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Separation of lipoproteins secreted from McA-RH7777 (rat hepatoma) cells by Superose 6 column size-exclusion chromatography, using PBS buffer (NaCl 150 mM, sodium phosphate 10 mM, pH 7.5, EDTA 1 mM), produced apolipoprotein (ape) ...
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Separation of lipoproteins secreted from McA-RH7777 (rat hepatoma) cells by Superose 6 column size-exclusion chromatography, using PBS buffer (NaCl 150 mM, sodium phosphate 10 mM, pH 7.5, EDTA 1 mM), produced apolipoprotein (ape) E or A-I profiles that did not correlate with lipoproteins separated by density ultracentrifugation. By density ultracentrifugation, apoE and apoA-I were mostly (>90%) confined to high-density lipoproteins (HDL, d=1.063-1.023 g/ml), but by chromatography apoE and apoA-I were recovered in all lipoprotein classes, including low-density lipoproteins (LDL), HDL, and post-HDL. Moreover, the elution volume of phenol red on Superose 6 greatly exceeded the total column volume. These discrepancies were attributable to pH and ionic strength effects, In low ionic strength, high pi-I buffer (Tris 25 mM, pH 8.3), elution volumes of lipoproteins, albumin, and phenol red were minimized. Elution volumes increased 25-70% when buffer pi-I was lowered at constant ionic strength (Tris 25 mM, pH 7.4) or when ionic strength was increased at constant pH (Tris 25 mM, pH 8.3, NaCl 500 mM). Altered phase partition appeared to cause the altered elution volumes, since recovery (measured as analyte peak area), resolution (measured as peak width at half height), and column void volume varied little from buffer to buffer. In Superose 6 size-exclusion chromatography with PBS buffer, then, elution volumes vary with pH and ionic strength. We propose that TEE buffer (Tris-borate 89 mM, pH 8.3, EDTA 2 mM) may produce fewer artefacts than PBS. With TEE there were (i) better correlation between size-exclusion and ultracentrifugal fractions, (ii) lower elution volumes, and (iii) less ''smearing'' of McA-RH7777 apoE and apoA-I containing lipoprotein bands.
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A simple and sensitive HPLC method for determination of metronidazole in human plasma has been developed. A step of freezing the protein precipitate allowed an efficient separation of aqueous and organic phases minimizing the nois...
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A simple and sensitive HPLC method for determination of metronidazole in human plasma has been developed. A step of freezing the protein precipitate allowed an efficient separation of aqueous and organic phases minimizing the noise level and improved therefore the limit of quantitation (10 ng ml-L using I mi of plasma sample). The separation of compounds was performed on a RP 18 column with acetonitrile-aqueous 0.01 M phosphate solution (15:85, v/v) as mobile phase. Detection was performed by UV absorbance at 318 nm. Metronidazole was well resolved from the plasma constituents and internal standard. An excellent linearity was observed between peak-height ratios plasma concentrations over a concentration range of 0.01 to 10 mu g ml(-1). Within-day and between-day precision (expressed by relative standard deviation) and accuracy (mean error in per cent) did not exceed 4% between 1 and 10 mu g ml(-1) and 8.3 and 7.2% respectively for the limit of quantitation. The method is suitable for bioavailability and pharmacokinetic studies in humans. (C) 1998 Elsevier Science BN. All rights reserved. [References: 13]
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Aim:Iohexol plasma clearance is used as an indicator of kidney function in clinical and preclinical settings. To investigate the pharmacokinetic profile of iohexol, a rapid, simple method for measurement of iohexol in different ma...
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Aim:Iohexol plasma clearance is used as an indicator of kidney function in clinical and preclinical settings. To investigate the pharmacokinetic profile of iohexol, a rapid, simple method for measurement of iohexol in different matrices and species was needed.Materials & methods:Iohexol was separated on an Accucore C18 column (Thermo Fisher Scientific, CA, USA). Detection was performed on a Thermo Scientific Quantiva tandem quadrupole mass spectrometer. The method was validated according to the requirements for bioanalytical methods issued by the US FDA and European Medicines Agency.Conclusion:We developed and validated a fast and efficient analytical method, suitable for analyzing iohexol in human EDTA plasma, human lithium-heparin plasma, human urine and goat- and pig EDTA plasma, using only one calibration line prepared in human EDTA plasma.
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Clinical metabolic phenotyping employs metabolomics and lipidomics to detect and measure hundreds to thousands of metabolites and lipids within human samples. This approach aims to identify metabolite and lipid changes between phe...
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Clinical metabolic phenotyping employs metabolomics and lipidomics to detect and measure hundreds to thousands of metabolites and lipids within human samples. This approach aims to identify metabolite and lipid changes between phenotypes (e.g. disease status) that aid understanding of biochemical mechanisms driving the phenotype. Sample preparation is a critical step in clinical metabolic phenotyping: it must be reproducible and give a high extraction yield of metabolites and lipids, and in high-throughput studies it needs to be rapid. Here, we assessed the extraction of polar metabolites from human urine and polar metabolites and lipids from human plasma for analysis by ultra-high-performance liquid chromatography- mass spectrometry (UHPLC-MS) metabolomics and lipidomics. We evaluated several monophasic (urine and plasma) and biphasic (plasma) extractions, and we also tested alterations to (a) solvent-biofluid incubation time and temperature during monophasic extraction, and (b) phase partitioning time during biphasic extraction. Extracts were analysed by three UHPLC-MS assays: (i) hydrophilic interaction chromatography (HILIC) for urine and plasma, (ii) C18 aqueous reversed phase for urine, and (iii) C18 reversed phase for plasma lipids, and the yield and reproducibility of each method was assessed. We measured UHPLC-MS injection reproducibility as well as sample preparation reproducibility to assess sample solvent composition compatibility with UHPLC-MS and to pinpoint the origin of variance within the methods. For HILIC UHPLC-MS plasma and urine analysis, monophasic 50 : 50 methanol : acetonitrile had the most detected putatively-identified polar metabolites with high method reproducibility. This method had the highest lipid yield for plasma extracts analysed by the HILIC method. If lipid removal from the plasma polar HILIC extract is required, then the biphasic methanol/chloroform/water method is recommended. For C18 (aqueous) UHPLC-MS urine analysis, 50 : 50 m
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The expression and activity of matrix metalloproteinases (MMPs) may be regulated by oxidative stress in various pathophysiological processes; therefore, the aim of the present study was to analyse the associations between the expr...
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The expression and activity of matrix metalloproteinases (MMPs) may be regulated by oxidative stress in various pathophysiological processes; therefore, the aim of the present study was to analyse the associations between the expression of the gelatinases MMP-9 and MMP-2 and their tissue inhibitors TIMP-1, TIMP-2 and levels of total antioxidant capacity (TAC) and advanced oxidation protein products (AOPP) in seminal plasma prepared for artificial insemination. Levels of MMPs and TIMPs were evaluated using ELISA, whereas TAC and AOPP in the seminal plasma of 131 childless men and 38 fertile volunteers were determined spectrophotometrically. Seminal MMP-9 expression was higher in childless men than in fertile subjects, whereas there was no significant differences in MMP-2 expression between the analysed seminal groups. TIMP-1 and TIMP-2 expression was similar in all groups. However, TAC expression was significantly higher in infertile normozoospermic and oligozoospermic men and AOPP expression was higher in astheno-, oligo- and normozoospermic infertile patients than in fertile men. High AOPP, together with an increased MMP-9:TIMP-1 ratio alters the oxidative-antioxidative balance of the ejaculate, thereby reducing male fertility, and therefore these parameters may serve as additional diagnostic markers of semen quality and male reproductive potential.
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Peroxisome proliferator-activated receptor gamma (PPAR gamma) agonists are used to treat type 2 diabetes mellitus (T2DM). Widespread use of PPAR gamma agonists has been prevented due to adverse effects including weight gain, edema...
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Peroxisome proliferator-activated receptor gamma (PPAR gamma) agonists are used to treat type 2 diabetes mellitus (T2DM). Widespread use of PPAR gamma agonists has been prevented due to adverse effects including weight gain, edema, and increased risk of congestive heart failure. Selective PPAR gamma modulators (SPPAR gamma Ms) have been identified that have antidiabetic efficacy and reduced toxicity in preclinical species. In comparison with PPAR gamma full agonists, SPPAR gamma M 6 (MK0533) displayed diminished maximal activity (partial agonism) in cell-based transcription activation assays and attenuated gene signatures in adipose tissue. Compound 6 exhibited comparable efficacy to rosiglitazone and pioglitazone in vivo. However, with regard to the induction of untoward events, 6 displayed no cardiac hypertrophy, attenuated increases in brown adipose tissue, minimal increases in plasma volume, and no increases in extracellular fluid volume in vivo. Further investigation of 6 is warranted to determine if the improvement in mechanism-based side effects observed in preclinical species will be recapitulated in humans.
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The simultaneous liquid chromatographic determination of oxcarbazepine and the enantiomers of its metabolites 10,11-dihydro-10-hydroxycarbamazepine and trans-10,11-dihydroxycarbamazepine in spiked human plasma is described. The co...
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The simultaneous liquid chromatographic determination of oxcarbazepine and the enantiomers of its metabolites 10,11-dihydro-10-hydroxycarbamazepine and trans-10,11-dihydroxycarbamazepine in spiked human plasma is described. The compounds are subjected to solid phase extraction before chromatography. The separation of the analytes is achieved using chiralcel OD column coupled on line with chiralcel ODH column and a mobile phase consisting of n-hexane-ethanol (70/30, v/v). The compound were detected by ultraviolet absorbance at 220 nm. The limit of quantification for each compound was 5 ng/ml.
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In this study, the structure and mechanical stability of human plasma fibronectin (HFN), a major protein component of blood plasma, have been evaluated in detail upon adsorption on the nonirradiated and irradiated Ti6Al4V material...
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In this study, the structure and mechanical stability of human plasma fibronectin (HFN), a major protein component of blood plasma, have been evaluated in detail upon adsorption on the nonirradiated and irradiated Ti6Al4V material through the use of atomic force microscopy. The results indicated that the material surface changes occurring after the irradiation process reduce the disulfide bonds that typically preclude the mechanical denaturation of individual HFN domains and interfere significantly with the intraionic interactions stabilizing the compact conformation of the adsorbed HFN molecules. In particular, upon adsorption on this material, the molecules adopt a more flexible conformation and become mechanically more compliant. Unexpected observations also indicated that, regardless the material surface, a single HFN molecule can be pulled into an extended conformation without the unfolding of its domains through a series of three unraveling steps. The forces involved in the unraveling process were found to be generally lower than the forces required to unfold the individual protein domains. This report is the first one to present the force displacement details associated to the straightening of a single compact protein at the molecular level.
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I read with great interest the recent work by Pavlovic and colleagues [1] reporting on the quantitative determination of free soluble 3-nitrotyrosine (NO2Tyr) in human plasma by a newly developed and validated GC–ECD method. This...
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I read with great interest the recent work by Pavlovic and colleagues [1] reporting on the quantitative determination of free soluble 3-nitrotyrosine (NO2Tyr) in human plasma by a newly developed and validated GC–ECD method. This analytical approach includes protein precipitation, SPE of NO2Tyr and the external standard 4-nitro-L-phenylalanine (NF),isolation of NO2Tyr and NF by LC, onestep derivatization to their O-heptafluorobutyryl ester N-heptafluorobutyryl amide derivatives and subsequent quantification by GC–ECD. This work is a considerable contribution to 3-nitrotyrosine quantification, represents an unequivocal proof of the outstanding challenge of reliable 3-nitrotyrosine analysis, and is a good example for how to address methodological problems. I agree with most of the points addressed by Pavlovic and colleagues [1], however there are a few issues that deserve a critical discussion.
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