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Analysis of r2u-Globulin in Rat Urine and Kidneys by Liquid Chromatography-Electrospray Ionization Mass Spectrometry Yan Mao,† Ronald J. Moore,† Kenneth B. Wagnon,† Judy T. Pierce,† Kathryn H. Debban,† Cynthia S. Smith,‡ Jeffrey A. Dill,† and Alfred F. Fuciarelli*,† Toxicokinetics and Bioanalytical Chemistry Technical Center, Battelle-Preclinical Drug Development-Northwest Operations, Richland, Washington 99352, and National Institute of Environmental Health Sciences, Research Triangle Park, Raleigh, North Carolina 27709 Received March 2, 1998
A quantitative method was developed for determination of R2u-globulin in urine and kidney samples collected from male rats using liquid chromatography-electrospray ionization mass spectrometry (LC-ESI/MS). Samples prepared from urine and kidney homogenates using size exclusion filters were subject to reversed-phase liquid chromatography and the effluent passed into an electrospray ionization source. Quantitative analysis using external standard calibration was based upon selected ion monitoring of protonated molecular ions by the mass spectrometer. Linear calibration curves were developed over the range of ∼4.6-370 µg of R2uglobulin/µL for spiked urine standards and over the range of ∼4.6-550 µg of R2u-globulin/µL for spiked kidney standards. The precision (relative standard deviation) for repeated injection (using urine samples) and intra-assay precision (using both urine and kidney samples) were within (10.4% and (13.2%, respectively. Using spiked urine standards, inter-assay precision, intra-assay accuracy, and inter-assay accuracy were within (20%, (20%, and (15%, respectively. Using spiked kidney standards, intra-assay accuracy was within (15%. The limits of detection (LOD) for the determination of R2u-globulin in urine and kidney samples were ∼0.41 pg/nL (1.0 fmol injected) and 25 pg/nL (∼13 fmol injected), respectively. The limits of quantitation (LOQ) for determination of R2u-globulin in urine and kidney samples were calculated as 1.4 pg/nL (3.7 fmol injected) and 83 pg/nL (45 fmol injected), respectively. Applicability of the LC-ESI/MS method was demonstrated by determination of R2u-globulin in both urine and kidney samples collected from male Fischer 344/N rats dosed intravenously with cis-Decalin at concentrations of 0, 2.5, 5.0, 10, and 20 mg/kg. A dose-dependent relationship was found between the amount of cis-Decalin administered and R2u-globulin accumulation in kidney samples, whereas no significant change in the urinary levels of R2uglobulin occurred. These observations are consistent with excessive accumulation of R2u-globulin occurring in protein droplets in renal proximal tubule epithelial cells as a result of decreased catabolic activity due to formation of ligand-protein complexs with Decalin and its metabolite(s). This report demonstrates that LC-ESI/MS may be routinely applied for quantitative analysis of R2u-globulin in rat urine and kidney samples to address R2u-globulin accumulation and its role in the development of nephrotoxicity associated with chemical exposures.
Introduction Exposure of male rats to a number of organic compounds (chemicals that induce R2u-globulin accumulation, CIGA ligands1 (1)) leads to a dose-dependent toxic syndrome known as protein (hyaline) droplet nephropathy, which is accompanied by excessive accumulation of * To whom correspondence should be addressed. Tel.: (509) 3752928. Fax: (509) 375-3649. E-Mail:
[email protected]. † Battelle-Preclinical Drug Development-Northwest Operations. ‡ National Institute of Environmental Health Sciences. 1 Abbreviations: ACN, acetonitrile; ALS, automated liquid sampler; BCA, bicinchoninic acid; CIGA, chemicals that induce R2u-globulin accumulation; ESI, electrospray ionization; ESI-MS, electrospray ionization-mass spectrometry; HP, Hewlett-Packard; LC-ESI/MS, liquid chromatography-electrospray ionization/mass spectrometry; LOD, limit of detection; LOQ, limit of quantitation; Mr, molecular mass calculated from ESI-MS; PBS, phosphate-buffered saline; % RSD, percent relative standard deviation; SIM, selected ion monitoring; TFA, trifluoroacetic acid.
R2u-globulin in kidney proximal tubule epithelial cells (16). CIGA ligands include petroleum-based and synthetic fuels (7, 8), military aviation propellants (9), Decalin (1012), 2,2,4-trimethylpentane (8), and methyl tert-butyl ether (13, 14). The protein droplet nephropathy associated with excessive R2u-globulin accumulation occurs specifically in the male rat, exhibiting both an age- and androgen-dependent onset, does not occur in male rats genetically deficient in their ability to produce R2uglobulin (i.e., NCI Black-Reiter [NBR] strain), does not occur in female rats or in any other laboratory animals tested, and does not occur in humans (1-6). Therefore, considerable commercial and regulatory interest is associated with identifying and establishing the mechanistic basis for protein droplet nephropathy occurring in male rats due to the implications that such studies have for human risk assessment.
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An understanding of the mechanistic basis underlying R2u-globulin nephropathy has been developing that facilitates a more accurate assessment of the carcinogenic potential of CIGA ligands in humans. On the basis of sequence homology, gene structure, and protein function, R2u-globulin and several other proteins have been grouped into a superfamily designated the lipocalins (15). X-ray crystallographic studies have revealed that lipocalins feature a barrel-shaped binding cavity formed from the interaction of eight antiparallel β-strands that contain hydrophobic amino acid residues that accommodate lipophilic ligands in reversible, noncovalent interactions (1, 15-17). On the basis of these structural features, CIGAinduced nephrotoxicity can be attributed to a lethal accumulation of the R2u-globulin-ligand complex that results from a conformational change in the protein upon ligand binding that inhibits lysosomal degradation (18). Due to its low molecular weight, R2u-globulin is normally freely filtered through the renal glomerulus and ∼50% is reabsorbed via endocytosis primarily in the P2 segment of the proximal tubular epithelium, while the remainder is excreted in the urine (1). Proteolytic degradation of R2u-globulin is relatively slow (plasma half-life of 5-8 h) compared to other low molecular weight proteins, and the noncovalent binding of R2u-globulin to CIGA ligands dramatically exacerbates the already limited catabolism of this protein (18). As a result, protein droplet accumulation in renal tubule epithelial cells occurs leading to a sequela of pathological events involving tubule cell degeneration, cell loss, and regenerative cellular proliferation, granular cast accumulation at the corticomedullary junction, mineralization of tubules within the renal medulla, single cell necrosis accompanied by cell proliferation, and ultimately, renal tubule hyperplasia and neoplasia (1). Therefore, histopathological and functional changes within the epithelial cells of proximal tubules can be traced back to molecular events occurring as a result of formation of R2u-globulin-ligand complexes, thus establishing a mechanistic basis for pathogenesis. Differentiating chemically induced renal tumors in male rats resulting from R2u-globulin-mediated nephropathy from renal tumors arising from an alternative etiology is vital for human risk assessment since chemically induced renal tumors occurring in male rats via R2uglobulin-mediated nephropathy are not used in hazard characterization for human risk assessment (19). Since R2u-globulin renal accumulation may serve as an early biochemical indicator of potential renal toxicity, accurate quantitation both in terms of the overall accumulation and catabolic rate is important for evaluating the CIGA potential of different organic compounds. Additionally, analysis of R2u-globulin in urine may serve as a useful indicator of organic compounds with severe CIGA potential or as an indicator of pathological changes in renal function in rats chronically exposed to CIGA ligands, without the need to sacrifice the animal as required for R2u-globulin determinations in kidneys. For these reasons, methods for determination of R2u-globulin in both urine and kidney samples are required. Along these lines, several approaches have been developed for assessing R2u-globulin. Immunochemical analyses include immunohistochemistry for localization of R2u-globulin (12, 14, 20, 21) and enzyme-linked immunosorbent assay (ELISA) (8, 14, 22, 23) for semiquantitative analyses in kidneys. Although high sensitivity and specificity are
Mao et al.
characteristic of immunochemical analyses, repetitive measurements of the same sample are necessary in an attempt to overcome their relatively poor precision. Additionally, immunochemical analyses are limited by the availability of suitable antibodies. Conventional high-performance liquid chromatography with ultraviolet detection (HPLC) has been successfully used for quantitative analysis of R2u-globulin in urine samples collected from male rats (24). However, this method is critically dependent upon glomerular filtration in vivo to provide sufficient purification to overcome the inherently poor selectivity and sensitivity associated with this analytical technique. Analysis of R2u-globulin in kidneys collected from male rats using conventional HPLC would be futile due to the plethora of proteins present in homogenates. Alternatively, combined chromatography-mass spectrometric methods are routinely used in toxicokinetic studies to overcome issues of selectivity and sensitivity for quantitative analysis of analytes in biological matricies. Application of mass spectrometry for analysis of biomolecules has rapidly developed since the introduction of electrospray ionization as a means to promote intact large molecules into the gas phase for subsequent mass spectrometric analyses (25-28). ESI spectra of proteins and peptides consist of a coherent series of multiply protonated molecules [M + nH]n+, permitting accurate mass determinations of high-mass proteins with a mass spectrometer of limited mass-to-charge (m/z) range. Using this technique, quadrupole instruments have been used for measurements of protein molecular weights in excess of 200 kDa (29). In addition to molecular mass determinations, characterization of posttranslational modifications and the determination of amino acid sequence using a combination of tandem mass spectrometry and digestion with specific endoproteinases are specific applications of ESI-MS that are routinely used for the analysis of proteins and peptides (30). Development of a liquid chromatography-electrospray ionization/mass spectrometry (LC-ESI/MS) method for quantitative analysis of R2u-globulin in urine and kidney samples collected from male rats was meritorious and offered the potential advantage of improvement of analytical precision and accuracy without the dependence on the availability of suitable antibodies for ELISA. In the LC-ESI/MS approach, size exclusion filters were used during sample preparation to select proteins with a molecular mass between 10K and 100K Da, and the retentates were chromatographed on-line using a reversedphase column with a mobile phase amenable for mass spectrometry. Selected-ion-monitoring mass spectrometry was used to identify R2u-globulin and enhance both sensitivity and selectivity for quantitative analyses. Applicability of the LC-ESI/MS technique was demonstrated by quantitative analysis of R2u-globulin in both urine and kidney samples collected from male Fischer 344/N rats dosed intravenously with cis-Decalin. This method represents a general approach based on LC-ESI/ MS for quantitative analysis of R2u-globulin in rat urine and kidney samples that can be routinely applied to study the role of R2u-globulin accumulation in chemically induced nephrotoxicity.
Methods and Materials Chemicals and Reagents. cis-Decalin (purity 99%) and trifluoroacetic acid (TFA) (purity 99+%) used in this study were
LC-ESI/MS Analysis of R2u-Globulin obtained from Aldrich Chemical Co., Inc. (Milwaukee, WI). Acetonitrile (ACN) (purity 99.9+%) was supplied by Burdick & Jackson (Research Triangle Park, NC). Apomyoglobin was supplied by Sigma Chemical Co. (St. Louis, MO). Bicinchoninic acid (BCA) protein assay reagents, pyrogallol red protein assay kit, and the reagent kit for creatinine determinations were purchased from Pierce Chemical Co. (Rockford, IL), Quantimetrix Corp. (Redondo Beach, CA), and Roche Diagnostic Systems, Inc. (Branchburg, NJ), respectively. Other buffers, reagents, and chemicals used in this study were obtained from Sigma Chemical Co. (St. Louis, MO) unless otherwise stated. Highpurity, deionized water with an electrical resistance of ∼18 MΩcm was obtained from a Barnstead Nanopure II deionization system (Barnstead/Thermolyne Corp., Dubuque, IA). Animals, Dosing, and Sample Collection. These studies were conducted under federal guidelines for the use and care of laboratory animals and were approved by Battelle’s Animal Care and Use Committee. Animals were housed in humidityand temperature-controlled, high-efficiency particulate airfiltered, mass air-displacement rooms in facilities accredited by the American Association for Accreditation of Laboratory Animal Care. Male Fischer 344/N rats (∼4 weeks) were purchased from Taconic (Germantown, NY) and housed in individual compartments of wire-mesh cage units (Lab Products Inc., Maywood, NJ) on open racks. During this period, animals had free access to NTP-2000 diet (irradiated pellets; Zeigler Bros.; Gardners, PA) and fresh tap water. They were maintained on a 12 h light/ dark cycle (light starting at 0600) at 24 ( 2 °C and 55 ( 15% relative humidity. For preparation of the R2u-globulin standard used for development of the LC-ESI/MS assay, urine was collected over ice for 16 h from 11-week-old male Fischer 344/N rats (Taconic, Germantown, NY) in plastic metabolic cages (Lab Products Inc., Maywood, NJ) that were fitted with fecal cups to prevent fecal contamination of urine and stored in 50-mL tissue culture tubes at -70 °C until required. Urine from age-matched female Fischer 344/N rats was also collected in a similar manner for comparison purposes. Male rats (221-272 g; 11 weeks) received cis-Decalin (0, 2.5, 5.0, 10, or 20 mg/kg) prepared in a mixture of Emulphor/ethanol/ water (1:1:8) via tail vein injection. Dosing solutions prepared at concentrations of 0, 1.27, 2.49, 5.16, and 10.2 mg/mL ensured that each animal received only between 0.42 and 0.56 mL of fluid during injection. Following injection, animals were transferred to plastic metabolic cages (Lab Products Inc., Maywood, NJ) that were fitted with fecal cups to prevent fecal contamination of urine. Urine was collected over ice for 16 h, assayed for creatinine concentrations using the creatinine reagent kit in an automated method developed on a Cobas Fara II Chemistry System (Montclair, NJ), and stored in 50-mL tissue culture tubes at -70 °C until required. During urine collection, animals were given water ad libitum but not food. Kidneys were harvested from 3 rats/exposure group 16 h following administration of cis-Decalin from animals that were deeply anesthetized with ∼70% CO2. Kidney weights were recorded at collection, and the kidneys were snap frozen in liquid nitrogen and stored at -70 °C. Preparation of r2u-Globulin Used as a Standard. R2uGlobulin was isolated and purified from the urine of 11-weekold male Fischer 344/N rats by HPLC. Aliquots (2 mL) of male urine were applied to Centricon-10 concentrators (Amicon, Inc., Beverly, MA) and centrifuged at 3000g at 4 °C for 3 h in an Avanti J-25 Centrifuge (Beckman Instruments Inc., Fullerton, CA) to remove low molecular weight (
