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1.
Glyoxal and methylglyoxal are two important markers of oxidative stress and both are involved in the evaluation of several diseases. A new HPLC method for determining glyoxal and methylglyoxal in urine was developed. The method is based on the reaction of alpha-dialdehydes, glyoxal and methylglyoxal, with 5,6-diamino-2,4-hydroxypyrimidine sulfate in basic medium to form highly fluorescent lumazine derivatives. Creatinine was also included in the method even though it does not react with the reagent. The derivatives and creatinine are separated on a C(18) reversed-phase column with a mobile phase consisting of acetonitrile:citrate buffer, pH 6.0 (3:97 v/v). The flow rate was 1.0mLmin(-1) and the effluent was monitored photometrically at 250 nm for determination of creatinine and fluorimetrically at 500 nm (exciting at 330 nm) for determination of glyoxal and methylglyoxal derivatives. Recording time of the separation is less than 10 min. Determination of the analytes is performed in urine after incubation of the sample, with the reagent in alkaline medium, for 30 min at 60 degrees C. Urinary levels of glyoxal and methylglyoxal, expressed as glyoxal/creatinine and methylglyoxal/creatinine ratios, in healthy young women and men were determined. For women, values of 0.80+/-0.37 and 0.60+/-0.22 microg/mg of creatinine were found for glyoxal and methylglyoxal, respectively. For men, values of 0.63+/-0.15 and 0.49+/-0.05 microg/mg of creatinine were found for glyoxal and methylglyoxal, respectively. These results were also related to the body mass index of each individual.  相似文献   

2.
3.
A simple and rapid reverse polar ionic LC method was developed and validated for simultaneous separation and determination of mirtazapine, an antidepressant drug, and its main metabolite N-desmethyl mirtazapine using fluorescence and polarimetric detectors connected in series. The chromatographic separation was achieved on Chirobiotic V column packed with vancomycin as a stationary phase in an isocratic mode of elution of methanol:glacial acetic acid:anhydrous triethyl amine (100:0.2:0.1, v/v/v) as a mobile phase. The compounds were detected by their excitation at 290nm and emission at 370nm using fluorescence detector while the optical rotation (+/-) of the enantiomers was identified by polarimetric detector. The analytes were extracted from rat plasma by precipitation of proteins and the average yield was 88-111% for mirtazapine and 85-123% for N-desmethyl mirtazapine. The method was linear over the concentration range of 20-5000ng/mL. The method was successfully applied on rat plasma spiked with the enantiomers of mirtazapine and N-desmethyl mirtazapine.  相似文献   

4.
A novel HPLC method, using UV and fluorimetric serial detection, for the simultaneous determination of methotrexate (MTX), five disease marker pteridines, and the reference metabolic subproduct creatinine (CREA) in human urine was established. A previous oxidation process using 10(-3) M KMnO4 (pH 5.0) and 35min of oxidation time was necessary to transform the analytes in the highly fluorescent pteridinic rings. CREA was not affected by the oxidative medium. Using Tris-HCl/NaCl buffer solution (pH 6.6) as mobile phase, MTX and the assayed pteridines were monitored by fluorescence at lambda(em) = 444 nm and lambda(ex) = 280 nm and creatinine was monitored by absorption measurements at lambda(abs) = 230 nm. All components were well resolved in approximately 7 min. Detection limits, according the criteria of Clayton and co-workers, were 10 ng ml(-1) for MTX, less than 1 ng ml(-1) for all of the pteridines, and 4 microg ml(-1) for CREA.  相似文献   

5.
A high performance liquid chromatography method with ultraviolet and fluorimetric detection has been developed for the simultaneous determination of urinary creatinine (Cr), tryptophan (Trp) and three Trp-related metabolites including kynurenine (Kyn), kynurenic acid (Kyna) and 5-hydroxyindole-3-acetic acid (5-HIAA). Samples were pretreated by centrifugation after a freeze-thaw cycle to remove protein and other precipitates. Separation was achieved by an Agilent HC-C18 (2) analytical column and a gradient elution program with a constant flow rate 1mL/min at an ambient temperature. Total run time was 30 min. Cr, Kyn and Kyna were measured by a variable wavelength detector at wavelengths 258 nm, 365 nm and 344 nm respectively. Trp and 5-HIAA were measured by a fluorescence detector with an excitation wavelength of 295 nm and an emission wavelength of 340 nm. This allowed the determination of Kyn/Cr, Kyna/Cr, Trp/Cr and 5-HIAA/Cr concentration ratios in a single run on the same urine sample. Good linear responses were found with correlation coefficient (r)>0.999 for all analytes within the concentration range of physiological level. The limit of detection of the developed method was: Cr, 0.0002 g/L; Kyn, 0.1 μmol/L; Kyna, 0.04 μmol/L; Trp, 0.02 μmol/L and 5-HIAA, 0.01 μmol/L. Recoveries from spiked human urine were: Cr, 93.0-106.4%; Kyn, 97.9-106.9%; Kyna, 98.5-105.6%; Trp, 96.7-105.2% and 5-HIAA, 96.1-99.7%. CVs of repeatability and intermediate precision of all analytes were less than 5%. This method has been applied to the analysis of urine samples from normal subjects.  相似文献   

6.
7.
The Escherichia coli gene rluA, coding for the pseudouridine synthase RluA that forms 23 S rRNA pseudouridine 746 and tRNA pseudouridine 32, was deleted in strains MG1655 and BL21/DE3. The rluA deletion mutant failed to form either 23 S RNA pseudouridine 746 or tRNA pseudouridine 32. Replacement of rluA in trans on a rescue plasmid restored both pseudouridines. Therefore, RluA is the sole protein responsible for the in vivo formation of 23 S RNA pseudouridine 746 and tRNA pseudouridine 32. Plasmid rescue of both rluA- strains using an rluA gene carrying asparagine or threonine replacements for the highly conserved aspartate 64 demonstrated that neither mutant could form 23 S RNA pseudouridine 746 or tRNA pseudouridine 32 in vivo, showing that this conserved aspartate is essential for enzyme-catalyzed formation of both pseudouridines. In vitro assays using overexpressed wild-type and mutant synthases confirmed that only the wild-type protein was active despite the overexpression of wild-type and mutant synthases in approximately equal amounts. There was no difference in exponential growth rate between wild-type and MG1655(rluA-) either in rich or minimal medium at 24, 37, or 42 degrees C, but when both strains were grown together, a strong selection against the deletion strain was observed.  相似文献   

8.
Pseudouridine synthase 3 from mouse modifies the anticodon loop of tRNA   总被引:2,自引:0,他引:2  
Chen J  Patton JR 《Biochemistry》2000,39(41):12723-12730
A cDNA encoding mouse pseudouridine synthase 3 (mPus3p) has been cloned. The predicted protein has 34% identity with yeast pseudouridine synthase 3 (Pus3), an enzyme known to form pseudouridine at positions 38 and 39 in yeast tRNA. The cDNA is 1.7 kb, and when used as a probe on a Northern blot of total RNA from mouse tissues or cells in culture, a band at 1.8 kb was observed. The open reading frame codes for a protein of 481 amino acids with a predicted molecular mass of 55 552 Da. When mPus3p was in vitro translated and used in reactions with tRNA substrates from both yeast and humans, uridines at position 39 were modified to pseudouridine. In a tRNA substrate with a uridine at position 38 (human tRNA(Leu)), there was very slight formation of pseudouridine at that position after incubation with mPus3p.  相似文献   

9.
The extracellular surface of the gap junction cell-to-cell channels was imaged in phosphate-buffered saline with an atomic force microscope. The fully hydrated isolated gap junction membranes adsorbed to mica were irregular sheets approximately 1-2 microns across and 13.2 (+/- 1.3) nm thick. The top bilayer of the gap junction was dissected by increasing the force applied to the tip or sometimes by increasing the scan rate at moderate forces. The exposed extracellular surface revealed a hexagonal array with a center-to-center spacing of 9.4 (+/- 0.9) nm between individual channels (connexons). Images of individual connexons with a lateral resolution of < 3.5 nm, and in the best case approximately 2.5 nm, were reliably and reproducibly obtained with high-quality tips. These membrane channels protruded 1.4 (+/- 0.4) nm from the extracellular surface of the lipid membrane, and the atomic force microscope tip reached up to 0.7 nm into the pore, which opened up to a diameter of 3.8 (+/- 0.6) nm on the extracellular side.  相似文献   

10.
Preliminary investigation of the phage phi X174 crystal structure   总被引:1,自引:0,他引:1  
Crystals of the single-stranded DNA bacteriophage phi X174 have been grown. They have a monoclinic unit cell with space group P2(1), unit cell dimensions of a = 306.0 (+/- 0.2) A, b = 361.1 (+/- 0.2) A, c = 299.7 (+/- 0.2 degrees) A, beta = 92.91 degrees (+/- 0.02 degrees) and diffract to at least 2.7 A resolution. There are two virus particles per unit cell. Packing considerations show that the mean diameter of the virus particles is 280 A. The virus separates into two bands in a sucrose gradient. The ratio between the absorbance at 260 nm and 280 nm is 1.45 to 1.65 for the faster and 1.15 to 1.35 for the slower bands, but both bands contain intact particles. Crystals derived from these bands are isomorphous and there is no detectable difference in their structure amplitudes.  相似文献   

11.
The biochemical parameters used in this study were: (1) serum pseudouridine, expressed as nmols/mL; (2) pseudouridine index, expressed as mol to mol ratio of serum pseudouridine versus serum creatinine concentration. The receiver operating characteristic (ROC) analysis has been used to exemplify the selection of discriminant values or "cut-off points" to maximize the diagnostic utility of a biochemical tumor marker, serum pseudouridine. This marker has been used in a variety of group population samples, i.e., normal subjects, subjects affected by several nonneoplastic diseases, subjects with neoplastic disorders in less advanced or more advanced stages, and finally in a sample population of patients affected by lymphomas and leukemias of different types. An analysis of the relative ROC curves allowed the selection of cut-off values that maximize the diagnostic efficiency or, alternatively, the diagnostic sensitivity or the diagnostic specificity for pseudouridine parameters, and has allowed the comparison of the two tests to answer the same clinical question.  相似文献   

12.
BackgroundBarricorTM Lithium heparin plasma tubes are new blood tubes that have been introduced to overcome the effects of gel in serum separator tubes (SST) and the shortcomings of standard Lithium heparin plasma. We aimed to evaluate BarricorTM tubes as an alternative to serum separator tubes and compare the stability between the tubes.MethodsForty-four paired samples were collected using both BarricorTM and SST. We compared five analytes at baseline (<6 h) and after every 24 h using the PassingBablok and Bland-Altman plots. Aspartate aminotransferase (AST), potassium (K), phosphate (PO4) , lactate dehydrogenase (LDH), and creatinine were analysed in both tubes. We calculated the percentage difference for each analyte between the baseline and time intervals to assess analyte stability. The percentage difference was compared to the desirable specification for bias and reference change value (RCV).ResultsAll analytes were comparable at baseline. Statistical differences (p<0.001) became evident after 24 h. PO4, K, and creatinine had a mean difference that exceeded the desirable specification for bias (-9.59%, - 9.35%, and -4.59%, respectively). Potassium was stable up to 24 h in both tubes. LDH showed better stability in SST (144 h vs 96 h). PO4 concentrations were more stable in both tubes with the SST (96 h vs 72 h). Creatinine and AST had the longest stability in both tubes compared to other analytes (144 h).ConclusionsData demonstrated variability and similarities in analyte concentrations and stability, respectively, in both tubes.  相似文献   

13.
A novel reversed-phase HPLC method was developed for the simultaneous determination of tacrine (THA) and the newly synthesized prodrug (N-butyramide-THA, BTHA) in mouse plasma and brain homogenate. The assay involves deproteinisation and subsequent detection at 240 nm with a gradient solvent system. Retention times were 18.5 and 9.3 min for BTHA and THA, respectively. Average recoveries for the analytes were 80.7% (BTHA) and 76.6% (THA) from plasma, and 75.0% (BTHA) and 68.4% (THA) from brain homogenate. Linear responses were observed over a wide range (0.25-20 microg/ml for BTHA in plasma and in brain homogenate, 0.025-20 microg/ml for THA in both matrices). Both BTHA and THA degraded from the prodrug can be detected even 12 h after intravenous administration of BTHA, indicating that BTHA is a promising prodrug for brain targeting.  相似文献   

14.
Variation in plasma urea and creatinine concentration and plasma urea:creatinine ratio (U:C) were studied in semidomestic free-ranging reindeer (Rangifer tarandus tarandus) on the Norwegian mainland, in wild Svalbard reindeer (Rangifer tarandus platyrhynchus), and in captive reindeer maintained either on a lichen-based diet or a protein-rich concentrate to investigate whether these parameters could be used as indicators of the nutritional status of reindeer. In the mainland animals, plasma creatinine concentration was high in winter and early spring and decreased by two-thirds toward the summer. The overall range in mean plasma creatinine concentration (+/-SE) was from 90+/-1.26 to 280+/-2.88 micromol/L. Mean plasma urea concentration (+/-SE) varied from 2.46+/-0.10 in winter up to 17.44+/-0.29 mmol/L in summer and autumn. Month of sampling explained 65% and 90% of the variation in plasma urea and creatinine concentrations, respectively, indicating that seasonality in the diet had the greatest influence on these parameters. Reindeer given lichens as the only feed showed an increase in plasma creatinine and a decrease in plasma urea concentration. Food restriction caused a temporary elevation in urea level but had no significant effect on plasma creatinine concentration. The slight effect of energy intake on urea and creatinine levels was supported by the fact that severe undernutrition in the Svalbard reindeer population had only a small effect on plasma urea and creatinine levels. Protein-rich pellet feed increased plasma urea from around 3 mmol/L to above 10 mmol/L and reduced creatinine concentrations to less than 100 micromol/L, suggesting that the protein content of forage is an important determinant of these blood parameters. Mean U:C ratio (+/-SE) in plasma varied from 8.9+/-0.28 to 120.8+/-1.88. Ratios above 20 appeared when protein intake was low and energy intake was restricted or when protein intake was high. Low ratios occurred when protein intake was low but energy intake adequate. Plasma urea and creatinine concentrations and the U:C ratio showed complex dynamics that were affected by both season and the protein and feed intake. We conclude that they appear to be difficult to interpret as single measures of nutritional status of reindeer.  相似文献   

15.
The pseudouridine synthases catalyze the isomerization of uridine to pseudouridine at particular positions in certain RNA molecules. Genomic data base searches and sequence alignments using the first four identified pseudouridine synthases led Koonin (Koonin, E. V. (1996) Nucleic Acids Res. 24, 2411-2415) and, independently, Santi and co-workers (Gustafsson, C., Reid, R., Greene, P. J., and Santi, D. V. (1996) Nucleic Acids Res. 24, 3756-3762) to group this class of enzyme into four families, which display no statistically significant global sequence similarity to each other. Upon further scrutiny (Huang, H. L., Pookanjanatavip, M., Gu, X. G., and Santi, D. V. (1998) Biochemistry 37, 344-351), the Santi group discovered that a single aspartic acid residue is the only amino acid present in all of the aligned sequences; they then demonstrated that this aspartic acid residue is catalytically essential in one pseudouridine synthase. To test the functional significance of the sequence alignments in light of the global dissimilarity between the pseudouridine synthase families, we changed the aspartic acid residue in representatives of two additional families to both alanine and cysteine: the mutant enzymes are catalytically inactive but retain the ability to bind tRNA substrate. We have also verified that the mutant enzymes do not release uracil from the substrate at a rate significant relative to turnover by the wild-type pseudouridine synthases. Our results clearly show that the aligned aspartic acid residue is critical for the catalytic activity of pseudouridine synthases from two additional families of these enzymes, supporting the predictive power of the sequence alignments and suggesting that the sequence motif containing the aligned aspartic acid residue might be a prerequisite for pseudouridine synthase function.  相似文献   

16.
Multiple assembly pathways underlie amyloid-beta fibril polymorphisms   总被引:1,自引:0,他引:1  
The amyloid beta-protein transiently forms low and high molecular mass oligomers and protofibrils in vitro, and after longer incubation times assembles into polymorphic mature fibrils. The precursor-to-product relationship of these species remains to be understood. Protofibrils are up to approximately 600 nm in length and have mass-per-lengths of 19(+/-2) kDa/nm measured by scanning transmission electron microscopy. Two predominant mature fibril types, several microns in length and with mass-per-lengths of 18(+/-3) and 27(+/-3) kDa/nm, are identified after longer incubation times. The difference of approximately 9 kDa/nm between the two fibril types indicates a bona fide elementary protofilament subunit of this mass-per-length. Fibrils in the 18(+/-3) kDa/nm group often exhibited distinct coiling with axial cross-over spacings of approximately 25 nm. Although strikingly different in morphology, the mass-per-length (MPL) of these coiled fibrils is equivalent to that measured for protofibrils. They could therefore arise from a conformational change in the protofibril concurrent with coiling and rapid elongation. Alternatively, we cannot rule out an assembly pathway not directly related to protofibrils. In contrast, the 27(+/-3) kDa/nm fibrils correspond to a MPL of approximately 1.5 x the protofibril and thus can neither arise from a simple conformational transition nor from lateral association of 19 kDa/nm protofibril precursors. Twisted ribbons with axial periodicities ranging from approximately 80 nm to 130 nm were prominent in the 27(+/-3) kDa/nm group as well as more tightly coiled fibrils. Individual fibril ribbons had elongation rates of 20(+/-12) nm/min when imaged by time-lapse atomic force microscopy. Protofibrils exhibited growth rates approximately 15 x slower at 1.3(+/-0.5) nm/min. The data support a model where concurrent multiple assembly pathways give rise to the various polymorphic fibril types.  相似文献   

17.
Two complementary methods for identifying and measuring sulfonamide residues in eggs were developed for use in surveying eggs for potential drug residues. The first method uses liquid chromatography-tandem mass spectrometry (LC-MS-MS) to confirm the presence of sulfonamide residues in eggs. During its validation the limit of confirmation was estimated to be 5-10 ng/g (ppb) depending on the drug. Also, a method for measuring residue level by liquid chromatography with ultraviolet detection (LC-UV) was validated using the same extraction procedure as the confirmatory method. The determinative method was validated over the 50-200 ppb range. Samples were prepared by homogenizing whole egg, extracting with acetonitrile, and cleaning up with a C(18) solid-phase extraction cartridge. For confirmation, analytes were separated by gradient LC on a C(18) column, ionized by electrospray ionization (ESI), and detected by MS-MS with an ion trap mass spectrometer. For determination, analytes were separated by a different gradient LC procedure and detected by UV at 287 nm. Fifteen drugs were dosed individually in laying hens, and residues of parent drug and/or metabolites were found in eggs for all the drugs. Validation was based on repetitive analyses of control samples, control samples fortified at 100 ppb sulfonamides, and samples of blended incurred eggs.  相似文献   

18.
A validated method for the determination of triethylenetetramine, a selective copper-chelator currently undergoing clinical trials for the treatment of diabetic heart failure, and its two major metabolites, N(1)-acetyltriethylenetetramine and N(1),N(10)-diacetyltriethylenetetramine in human serum using HPLC is reported. The method used 9-flouorenylmethylchloroformate chloride to label all three analytes. The fluorescence labeled analytes were then separated chromatographically using a reversed phase C18 column under a gradient elution program and detected spectrofluorometrically at 317 nm with excitation at 263 nm. Application of the method is demonstrated by pharmacokinetic measurement in one healthy volunteer taking the drug orally.  相似文献   

19.
An optical array biosensor encapsulated with hydrolase and oxidoreductase using sol-gel immobilization technique has been fabricated for simultaneous analysis and screening of multiple samples to determine the presence of multianalytes which are clinically important in relation to renal failure. Urease and creatinine deiminase were used to detect urea and creatinine, while glucose oxidase and uricase were coimmobilized with horseradish peroxidase to quantify glucose and uric acid. Moreover, the concentrations of analytes in fetal calf serum were measured and quantified using the developed sensing system. The array biosensor showed good specificity for the simultaneous analysis of multiple samples for multianalytes without obvious cross-interference. The analytical ranges of the four analytes were between 0.01 and 10mM with detection limits of 2.5-80 microM. High precision with relative standard deviations of 3.8-9.2% (n=45) was also demonstrated. The reproducibility of array-to-array in 3 consecutive months was 5.4% (n=3). Moreover, the concentrations of analytes in fetal calf serum were 5.9 mM for urea, 0.13 mM for creatinine, 3.3mM for glucose, and 0.15 mM for uric acid, which were in good agreement with results obtained using the traditional spectroscopic methods. These results demonstrate the first use of a sol-gel-derived optical array biosensor for simultaneous analysis of multiple samples for the presence of multiple clinically important renal analytes.  相似文献   

20.
A high-performance liquid chromatographic (HPLC) method with electrochemical detection and solid-phase extraction (SPE) using cartridges of weak cation-exchange capacity as the primary retention mechanism is described for the separation and determination of methylnaltrexone (MNTX) in small clinical samples of plasma or urine. The procedure was performed using a Phenomenex Prodigy ODS-2, 5 microm, 150x3.2 mm analytical column and 50 mM potassium acetate buffer, with 11% methanol as organic modifier at pH* 4.5 at a flow-rate of 0.5 ml/min. The detection potential was 700 mV. The six-point standard calibration curves were linear over three consecutive days in the range from 2 to 100 ng/ml. The average goodness of fit (r) was 0.9993. The lower limit of detection (LOD) and limit of quantification (LOQ) were found to be 2.0 and 5.0 ng/ml, respectively. At the LOQ, the coefficient of variation for the entire method was 8.0% and the accuracy was 10.0% (n = 10). Recovery of the drug from plasma was in the region of 94%. The method was applied to a pharmacokinetics study of methylnaltrexone after subcutaneous administration and in numerous assays of analytes in blood plasma and urine. The pharmacokinetics parameters for a single dose of 0.1 or 0.3 mg/kg in plasma were C(max) = 110 (+/-55) and 287 (+/-101) ng/ml and t(max) = 16.7 (+/-10.8) and 20.0 (+/-9.5) min, respectively. The method is simple, yet sensitive for the detection and determination of methylnaltrexone in biological samples at the level of the physiological response.  相似文献   

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