy, because of time-dependent increases in hepatic clearance (autoinduction). Although methadone N-demethylation is catalyzed in vitro by cytochrome P4502B6 (CYP2B6) and CYP3A4, and clearance in vivo depends on CYP2B6, mechanism(s) of autoinduction are incompletely understood. In this investigation, we determined mechanism(s) of methadone autoinduction using human hepatocytes.METHODS:Fresh human hepatocytes were exposed to 0.1 to 10 µM methadone for 72 hours. Cells were washed and methadone N-demethylation assessed. CYP2B6, CYP3A4, and CYP3A5 messenger RNA (mRNA), protein expression (by gel-free high-performance liquid chromatography mass spectrometry) and catalytic activity (bupropion hydroxylation and alfentanil dealkylation for CYP2B6 and CYP3A4/5, respectively) were measured. Mechanisms of CYP induction were characterized using pregnane X receptor and constitutive androstane receptor reporter gene assays.RESULTS:Methadone (10 µM) increased methadone N-demethylation 2-fold, CYP2B6 and CYP3A4 mRNA 3-fold, and protein expression 2-fold. CYP3A5 mRNA was unchanged. CYP2B6 and CYP3A4/5 activities increased 2-fold. Induction by methadone enantiomers (R-methadone versus S-methadone) did not differ. Induction was relatively weak compared with maximum induction by phenobarbital and rifampin. Lower methadone concentrations had smaller effects. Methadone was an agonist for the pregnane X receptor but not the constitutive androstane receptor.CONCLUSIONS:Methadone caused concentration-dependent autoinduction of methadone N-demethylation in human hepatocytes, related to induction of CYP2B6 and CYP3A4 mRNA expression, protein expression, and catalytic activity. Induction was related to pregnane X receptor but not constitutive androstane receptor activation. These in vitro findings provide mechanistic insights into clinical autoinduction of methadone metabolism and clearance.
    Anesthesia and analgesia 06/2013; · 3.08 Impact Factor
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    C M Blake, E D Kharasch, M Schwab, P Nagele
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    ABSTRACT: Metoprolol, a commonly prescribed beta-blocker, is primarily metabolized by cytochrome P450 2D6 (CYP2D6), an enzyme with substantial genetic heterogeneity. Several smaller studies have shown that metoprolol pharmacokinetics is influenced by CYP2D6 genotype and metabolizer phenotype. To increase robustness of metoprolol pharmacokinetic estimates, a systematic review and meta-analysis of pharmacokinetic studies that administered a single oral dose of immediate release metoprolol was performed. Pooled analysis (n= 264) demonstrated differences in peak plasma metoprolol concentration, area under the concentration-time curve, elimination half-life, and apparent oral clearance that were 2.3-, 4.9-, 2.3-, and 5.9-fold between extensive and poor metabolizers, respectively, and 5.3-, 13-, 2.6-, and 15-fold between ultra-rapid and poor metabolizers (all p<0.001). Enantiomer-specific analysis revealed genotype-dependent enantio-selective metabolism, with nearly 40% greater R- vs S-metoprolol metabolism in ultra-rapid and extensive metabolizers. This study demonstrates a marked effect of CYP2D6 metabolizer phenotype on metoprolol pharmacokinetics and confirms enantiomer specific metabolism of metoprolol.Clinical Pharmacology & Therapeutics (2013); accepted article preview online 9 May 2013; doi:10.1038/clpt.2013.96.
    Clinical Pharmacology &#38 Therapeutics 05/2013; · 6.85 Impact Factor
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    ABSTRACT: The development of biomolecular imprinting over the last decade has raised promising perspectives in replacing natural antibodies with artificial antibodies. A significant number of reports have been dedicated to imprinting of organic and inorganic nanostructures, but very few were performed on nanomaterials with a transduction function. Herein we describe a relatively fast and efficient plasmonic hot spot-localized surface imprinting of gold nanorods using reversible template immobilization and siloxane co-polymerization. The technique enables a fine control of the imprinting process at the nanometer scale and provides a nanobiosensor with high selectivity and reusability. Proof of concept is established by the detection of neutrophil gelatinase-associated lipocalin (NGAL), a biomarker for acute kidney injury, using localized surface plasmon resonance spectroscopy. The work represents a valuable step towards plasmonic nanobiosensors with synthetic antibodies for label-free and cost-efficient diagnostic assays. We expect that this novel class of surface imprinted plasmonic nanomaterials will open up new possibilities in advancing biomedical applications of plasmonic nanostructures.
    Advanced Functional Materials 04/2013; 23(14):1789-1797. · 10.44 Impact Factor
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    ABSTRACT: Polymorphisms in CYP2D6 and CYP2C19 affect the efficacy and safety of tricyclics, with some drugs being affected by CYP2D6 only, and others by both polymorphic enzymes. Amitriptyline, clomipramine, doxepin, imipramine, and trimipramine are demethylated by CYP2C19 to pharmacologically active metabolites. These drugs and their metabolites, along with desipramine and nortriptyline, undergo hydroxylation by CYP2D6 to less active metabolites. Evidence from published literature is presented for CYP2D6 and CYP2C19 genotype-directed dosing of tricyclic antidepressants.Clinical Pharmacology & Therapeutics (2013); advance online publication 13 March 2013. doi:10.1038/clpt.2013.2.
    Clinical Pharmacology &#38 Therapeutics 01/2013; · 6.85 Impact Factor
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    ABSTRACT: The long-acting opioid methadone displays considerable unexplained interindividual pharmacokinetic variability. Methadone metabolism clinically occurs primarily by N-demethylation to 2-ethyl-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), catalyzed predominantly by cytochrome P450 2B6 (CYP2B6). Retrospective studies suggest an influence of the common allele variant CYP2B6*6 on methadone plasma concentrations. The catalytic activity of CYP2B6.6, encoded by CYP2B6*6, is highly substrate-dependent. This investigation evaluated methadone N-demethylation by CYP2B6.6, and in comparison to that by wild-type CYP2B6.1. Methadone enantiomer and racemate N-demethylation by recombinant expressed CYP2B6.6 and CYP2B6.1 was determined. At substrate concentrations (0.25-2 μM) approximating plasma concentrations occurring clinically, rates of methadone enantiomer N-demethylation by CYP2B6.6, incubated individually or as the racemate, were one-third to one-fourth those by CYP2B6.1. For methadone individual enantiomers metabolism by CYP2B6.6 compared with CYP2B6.1, V(max) was diminished, K(s) was greater, the in vitro intrinsic clearance was diminished 5- to 6-fold. The intrinsic clearance for R- and S-EDDP formation from racemic methadone was diminished approximately 6-fold and 3-fold for R- and S-methadone. Both CYP2B6.6 and CYP2B6.1 showed similar stereoselectivity (S>R-methadone). Human liver microsomes with diminished CYP2B6 content due to a CYP2B6*6 allele had lower rates of methadone N-demethylation. Results show that methadone N-demethylation catalyzed by CYP2B6.6, the CYP2B6 variant encoded by the CYP2B6*6 polymorphism, is catalytically deficient compared with wild-type CYP2B6.1. Diminished methadone N-demethylation by CYP2B6.6 may provide a mechanistic explanation for clinical observations of altered methadone disposition in individuals carrying the CYP2B6*6 polymorphism.
    Drug metabolism and disposition: the biological fate of chemicals 01/2013; · 3.74 Impact Factor
  • Evan D Kharasch, Kristi Stubbert
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    ABSTRACT: Methadone N-demethylation in vitro is catalyzed by hepatic cytochrome P4502B6 (CYP2B6) and CYP3A4, but clinical disposition is often attributed to CYP3A4. This investigation tested the hypothesis that CYP2B6 is a prominent CYP isoform responsible for clinical methadone N-demethylation and clearance, using the in vivo mechanism-based CYP2B6 inhibitor ticlopidine, given orally for 4 days. A preliminary clinical investigation with the CYP3A4/5 substrate probe alfentanil established that ticlopidine did not inhibit intestinal or hepatic CYP3A4/5. Subjects received intravenous plus oral (deuterium-labeled) racemic methadone before and after ticlopidine. Ticlopidine significantly and stereoselectively (S > R) inhibited methadone N-demethylation, decreasing plasma metabolite/methadone area under the curve ratios and metabolite formation clearances. Ticlopidine also significantly increased the dose-adjusted plasma area under the curve for R- and S-methadone by 20% and 60%, respectively, after both intravenous and oral dosing. CYP2B6 inhibition reduces methadone N-demethylation and clearance, and alters methadone concentrations, demonstrating an important role for CYP2B6 in clinical methadone disposition.
    The Journal of Clinical Pharmacology 01/2013; · 2.84 Impact Factor
  • Jeremiah J Morrissey, Evan D Kharasch
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    ABSTRACT: PURPOSE: Renal cancer is frequently asymptomatic until late stages of disease and has poor prognosis when not discovered early. Aquaporin 1 (AQP1) and perilipin 2 (PLIN2) are recently discovered sensitive urine biomarkers of clear cell and papillary kidney cancer. This investigation sought to validate these biomarkers in a second cohort of patients and determine the effect of common kidney diseases on their specificity. MATERIALS AND METHODS: Urine samples were obtained from 36 patients with clear cell or papillary kidney cancer, 43 controls, 44 patients with documented urinary tract infection, 24 patients diagnosed with diabetic nephropathy, and 18 patients diagnosed with glomerulonephritis. Urinary levels of AQP1 and PLIN2 (normalized to urine creatinine) were determined by a sensitive and specific Western blot procedure. RESULTS: Urine AQP1 and PLIN2 levels in patients with kidney cancer were 23- and 4-fold greater than controls and decreased 83-84% after tumor excision. There was a linear correlation between urinary AQP1 and PLIN2 levels and tumor size (each P<0.001). Urine AQP1 and PLIN2 levels of patients with kidney cancer were 11- to 23-fold and 17- to 25-fold greater, respectively, than in patients with the common kidney diseases. CONCLUSIONS: The ability of urinary AQP1 and PLIN2 to identify patients with kidney cancer compared to controls was validated in a second cohort of patients. Common kidney diseases do not adversely increase urinary AQP1 and PLIN2 levels or reduce their specificity to screen for renal cancer.
    The Journal of urology 11/2012; · 3.75 Impact Factor

Publication Stats

5k Citations
1,026.71 Total Impact Points

Institutions

  • 2005–2014
    • Washington University in St. Louis
      • Department of Anesthesiology
      San Luis, Missouri, United States
  • 2010
    • University of North Carolina at Chapel Hill
      • Department of Medicine
      Chapel Hill, NC, United States
  • 2009
    • Mayo Foundation for Medical Education and Research
      • Department of Anesthesiology
      Scottsdale, AZ, United States
    • University of Missouri - St. Louis
      Saint Louis, Michigan, United States
  • 1988–2007
    • University of Washington Seattle
      • • Department of Medicinal Chemistry
      • • Department of Pharmacy
      • • Department of Pharmaceutics
      Seattle, WA, United States
  • 2002–2005
    • Norwegian University of Science and Technology
      • Department of Circulation and Medical Imaging
      Trondheim, Sor-Trondelag Fylke, Norway
  • 2003–2004
    • Ankara University
      • Department of Pharmaceutical Chemistry
      Ankara, Ankara, Turkey
  • 2000
    • University of New Mexico
      • School of Medicine
      Albuquerque, NM, United States
  • 1997–1999
    • VA Puget Sound Health Care System
      Washington, Washington, D.C., United States
  • 1998
    • Spokane VA Medical Center
      Spokane, Washington, United States