Characterization of fibronectin metabolites in normal rat urine

Fibronectin fragments showing immunoreactivity against an anti-rat plasma fibronectin antibody were detected in normal rat urine. Untreated urine showed immunoreactivity and this reactivity could not be dialyzed. These rat urinary fibronectin-related substances (RUF) were adsorbed on heparin-immobilized gel but hardly at all on gelatin-immobilized gel. They were separated into three fractions by DEAE-Sephadex ion exchange chromatography and each fraction was further purified by heparin-Sepharose affinity chromatography. The partially purified preparations, RUF-1, RUF-2 and RUF-3, were analyzed by the sandwich-type enzyme-linked immunosorbent assay (ELISA) method, and it was found their maximum reactivities were about 30%, 45% and 20% that of the intact plasma fibronectin, respectively. Plasma fibronectin was digested with elastase and then analyzed by the ELISA method to elucidate the patterns shown by the fragments. The fragments showed almost the same patterns on ELISA analysis, and the maximum immunoreactivity decreased in parallel with the degradation. On SDS-polyacrylamide gel electrophoresis, the RUF preparations were found to be composed of multiple forms of molecules, Mr 15,000-100,000, which reacted with the antibody. These results suggest that various forms of fibronectin metabolites are excreted in normal rat urine.     

Negative chemical ionization gas chromatography/mass spectrometry to quantify urinary 3-methylhistidine: application to burn injury

A rapid method for measuring 3-methylhistidine (3MH) in rat and human urine with higher sensitivity and precision than any previously reported method is described using internal standard [1-(13)C]3MH (M+1) and negative chemical ionization (NCI) gas chromatography/mass spectrometry (GC/MS). Internal standard [1-(13)C]3MH (M+1) was added to rat and human urine samples, hydrolyzed, and absorbed onto cation exchange columns. The column eluent was dried and derivatized for GC/MS analysis. Quantification of 3MH levels was accomplished by monitoring the m/z 204 fragment. The m/z 204 fragment was chosen due to the fragment's abundance and stability as determined by analysis of [methyl-(2)H(3), (18)O(2)]3MH (M+7) and [methyl-(13)C]3MH (M+1) fragmentation patterns under NCI conditions. This method shows excellent linearity (0.9989) over the range studied (0-0.5 mol), high recovery (95.9%), and low coefficient of variation (4.7%). The described method is sensitive enough to detect 6.8 pmol amount of urinary 3MH with a precision of 9.1%. The in vivo utility of this method to quantify urinary 3MH was tested in a burn injury rat model and on urine specimens from pediatric burn patients. Data obtained from the urine of burn-injured rats and pediatric burn patients match previously reported trends and validate the in vivo utility of this method.     

LC-MS/MS characterization of the urinary excretion profile of the mycotoxin deoxynivalenol in human and rat

The understanding of mycotoxins transfer to biological fluids is challenged by the difficulties in performing and replicating in vivo experiments as well as the lack of suitable methods of analysis to detect simultaneously a range of chemically different metabolites at trace levels. LC-MS/MS has been used herein to study the urinary excretion profile of the mycotoxin deoxynivalenol in human and Wistar rat. Deoxynivalenol and deoxynivalenol glucuronide were found in both human and rat urines, whereas de-epoxydeoxynivalenol and its glucuronide conjugate were only detected in rat urine. The presence of two deoxynivalenol glucuronide isomers in Wistar rat urine has been shown for the first time. Structure confirmation of the detected metabolites was provided by the analysis of fragmentation patterns. A solid phase extraction clean up procedure allowing recoveries in the range 72-102% for deoxynivalenol, de-epoxydeoxynivalenol, and their glucuronide conjugates was optimized. A multiple reaction monitoring method for the simultaneous determination of all investigated metabolites was elaborated allowing the direct detection of deoxynivalenol metabolites without the hydrolysis step. Deoxynivalenol urinary levels in the range 0.003-0.008 μg/ml were detected in healthy human subjects, whereas deoxynivalenol and de-epoxynivalenol levels between 1.9-4.9 μg/ml and 1.6-5.9 μg/ml, respectively were found in administered rat urine. These findings emphasize the relevance of the highly selective and sensitive LC-MS/MS technique for the direct detection and characterization of deoxynivalenol metabolites in complex biological matrices.     

Metabonomic study of aristolochic acid-induced nephrotoxicity in rats

This paper describes a metabonomic study characterizing the nephrotoxicity induced by aristolochic acid (AA), a suspected kidney toxicant. For these studies, we examined the biochemical compositions of AA-treated rat urine using LC-MS and pattern recognition methods. The biochemical and histological patterns of rat groups treated with different AA sources showed distinct differences from those of the control group. Certain metabolic pathways, such as homocysteine formation and the folate cycle were significantly accelerated, while others, including arachidonic acid biosynthesis, were decreased. A subset-validation procedure using linear discriminant analysis (LDA) and selected predictive variables indicated that approximately 95% of the treated and nontreated rat urine samples were classified correctly into their respective treatment groups. The results suggested that this metabonomic approach is a promising methodology for the rapid in vivo screening of nephrotoxicity associated with ingesting multi-ingredient medicinal herb supplements, and provides a valid method for comprehending the chemical-induced perturbations in the metabolic network and the networked lesions.     

Identification of amygdalin and its major metabolites in rat urine by LC-MS/MS

Amygdalin and its metabolites in rat urine were identified using liquid chromatography-electrospray ionization (ESI) tandem ion-trap mass spectrometry. The purified rat urine sample was separated using a reversed-phase C18 column with 10 mM sodium phosphate buffer (pH 3.1) containing 30% methanol as the mobile phase, amygdalin and its metabolites were detected by on-line mass detector in selected ion monitoring (SIM) mode. The identification of the metabolites and elucidation of their structure were performed by comparing the changes in molecular masses (DeltaM), retention times and MS(2) spectral patterns of metabolites with those of parent drug. At least seven metabolites and the parent drug were found in rat urine after i.v. injection of 100 mg/kg doses of amygdalin. Among them, six metabolites were reported for the first time.     

Identification of a pyrovalerone metabolite in the rat by gas chromatography-mass spectrometry and determination of pyrovalerone by gas chromatography-nitrogen-phosphorus detection

Pyrovalerone and its hydrolated metabolite have been identified by gas chromatography-mass spectrometry in rat urine and plasma. A sensitive gas chromatographic method for the quantitative analysis of pyrovalerone in rat urine and plasma is described. The method also permits the quantitative monitoring of the urinary excretion of the drug and its metabolite. Pyrovalerone and its hydroxylated metabolite are detected up to 18 h after a single oral administration to the rat at a dose of 20 mg/kg.     

An isotope dilution gas chromatographic-mass spectrometric method for the simultaneous assay of estrogens and phytoestrogens in urine

The metabolism of endogenous estrogens is complicated and certain metabolic patterns may reflect an individual risk of estrogen-dependent diseases such as breast cancer. Since the 1960s we have been constantly involved in developing estrogen profiling methods, in the beginning using gas chromatography and later gas chromatography–mass spectrometry (GC–MS) in the selected ion monitoring mode (SIM) and finally utilizing isotope dilution (ID–GC–MS–SIM). The addition of the dietary phytoestrogens to the profile rendered the method even more complicated. The present work presents the final estrogen profile method for 15 endogenous estrogens, four lignans, seven isoflavonoids and coumestrol in one small urine sample (1/150th of a 24 h human urine sample, minimum 2.5–5 ml) with complete validation including investigations as to the precision, sensitivity, accuracy and specificity. The method does not include the minimal amounts of unconjugated estrogens in urine. It may also be used for animal (e.g. rat and mouse) urine using a minimum of 2 ml of usually pooled sample. Despite its complexity it was found to fulfill the reliability criteria, resulting in highly specific and accurate results.     

Formation and excretion of pyrrole-2-carboxylic acid. Whole animal and enzyme studies in the rat.

A corrected method for the measurement of pyrrole-2-carboxylate in rat urine was used in studies of its excretion under various experimental conditions. The findings implicated administered hydroxy-L-proline as a relatively efficient source of urinary pyrrole-2-carboxylate and tended to exclude administered L-proline as a significant direct source. Removal of aerobic gut flora had no influence on the excretion of pyrrole-2-carboxylate either endogenously or following hydroxy-L-proline administration. Related studied showed that rat kidney L-amino acid oxidase catalyzes oxidation of hydroxy-L-proline to delta1-pyrroline-4-hydroxy-2-carboxylate, which is converted to pyrrole-2-carboxylate on acidification of reaction mixtures. All findings were consistent with hydroxy-L-proline as the source of endogenous pyrrole-2-carboxylate excretion. Excretion patterns and labeling patterns were compared after administration of pyrrole-2-carboxylate or of hydroxy-proline epimers. From these data, the true excretion product of hydroxy-L-proline oxidation by L-amino acid oxidase appeared to be the unstable oxidation product, delta1-pyrroline-4-hydroxy-2-carboxylate, which is converted to pyrrole-2-carboxylate in urine. The capacity of homogenates of guinea pig kidney and human kidney to carry out oxidation of hydroxy-L-proline to pyrrole-2-carboxylate was much less than that of rat kidney, consistent with the lower levels of urinary pyrrole-2-carboxylate in these species. Experiments designed to examine the modest increase of pyrrole-2-carboxylate excretion after proline loads led to new observations on tissue levels of hydroxy-L-proline following proline administration and on the inhibition by L-proline of hydroxy-L-proline oxidase.     

Determination of a guanosine—malonaldehyde adduct in urine by high-performance liquid chromatography with a thiobarbituric acid reaction detector

A new method for the determination of a guanosine—malonaldehyde adduct, β- -ribofuranosylpyrimido[1,2-a]purin-10(3H)-one (GMA), in rat and human urine is described. The method involves rapid pretreatment using, in sequence, polyamide, ion-exchange and reversed-phase cartridges; determination is by means of high-performance liquid chromatography with a thiobarbituric acid reactor in series with a fluorescence detector. This device can quantitatively determine the adduct at the sub-picomole level. This rapid, selective and sensitive method is suitable for the determination of guanine—malonaldehyde adducts in biological samples, such as human and rat urine. A semi-preparative method for the extraction and purification of these adducts from rat urine and for their identification by mass spectrometry and high-performance liquid chromatography with ultraviolet detection is also reported.     

Excretion in rat urine of substances derived from the basal glomerular membrane]

We have prepared in rabbits antibodies against rat GBM, with GBM prepared from isolated glomeruli issued from rat renal cortex. It is shown with immunodiffusion technique the excretion of basement membrane-like fragments into the normal rat urine. This result confirms the earlier works studying the human and rabbit urines. The origin and the nature of this patterns, the signification of this presence into the mammalian urines are discussed.     

Genetic analysis of human deoxyribonuclease I by immunoblotting and the zymogram method following isoelectric focusing

We have devised two independent detection methods for investigating possible molecular heterogeneity and genetic polymorphism in human DNase I, in terms of both its antigenicity and enzymatic activity. One was an immunoblotting method using an antibody specific to DNase I following polyacrylamide gel isoelectric focusing (IEF-PAGE). The DNase I-specific antibody was raised in a rabbit using purified enzyme from human urine as the immunogen. DNase I in urine was found to exist in multiple forms with different pI values separable by IEF-PAGE within a pH range of 3.5-4.0. This method was able to detect as little as 0.1 micrograms of the purified DNase I and facilitated classification of desialylated urine samples from different individuals into several groups according to differences in DNase I isozyme patterns. About 0.5 ml of the original urine was sufficient for analysis of the isozyme patterns. The other method was the zymogram method, which had a high sensitivity and resolution almost identical to those of the immunoblotting method for analysis of DNase I patterns. It was easier to perform, more time-saving, and more useful since it did not require antibody specific to DNase I. These two methods should prove valuable for biochemical and genetic analysis of DNase I isozymes.     

Structural characterization of metabolites of salvianolic acid B from Salvia miltiorrhiza in normal and antibiotic-treated rats by liquid chromatography-mass spectrometry

This study was conducted to compare the in vivo metabolites of salvianolic acid B (Sal B) between normal rats and antibiotic-treated rats and to clarify the role of intestinal bacteria on the absorption, metabolism and excretion of Sal B. A valid method using LC-MS(n) analysis was established for identification of rat biliary and fecal metabolites. And isolation of normal rat urinary metabolites by repeated column chromatography was applied in this study. Four biliary metabolites and five fecal metabolites in normal rats were identified on the basis of their MS(n) fragmentation patterns. Meanwhile, two normal rat urinary metabolites were firstly identified on the basis of their NMR and MS data. In contrast, no metabolites were detected in antibiotic-treated rat urine and bile, while the prototype of Sal B was found in antibiotic-treated rat feces. The differences of in vivo metabolites between normal rats and antibiotic-treated rats were proposed for the first time. Furthermore, it was indicated that the intestinal bacteria showed an important role on the absorption, metabolism and excretion of Sal B. This investigation provided scientific evidence to infer the active principles responsible for the pharmacological effects of Sal B.     

Simultaneous determination of four active alkaloids from a traditional Chinese medicine Corydalis saxicola Bunting. (Yanhuanglian) in plasma and urine samples by LC-MS-MS

A sensitive rapid method for the simultaneous determination of four major active alkaloids (dehydrocavidine, coptisine, dehydroapocavidine, and tetradehydroscoulerine, in abbreviation thereafter called YHL-I, YHL-II, YHL-III, and YHL-IV, respectively) from a Chinese traditional medicine Corydalis saxicola Bunting. (Yanhuanglian) in rat plasma and urine was established and validated. The assay for these substances in plasma and urine was based on HPLC coupled with tandem mass spectrometry (MS/MS) detection using multiple reaction monitoring mode (MRM) with berberine and clenbuterol as internal standards. The plasma and urine sample were deproteinated by adding methanol prior to liquid chromatography where separation was performed on a Luna column (5 microm, 100 x 2.00 mm) and an Agilent Zorbax SB-C18 guard column (5 microm, 20 x 4 mm). The method was validated with the concentration range 1-1000 ng/mL in plasma and 10-1000 ng/mL in urine for the four test compounds, and the calibration curves were linear with correlation coefficients >0.999. The lowest limits of quantitation for all four substances were 1 ng/mL in 0.1 mL rat plasma and 10 ng/mL in 0.1 mL urine. The intra-assay accuracy and precision in plasma ranged from 88.1 to 115.7% and 1.4 to 10.8%, respectively, while inter-assay accuracy and precision for YHL-I, YHL-II, YHL-III, and YHL-IV ranged from 96.2 to 113.2% and 0.4 to 16.9%, respectively. The intra-assay accuracy and precision for YHL-I, YHL-II, YHL-III, and YHL-IV in rat urine ranged from 96.1 to 112.9% and 1.2 to 8.3%, respectively, while inter-assay accuracy and precision ranged from 95.0 to 106.8% and 2.2 to 10.3%, respectively. The method was further applied to assess pharmacokinetics and urine excretion of the four alkaloids after oral and intravenous administration to rats. Practical utility of this new LC-MS-MS method was confirmed in pilot pharmacokinetic studies in rats following both intravenous and oral administration.     

Semi-automated solid-phase extraction procedure for the high-performance liquid chromatographic determination of alinastine in biological fluids

A solid-phase extraction (SPE) method for sample clean-up followed by a reversed-phase HPLC procedure for the assay of alinastina (pINN) in biological fluids is reported. The effects of the sample pH, composition of the washing and elution solvents and the nature of the SPE cartridge on recovery were evaluated. The selectivity of SPE was examined using spiked rat urine and plasma samples and the CH and PH cartridges gave rise to the cleanest extracts. The recoveries obtained in spiked rat urine and plasma samples were 91.2±2.7 and 99.9±2.8%, respectively. The proposed SPE method coupled off-line with a reserved-phase HPLC system with fluorimetric detection was applied to the quantitation of alinastine in real rat urine samples. The analytical method was also applied and validated for the determination of alinastine in dog plasma. The recovery from spiked dog plasma samples using the PH cartridge was around 65%. The within-day and between-day precisions were 7 and 12%, respectively. The detection and quantitation limits in dog plasma were 0.024 and 0.078 μg/ml, respectively.     

Quantification of trimesic acid in liver, spleen and urine by high-performance liquid chromatography coupled to a photodiode-array detection

The quantification of trimesic acid, a constitutive organic linker from the biodegradable porous iron(III) trimesate MIL-100(Fe) (MIL stands for Materials from Institut Lavoisier), has been performed in different biological complex media (liver, spleen and urine) using a liquid-liquid extraction procedure. A recovery exceeding 92 wt% was achieved from rat tissues and urine spiked with trimesic acid. After extraction, the determination of the trimesic acid concentration was realised by using a simple and accurate high-performance liquid chromatography (HPLC) method using photodiode-array detection (PDA) and aminosalicylic acid, as internal standard. Linearity of this method was kept from 0.01 to 100mg of trimesic acid per liter of urine and from 0.05 to 5.00 wt% of trimesic acid per tissue weight. The limit of detection of the method was 0.01 μg per injection. This method was finally applied to analyze and quantify the amount of trimesic acid in rat urine and tissue samples at the different stages of degradation of MIL-100(Fe).     

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.     

Determination of a major metabolite of tipredane in rat urine by high-performance liquid chromatography with column switching

An automated method, based on column-switching reversed-phase high-performance liquid chromatography, has been developed for the determination of a major metabolite of tipredane in rat urine. Samples are injected directly onto a cyanopropyl extraction column. The portion of eluate containing the metabolite is switched, via an injection loop, onto an octadecylsilane analytical column. The limit of quantification of the method was 25 ng/ml for a 20 μl injection volume of urine. The intra-assay precision (0.7–4.8%) and accuracy (94–105%), and the inter-assay precision (2.7–12.6%) and accuracy (94–105%), were acceptable. The analyte was found to be stable in rat urine when stored at room temperature for six days, in a freezer at or below −20°C for twelve weeks, and when the samples were subjected to two freeze–thaw cycles. No significant interference was observed from tipredane and its major human metabolites, or urine constituents in male and female rats. The method was successfully used to analyse samples from a long-term toxicology study.     

Proteomics of rat biological fluids--the tenth anniversary update

In addition to a remarkable sexual dimorphism of serum and urine proteomes, the rat is exceptional for the wide difference between the serum patterns during an acute phase reaction vs baseline conditions. This feature allows monitoring with high sensitivity onset and progression of any pathological state that involves an inflammatory component as well as assessing the outcome of any therapeutic intervention. Reference maps have been defined for the proteomes of serum, urine, cerebrospinal fluid and bronchoalveolar lavage fluid. For both serum and urine most of the proteomic investigations have dealt with toxicological testing, for BALF with allergic or irritative reactions, whereas with CSF the main aim was the characterization of rat models of neurological disorders. When surveying more than ten years of literature on rat biological fluid proteomics, it is puzzling to see how seldom a consistent analytical plan has been set up for the comparative investigation on two or more types of sample, whether to fully characterize a disease model or to evaluate pharmacological/toxicological effects of a drug. It is also regrettable that in several cases only a negligible part of the results is discussed at length whereas most data are not even made known to the scientific community.     

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.     

Measurement of dimethylglycine in biological fluids

The method of quantitating N,N-dimethylglycine involves cation-exchange high-performance liquid chromatography and detection of dimethylglycine with dimethylglycine dehydrogenase. Dimethylglycine was added to plasma and urine and samples were assayed for dimethylglycine. Plasma and urine to which no dimethylglycine was added were also assayed. Recoveries of added dimethylglycine were 99 to 104% with no endogenous dimethylglycine found in rat plasma or normal human urine. The human plasma used contained a small amount of endogenous dimethylglycine. The cation-exchange chromatography separates dimethylglycine from other compounds which can serve as substrates for dimethylglycine dehydrogenase. Repeatability of the assay is +/- 10%. Using this method we have identified dimethylglycine in the urine of a 1-month-old female human patient.