Determination of cediranib in mouse plasma and brain tissue using high-performance liquid chromatography-mass spectrometry

A new high performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) assay for cediranib, a tyrosine kinase inhibitor for VEGFRs, was developed and validated, for the determination of plasma and brain levels of cediranib in small specimen volumes. Tyrphostin (AG1478) was used as internal standard. Mouse plasma and brain homogenate samples were prepared using liquid-liquid extraction. The assay was validated for a 2.5-2500 ng/mL concentration range for plasma, and for 1-2000 ng/mL range for brain homogenate. For these calibration ranges, within-assay variabilities were 1.1-14.3% for plasma and 1.5-9.4% for brain homogenate; between-assay variabilities were 2.4-9.2% for plasma, and 4.9-10.2% for brain homogenate. Overall accuracy ranged from 101.5 to 107.0% for plasma and 96.5 to 100.2% for brain homogenate, for all target concentrations. The developed assay has been successfully applied for a brain distribution study in mice at an oral dose of 5 mg/kg.     

Determination of 1,2-bis (2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA free acid) in rat plasma, urine and feces by liquid chromatography with UV and tandem mass spectrometric detection

BAPTA free acid was identified as the main metabolic product of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(actoxymethyl ester) (BAPTA-AM), a neuroprotective agent in cerebral ischemia, in rats. In this paper, liquid chromatography-ultraviolet (LC-UV) and mass spectrometry/mass spectrometry (LC-MS/MS) methods were employed for the determination of BAPTA free acid in rat urine and feces and rat plasma, respectively. By liquid-liquid extraction and LC-UV analysis, a limit of quantitation of 1000 ng/ml using 0.2 ml rat urine for extraction and 250 ng/ml using 1 ml rat fecal homogenate supernatant for extraction could be reached. The assay was linear in the range of 1000-50,000 ng/ml for rat urine and 250-10,000 ng/ml for rat fecal homogenate supernatant. Because the sensitivity of the LC-UV method was apparently insufficient for evaluating the pharmacokinetic profile of BAPTA in rat plasma, a LC-MS/MS method was subsequently developed for the analysis of BAPTA free acid. By protein precipitation and LC-MS/MS analysis, the limit of quantitation was 5 ng/ml using 0.1 ml rat plasma and the linear range was 5.0-500 ng/ml. Both methods were validated and can be used to support a thorough preclinical pharmacokinetic evaluation of BAPTA-AM liposome injection.     

Simultaneous quantitative analysis of methyl salicylate,ethyl salicylate and salicylic acid from biological fluids using gas chromatography–mass spectrometry

A gas chromatographic–mass spectrometric (GC–MS) assay was developed for the quantitative analysis of methyl salicylate (MeS), ethyl salicylate (ES) and salicylic acid (SA) from biological fluids. The method was validated from 100-μl rat liver homogenate preparations (5 mg/ml protein) in 70 mM KH₂PO₄ (pH 7.4) buffer and from 100 μl rat plasma. The samples were extracted with chloroform, derivatized with BSTFA and quantitated by GC–MS in the SIM mode. The standard curves ranged from 31 ng/ml to 800 or 1250 ng/ml. Relative standard deviations and bias were less than 11% in plasma and homogenate for all compounds except SA which evidenced greater variability. The assay was used in preliminary experiments to characterize the pharmacokinetics of MeS in rats.     

三聚氰胺胶体金免疫层析试纸条的研制

通过胶体金免疫层析技术建立一种特异、便捷、快速的三聚氰胺抗原检测方法,对奶制品及饲料中的三聚氰胺残留水平监测提供参考。用柠檬酸三钠还原法制备的胶体金,标记纯化的三聚氰胺单克隆抗体,喷于试纸的金标垫。将MEL-OVA(三聚氰胺和卵清白蛋白的偶练物)和纯化的羊抗鼠IgG分别喷于试纸的T(检测线)处和C(质控线)处,通过挑选试纸条材料和调试工艺参数,并最终组装成试纸条。结果显示,制备的试纸监测体系方法检出限为50 g/L。试纸条对牛奶、奶粉和饲料中的三聚氰胺残留的检出限分别为100 g/L、100 ng/g和200 ng/g。将该法与LC-MS/MS法对比检测牛奶、奶粉和饲料样品,在试纸条检测范围内,与LC-MS/MS法检测结果一致性好,从而验证了该方法的有效性。制备的三聚氰胺胶体金检测试纸在常温干燥环境下至少可保质6个月,能够检测出三聚氰胺含量大于50 g/L的样品,适用于现场实际样品三聚氰胺残留水平监测,具有良好的应用前景。     

A method for selective enrichment and analysis of nitrotyrosine-containing peptides in complex proteome samples

Elevated levels of protein tyrosine nitration have been found in various neurodegenerative diseases and age-related pathologies. Until recently, however, the lack of an efficient enrichment method has prevented the analysis of this important low-level protein modification. We have developed a method that specifically enriches nitrotyrosine-containing peptides so that both nitrotyrosine peptides and specific nitration sites can be unambiguously identified with LC-MS/MS. The procedure consists of the derivatization of nitrotyrosine into free sulfhydryl groups followed by high efficiency enrichment of sulfhydryl-containing peptides with thiopropyl sepharose beads. The derivatization process includes: (1) acetylation with acetic anhydride to block all primary amines, (2) reduction of nitrotyrosine to aminotyrosine, (3) derivatization of aminotyrosine with N-Succinimidyl S-Acetylthioacetate (SATA), and (4) deprotection of S-acetyl on SATA to form free sulfhydryl groups. The high specificity of this method is demonstrated by the contrasting percentage of nitrotyrosine-derivatized peptides in the identified tandem mass spectra between enriched and unenriched samples. Global analysis of unenriched in vitro nitrated human histone H1.2, bovine serum albumin (BSA), and mouse brain homogenate samples had 9%, 9%, and 5.9% of identified nitrotyrosine-containing peptides, while the enriched samples had 91% , 62%, and 35%, respectively. Duplicate LC-MS/MS analyses of the enriched mouse brain homogenate identified 150 unique nitrated peptides covering 102 proteins with an estimated 3.3% false discovery rate.     

Analysis of antibiotics in urine and wipe samples from environmental and biological monitoring--comparison of HPLC with UV-, single MS- and tandem MS-detection

Results of the simultaneous determination of the structurally different antibiotics cefazoline, cefotiame, cefuroxime, chloramphenicol, ciprofloxacin, ofloxacin, sulfamethoxazole and trimethoprim from environmental and biological monitoring using high-performance liquid chromatography with UV, single mass and tandem mass spectrometry were compared. For sample enrichment and clean-up a SPE method using bakerbond C18 cartridges was developed. Mean recovery rates were above 70%. Because of the complex urine matrix, only the wipe samples could be analyzed by UV-detection. However, UV-detection and single MS-detection are useful for control measurements after spillage, e.g. (LOD=1-2 ng/cm(2)). Samples from biological monitoring of occupational uptake should be analyzed by LC-MS/MS. The limits of detection (LOD) in urine ranged from 0.4 to 70 microg/L for LC-MS and 0.01 to 0.9 microg/L for LC-MS/MS detection. The limits of detection in wipe samples ranged from 0.003 to 0.13 ng/cm(2).     

Confident identification of 3-nitrotyrosine modifications in mass spectral data across multiple mass spectrometry platforms

3-nitrotyrosine (3NT) is an oxidative posttranslational modification associated with many diseases. Determining the specific sites of this modification remains a challenge due to the low stoichiometry of 3NT modifications in biological samples. Mass spectrometry-based proteomics is a powerful tool for identifying 3NT modifications, however several reports identifying 3NT sites were later demonstrated to be incorrect, highlighting that both the accuracy and efficiency of these workflows need improvement. To advance our understanding of the chromatographic and spectral properties of 3NT-containing peptides we have adapted a straightforward, reproducible procedure to generate a large set of 3NT peptides by chemical nitration of a defined, commercially available 48 protein mixture. Using two complementary LC-MS/MS platforms, a QTOF (QSTAR Elite) and dual pressure ion trap mass spectrometer (LTQ Velos), we detected over 200 validated 3NT-containing peptides with significant overlap in the peptides detected by both systems. We investigated the LC-MS/MS properties for each peptide manually using defined criteria and then assessed their utility to confirm that the peptide was 3NT modified. This broad set of validated 3NT-containing peptides can be utilized to optimize mass spectrometric instrumentation and data mining strategies or further develop 3NT peptide enrichment strategies for this biologically important, oxidative posttranslational modification.     

A LC-MS/MS method to quantify the novel cholesterol lowering drug ezetimibe in human serum, urine and feces in healthy subjects genotyped for SLCO1B1

Ezetimibe (Ezetrol) is a novel cholesterol lowering drug which disposition is not fully understood in man. We developed a selective and high-sensitive assay to measure serum concentration-time profiles, renal and fecal elimination of ezetimibe in pharmacokinetic studies. Ezetimibe glucuronide, the major metabolite of ezetimibe was determined by enzymatic degradation to the parent compound. Ezetimibe was measured after extraction with methyl tert-butyl ether using 4-hydroxychalcone as internal standard and liquid chromatography coupled via an APCI interface with tandem mass spectrometry (LC-MS/MS) for detection. The chromatography (column XTerra) MS, C(18), 2.1 mm x 100 mm, particle size 3.5 microm) was done isocratically with acetonitrile/water (60/40, v/v; flow rate 200 microl/min). The MS/MS analysis was performed in the negative ion mode (m/z transition: ezetimibe 408-271, internal standard 223-117). The validation ranges for ezetimibe and total ezetimibe were as follows: serum 0.0001-0.015 microg/ml and 0.001-0.2 microg/ml; urine and fecal homogenate 0.025-10 microg/ml and 0.1-20 mg/ml, respectively. The assay was successfully applied to measure ezetimibe disposition in two subjects genotyped for the hepatic uptake transporter SLCO1B1.     

Quantification of fluorotelomer-based chemicals in mammalian matrices by monitoring perfluoroalkyl chain fragments with GC/MS

Perfluorocarboxylic acids (PFCAs), namely perfluorooctanoic acid (PFOA) and perfluorononanoic acid (PFNA), have been identified as persistent, bioaccumulative and potentially toxic compounds. The structural analog, 8-2 fluorotelomer alcohol (8-2 fTOH) is considered the probable precursor of these stable metabolites. Because simultaneous quantification is needed for volatile and non-volatile perfluorinated chemicals (PFCs) in complex matrices, a GC/MS method was developed and tested based on selected ion monitoring of perfluorinated alkyl parent chain fragment ions. Although the method requires a derivatization step, combined GC/MS analysis of PFCA-me's and FTOHs increases analytical efficiency and decreases sample analysis time. The method instrument detection limits are between 7.1 and 24.5 ng/mL extract (MTBE), and the method quantification limits are below 50 ng/mL serum or ng/g liver for all PFCs investigated. Recoveries from mouse serum and liver homogenates, which were spiked with FTOHs and PFCAs at levels of 25 and 200 ng/mL or ng/g, ranged from 81 to 101%. Finally, the utility of the method was demonstrated by dosing male CD-1 mice with 30 mg/kg-BW of 8-2 fTOH and quantifying PFCs 6h post-treatment. The advantages of this method are (1) the simultaneous detection of both volatile and non-volatile fluorotelomer-based chemicals in complex matrices, such as mammalian tissues, (2) as a confirmatory method to LC-MS/MS, and (3) as an alternative method of analysis for laboratories without access to LC-MS/MS.     

Liquid chromatography-electrospray tandem mass spectrometric assay suitable for quantitation of halofuginone in plasma

An LC-MS/MS method was developed to quantitate the potential antitumor agent halofuginone in plasma. The assay uses 0.2 ml of plasma; chlorohalofuginone internal standard; acetonitrile for protein precipitation; a Phenomenex SYNERGI 4 micro Polar RP 80A (4 microm, 100 mm x 2 mm) column; an isocratic mobile phase of methanol:water:formic acid (80:20:0.02, v/v/v); and positive-ion electrospray ionization with selective reaction monitoring detection. Halofuginone eluted at approximately 2.4 min, internal standard eluted at approximately 2.9 min, and no endogenous materials interfered with their measurement. The assay was accurate, precise, and linear between 0.1 and 100 ng/ml. Halofuginone could be quantitated in dog plasma for at least 24 h after an i.v. dose of 0.1mg/kg. The assay is being used in ongoing pharmacokinetic studies of halofuginone.     

Determination of HS270, a new histone deacetylase inhibitor, in rat plasma by LC-MS/MS--application to a preclinical pharmacokinetic study

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC-MS/MS) method has been developed and fully validated to determine HS270, a new histone deacetylase (HDAC) inhibitor, in rat plasma using SAHA as the internal standard (IS). After a single step liquid-liquid extraction with acetoacetate, analytes were subjected to LC-MS/MS analysis using positive electro-spray ionization (ESI(+)) under selected reaction monitoring mode (SRM). The chromatographic separation was achieved on a Hypurity C(18) column (50 mm × 2.1 mm, i.d., 5 μm). The MS/MS detection was conducted by monitoring the fragmentation of m/z 392.3→100.1 for HS270, m/z 265.1→232.1 for IS. The method had a chromatographic running time of 2.5 min and linear calibration curves over the concentrations of 0.5-1000 ng/mL. The recovery of the method was 70.8-82.5% and the lower limit of quanti?cation (LLOQ) was 0.5 ng/mL. The intra- and inter-batch precisions were less than 15% for all quality control samples at concentrations of 1.0, 100.0, and 750.0 ng/mL. The validated LC-MS/MS method has successfully applied to a HS270 pharmacokinetic study after oral doses of 25, 50, 100, 200 mg/kg, and i.v. dose of 5 mg/kg to rats.     

Quantitative analysis of thymosin alpha1 in human serum by LC-MS/MS

A high performance liquid chromatography with tandem mass spectrometry (LC-MS/MS) method was developed to measure the thymosin alpha 1 (Talpha1) concentration in human serum. Tá1 in human serum was determined by solid phase extraction and reverse phase LC-MS/MS. The high-performance liquid chromatography (HPLC) system interfaced with the MS/MS system with a Turbo Ion spray interface. Positive ion detection and multiple reaction monitoring (MRM) mode were used for this human serum quantitation. Eight different concentration standards were used to establish the detection range. Six quality control (QC) and 2 matrix blanks were checked by calibration curves performed on the same day. The lower quantitation limit was 0.5 ng/mL Talpha1 in human serum. Calibration curves were established between 0.5 to 100 ng/mL by weighted linear regression. The correlation coefficients for different days were 0.9955 or greater. Quantitation of Talpha1 by the LC-MS/MS method is fast, accurate, and precise.     

A liquid chromatography-electrospray ionization tandem mass spectrometric assay for quantitation of the histone deacetylase inhibitor, vorinostat (suberoylanilide hydroxamicacid, SAHA), and its metabolites in human serum

Vorinostat (suberoylanilide hydroxamic acid, SAHA) is undergoing evaluation as an antineoplastic agent. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for quantitating vorinostat and its major metabolites, vorinostat glucuronide and 4-anilino-4-oxobutanoic acid, in human serum. The assay uses: deuterated internal standards; acetonitrile protein precipitation; a BDS Hypersil C18 (3 microm, 100 mm x 3 mm) column; a gradient mobile phase of 0.5% acetic acid in acetonitrile and water; and electrospray positive-mode ionization with selected reaction monitoring (SRM) detection. The lower limit of quantitation was 3.0 ng/ml for each analyte. The assay is being employed in at least 12 clinical studies of vorinostat-containing regimens.     

Evaluation of enzyme-linked immunosorbent assay and liquid chromatography-tandem mass spectrometry methodology for the analysis of 8-oxo-7,8-dihydro-2'-deoxyguanosine in saliva and urine

While ELISA is a frequently used means of assessing 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG) in biological fluids, differences in baseline urinary 8-oxodG levels, compared to chromatographic techniques, have raised questions regarding the specificity of immunoassays. Recently, ELISA of salivary 8-oxodG has been used to report on periodontal disease. We compared salivary 8-oxodG levels, determined by two commercial ELISA kits, to liquid chromatography-tandem mass spectrometry (LC-MS/MS) with prior purification using solid-phase extraction. While values were obtained with both ELISA kits, salivary 8-oxodG values were below or around the limit of detection of our LC-MS/MS assay. As the limit of detection for the LC-MS/MS procedure is much lower than ELISA, we concluded that the assessment of salivary 8-oxodG by ELISA is not accurate. In contrast to previous studies, ELISA levels of urinary 8-oxodG (1.67 ± 0.53 pmol/μmol creatinine) were within the range reported previously only for chromatographic assays, although still significantly different than LC-MS/MS (0.41 ± 0.39 pmol/μmol creatinine; p = 0.002). Furthermore, no correlation with LC-MS/MS was seen. These results question the ability of ELISA approaches, at present, to specifically determine absolute levels of 8-oxodG in saliva and urine. Ongoing investigation in our laboratories aims to identify the basis of the discrepancy between ELISA and LC-MS/MS.     

Quantitative analysis of thymosin α1 in human serum by LC-MS/MS

1 high performance liquid chromatography with tandem mass spectrometry (LC-MS/MS) method was developed to measure the thymosin alpha 1 (Tα1) concentration in human serum. Tα1 in human serum was determined by solid phase extraction and reverse phase LC-MS/MS. The high-performance liquid chromatography (HPLC) system interfaced with the MS/MS system with a Turbo Ion spray interface. Positive ion detection and multiple reaction monitoring (MRM) mode were used for this human serum quantitation. Eight different concentration standards were used to establish the detection range. Six quality control (QC) and 2 matrix blanks were checked by calibration curves performed on the same day. The lower quantitation limit was 0.5 ng/mL T α1 in human serum. Calibration curves were established between 0.5 to 100 ng/mL by weighted linear regression. The correlation coefficients for different days were 0.9955 or greater. Quantitation of Tα1 by the LC-MS/MS method is fast accurate, and precise.     

Accurate inclusion mass screening: a bridge from unbiased discovery to targeted assay development for biomarker verification

Verification of candidate biomarker proteins in blood is typically done using multiple reaction monitoring (MRM) of peptides by LC-MS/MS on triple quadrupole MS systems. MRM assay development for each protein requires significant time and cost, much of which is likely to be of little value if the candidate biomarker is below the detection limit in blood or a false positive in the original discovery data. Here we present a new technology, accurate inclusion mass screening (AIMS), designed to provide a bridge from unbiased discovery to MS-based targeted assay development. Masses on the software inclusion list are monitored in each scan on the Orbitrap MS system, and MS/MS spectra for sequence confirmation are acquired only when a peptide from the list is detected with both the correct accurate mass and charge state. The AIMS experiment confirms that a given peptide (and thus the protein from which it is derived) is present in the plasma. Throughput of the method is sufficient to qualify up to a hundred proteins/week. The sensitivity of AIMS is similar to MRM on a triple quadrupole MS system using optimized sample preparation methods (low tens of ng/ml in plasma), and MS/MS data from the AIMS experiments on the Orbitrap can be directly used to configure MRM assays. The method was shown to be at least 4-fold more efficient at detecting peptides of interest than undirected LC-MS/MS experiments using the same instrumentation, and relative quantitation information can be obtained by AIMS in case versus control experiments. Detection by AIMS ensures that a quantitative MRM-based assay can be configured for that protein. The method has the potential to qualify large number of biomarker candidates based on their detection in plasma prior to committing to the time- and resource-intensive steps of establishing a quantitative assay.     

Identification of Pin1-binding phosphorylated proteins in the mouse brain

To determine the role of Pin1 in the neurotransmission pathway, Pin1-binding proteins in mouse brain extract were identified. The Pin1-binding proteins were extracted from mouse brain homogenate, and the trypsin-digested peptides were analyzed by nano-liquid chromatography tandem mass spectrometry (LC-MS/MS). Proteins that involve the neurotransmission pathway, such as synapsin I, synapsin II, and calcium/calmodulin-dependent protein kinase type II (CaMKII), were identified in a Mascot search. Pull-down and immunoprecipitation assay indicated that Pin1 binds CaMKII in a phosphorylation-specific manner. It was assumed that Pin1 participates in the neurotransmission pathway involving the phosphorylation signal by CaMKII.     

Identification of Pin1-Binding Phosphorylated Proteins in the Mouse Brain

To determine the role of Pin1 in the neurotransmission pathway, Pin1-binding proteins in mouse brain extract were identified. The Pin1-binding proteins were extracted from mouse brain homogenate, and the trypsin-digested peptides were analyzed by nano-liquid chromatography tandem mass spectrometry (LC-MS/MS). Proteins that involve the neurotransmission pathway, such as synapsin I, synapsin II, and calcium/calmodulin-dependent protein kinase type II (CaMKII), were identified in a Mascot search. Pull-down and immunoprecipitation assay indicated that Pin1 binds CaMKII in a phosphorylation-specific manner. It was assumed that Pin1 participates in the neurotransmission pathway involving the phosphorylation signal by CaMKII.     

Quantification of residual EDU (N-ethyl-N'-(dimethylaminopropyl) carbodiimide (EDC) hydrolyzed urea derivative) and other residual by LC-MS/MS

An LC-MS/MS method for determination of the break down product of N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide (EDC) urea derivative, EDU, has been developed and validated for monitoring the residual coupling reagents. Results indicate that the method exhibits suitable specificity, sensitivity, precision, linearity and accuracy for quantification of residual EDU in the presence of meningococcal polysaccharide-diphtheria toxoid conjugate vaccine and other vaccine matrix compounds. The assay has been validated for a detection range of 10-100 ng/mL and then successfully transferred to quality control (QC) lab. This same method has also been applied to the determination of residual diaminohexane (DAH) in the presence of EDU. LC-MS/MS has proven to be useful as a quick and sensitive approach for simultaneous determination of multiple residual compounds in glycoconjugate vaccine samples.     

Simultaneous quantification of nicotine and metabolites in rat brain by liquid chromatography-tandem mass spectrometry

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous quantification of nicotine (NIC), cotinine (COT), nornicotine (NNIC), norcotinine (NCOT), nicotine-N-β-D-glucuronide (NIC GLUC), cotinine-N-β-D-glucuronide (COT GLUC), nicotine-1'-oxide (NNO), cotinine-N-oxide (CNO), trans-3'-hydroxycotinine (3-HC), anabasine (AB) and anatabine (AT) was modified and validated for quantification of these selected analytes in rat brain tissue. This analytical method provides support for preclinical NIC pharmacokinetic and toxicological studies after controlled dosing protocols. After brain homogenization and solid-phase extraction, target analytes and corresponding deuterated internal standards were chromatographically separated on a Discovery(?) HS F5 HPLC column with gradient elution and analyzed by LC-MS/MS in positive electrospray ionization (ESI) mode with multiple reaction monitoring (MRM) data acquisition. Method linearity was assessed and calibration curves were determined over the following ranges: 0.1-7.5 ng/mg for NIC, COT GLUC and AB; and 0.025-7.5 ng/mg for COT, NNIC, NCOT, NIC GLUC, NNO, CNO, 3-HC and AT (R(2)≥0.99 for all analytes). Extraction recoveries ranged from 64% to 115%, LC-MS/MS matrix effects were ≤21%, and overall process efficiency ranged from 57% to 93% at low and high quality control concentrations. Intra- and inter-assay imprecisions and accuracy for all analytes were ≤12.9% and ≥86%, respectively. The method was successfully applied to quantification of NIC and metabolites in the brain of post-natal day 90 rats that were sacrificed 2-h after a single 0.8 mg/kg s.c. administration of (-)NIC. In these tissues, striatal concentrations were 204.8±49.4, 138.2±14.2 and 36.1±6.1 pg/mg of NIC, COT and NNIC, respectively. Concentrations of NIC, COT and NNIC in the remaining whole brain (RWhB) were 183.3±68.0, 130.0±14.1 and 46.7±10.3 pg/mg, respectively. Quantification of these same analytes in plasma was also performed by a previously validated method. NIC, COT, NNIC, NCOT, NNO and CNO were detected in plasma with concentrations comparable to those reported in previous studies. However, and in contrast to brain tissues, COT concentrations in plasma were significantly higher than were those of NIC (194.6±18.6 ng/mL versus 52.7±12.9 ng/mL). Taken together, these results demonstrate that a sensitive and selective method has been developed for the determination of NIC biomarkers in rat brain.