1,720,961 research outputs found
Validation of an HPLC-fluorescence method for the simultaneous free and glycosylated pyridinium crosslinks determination in urine
No full textBACKGROUND: Pyridinium crosslinks, released during bone resorption, are excreted in urine as free pyridinoline (Pyr) and deoxypyridinoline (DPyr), or bound to peptide or to sugars, as galactosyl-pyridinoline (Gal-Pyr) and glucosyl-galactosyl pyridinoline (GluGal-Pyr). Commonly, only total Pyr and D-Pyr urinary amounts (free + bound forms) are evaluated.
METHOD: We developed and validated an analytical method based on HPLC-fluorescence for the evaluation of the collagen crosslinks Pyr and DPyr (free and total), GluGal-Pyr and Gal-Pyr in the urine of healthy women (n = 20; aged 27-41) and girls (n = 20; aged 5-10). Urine, spiked with an unnatural D-Pyr homologue, as IS, was solid-phase extracted prior to HPLC analysis. The use of this IS and of pure Pyr, D-Pyr, GluGal-Pyr and Gal-Pyr, synthesized to be used as primary calibrators, guarantees the specificity of the method and the correct crosslinks quantification. Total Pyr and D-Pyr amounts were also evaluated after urine hydrolysis.
RESULTS: The method demonstrates good selectivity, sensitivity, linearity, precision, accuracy, recovery and stability for all measured crosslinks. Pyr and D-Pyr, both free and total, and GluGal-Pyr amounts were significantly higher in girls than in women (p < 0.0001). Gal-Pyr, evaluated in girls for the first time, was under its lower quantification limit (< 21.20 pmol/mL) in women.
CONCLUSIONS: The quantification of free and glycosylated pyridinium crosslinks might provide more information on the degradation of various types of collagen, respect to the measurement of total Pyr and D-Pyr alone. Moreover, this validated method could be a useful non-invasive technique for studying pathological conditions characterized by modified glycosylation enzyme activity and for other clinical investigations on bone fragility
Validation of methyl malondialdehyde as internal standard for malondialdehyde detection by capillary electrophoresis
No full textThe aim of this study was to validate, by capillary electrophoresis, the use of synthesized methyl malondialdehyde as the internal standard for the direct quantification of free and total (free+bound) malondialdehyde in biological samples. All analyses were performed in 20 cm x 50 microm uncoated capillaries at 20 degrees C, using 25 mmol/L borax (pH 9.3) and 5 mmol/L tetradecyltrimethylammonium bromide as running buffer. The applied voltage was -4kV (about 8 microA), the detector being set at 260 nm for a total run time of 8 min per sample. Free malondialdehyde was evaluated after acetonitrile extraction, while the samples evaluated for total malondialdehyde were, before extraction, hydrolyzed for 1h at 60 degrees C in the presence of 1 mol/L NaOH. The detection threshold was 0.2 micromol/L in microsomes and 0.4 micromol/L in plasma. As an application of the method, three pools of rat liver microsomes were quantified before (0.35+/-0.1 and 1.1+/-0.5 nmol/mg protein, free and total malondialdehyde, respectively, mean+/-SD) and after lipoperoxidation induction using systems able to generate oxygen free radicals (18.4+/-3.2 and 19.7+/-2.0 nmol/mg protein). The results were confirmed by isotopic dilution gas chromatography-mass spectrometry, used as the reference method. The feasibility of capillary electrophoresis for malondialdehyde determination in normal and pathological human plasma was also investigated
Chirality of 3 hydroxyoctadecanoic acid from stearoyl CoA by rat liver soluble enzymes
No full textOne of the transformation products formed from stearoyl CoA by the soluble enzymes of rat liver homogenate has been identified as 3 hydroxyoctadecanoic acid. The chirality of this acid obtained from incubation of [1 14C]stearoyl CoA with the 105,000 g soluble enzymes of rat liver has been determined. After 10 and 20 min of incubation 70% of the L(+) and 30% of the D(-) enantiomer are formed, whereas racemic 3 hydroxyoctadecanoic acid is isolated after 90 min of incubation. These results suggest either that an epimerizing enzyme is present in the soluble fraction or that 2 enzymes, each specifically forming one of the 2 enantiomers, are present
Development of a multi-analyte method for the determination of anabolic hormones in bovine urine by isotope-labelled GC-MS/MS
No full textThe use of natural and synthetic hormones for growth promotion purposes in meat producing animals is prohibited in the European Community since 1986 to protect consumers from possible developmental, neurobiological, genotoxic and carcinogenic effects due to the intake of hormone residues and their metabolites. Consequently, every EU Member State has to monitor a set proportion of the total annual production of different animal food commodities for their anabolic residues, determining them either in biological fluids (blood or urine) or muscle tissue and edible organs.
Screening analysis of anabolic hormones using immunoassays suffer from cross-reactivity with structurally related hormones. In order to reduce analytical interferences and improve accuracy, we developed a method using gas chromatography coupled to ion trap tandem mass spectrometry (GC-MS/MS) for the simultaneous determination of 11 anabolics in bovine urine (hexestrol, diethylstilbestrol, dienestrol, 17α-estradiol, 17β-estradiol, 17α-ethynylestradiol, 19-nortestosterone, 17α-methyltestosterone, β-testosterone, α-zeranol and β-zeranol). The assay consists of a simple sample preparation procedure, which utilizes only one solid-phase extraction step and one liquid-liquid extraction step for sample clean-up prior to derivatization for positive electron impact GC-MS/MS analysis. Two types of derivatization agents were assayed, N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane and heptafluorobutyric anhydride (HFBA). The three possible acquisition modes of the GC-MS system, Full Scan, selective ion monitoring (SIM) and tandem mass spectrometry (MS/MS), were compared and best results were obtained in the MS/MS mode in the narrow concentration range of 0.25 to 8.0 ng/mL. Better results and good linearity were obtained using BSTFA as a derivatization agent by which no matrix effects were observed. The limits of detection were in the range of 0.005 ng/mL (diethylstilbestrol) to 0.38 ng/mL (17α-methyltestosterone and 17α-ethynylestradiol). Recoveries varied from 81 ± 4% (α-zeranol) to 149 ± 24% (17α-methyltestosterone) using the MS/MS mode. Repeatability values were obtained from 4 replicate analysis of spiked urine samples at 1 ng/mL and ranged from 2.1 to 22.7%
Evaluation of oxidative status in human serum: comparison of different methodological approaches
No full textDifferent biomarker assays have been developed for assessing oxidative stress in human serum. In this retrospective study the analytical performance of two different methodological approaches was evaluated in subjects with known increased oxidative stress to measure serum peroxidation indices: mild (n=35) and heavy (n=61) smokers, chronic renal failure (n=19) and kidney transplanted patients (n=59) compared to healthy controls (n=56).
Serum oxidative stress, assessing Reactive Oxygen Metabolite derivatives (d-ROMs, Diacron International, Italy) levels and Total Antioxidant Capacity (TAC, Diacron International, Italy) by commercial spectrophotometric assays, was compared with malondialdehyde (MDA) concentrations, quantified both in free (FMDA) and total (T-MDA) forms, by isotope-dilution gas chromatography-mass spectrometry (GC-MS) technique (“gold standard” reference method), generally unsuitable for routine use.
Sensitivity, specificity and cut-off points of T-MDA and F-MDA, d-ROMs, TAC assays were evaluated by receiver-operator characteristic (ROC) analyses together with the area under ROC curve (AUC).
ROC analyses accuracy: the best for T-MDA (AUC: 1; sensitivity and specificity: 100%), good for d-ROMs (AUC: 0.87; sensitivity 72.8%, specificity: 100%) and F-MDA (AUC: 0.82; sensitivity 74.7%, specificity: 100%), and not as good as the previous for TAC (AUC: 0.66; sensitivity: 52% and specificity: 92.9%). The increased peroxidative damage was best proved only by T-MDA levels. The assessment of d-ROMS concentrations and TAC by reliable assays is useful for routine clinical purposes; even if less sensitive, it determines the balance of oxidative status. The comparison between “gold standard” and routine methods allows biochemists and clinicians to evaluate data more thorough
Dimethylarginines in complicated type 1 diabetes: roles of insulin, glucose, and oxidative stress
No full textTo investigate the roles of insulin, glucose, and oxidative stress on plasma asymmetric and symmetric dimethylarginine (ADMA, SDMA) levels in complicated diabetes, we studied patients with type 1 diabetes (T1D; n = 20), T1D + end-stage renal disease under hemodialysis (T1D + ESRD; n = 12), T1D + ESRD who received kidney transplant (KD; n = 16), and T1D + ESRD who received kidney-pancreas transplant (KP; n = 20) and healthy controls (n = 50). Levels of ADMA, SDMA, and free and total malondialdehyde (MDA) were increased in all patients, with the highest rises for SDMA and free MDA in T1D+ESRD. In KP, the normalized glycemia contributes to the recovery of ADMA, SDMA, and MDA levels toward normal values. From the covariance analyses, both glucose and insulin relate significantly to ADMA in T1D + ESRD (beta = +0.004, beta = -0.038, respectively) and in KP (beta = +0.032, beta = +0.032, respectively). Creatinine clearance and insulin relate to SDMA in all patient groups (beta = -0.006). Our results provide evidence for the effect of kidney-pancreas transplant on the recovery of ADMA, SDMA, and indexes of oxidative stress toward normal values. Only free MDA allows one to discriminate the magnitude of the oxidative status, as increased total MDA could also be attributable to a reduced renal function
Evaluation of enzyme activities by gas chromatography-mass spectrometry : HMG-CoA reductase and cholesterol 7-hydroxylase
No full textMethods are described for the evaluation of HMGCoA reductase and cholesterol 7 alpha-hydroxylase activities. The methods are based on the measurement by selected-ion monitoring of mevalonate, formed from HMGCoA and of 7 alpha-hydroxycholesterol formed from cholesterol, respectively. The methods are as sensitive as those based on the use of radioisotopes but less time-consuming because quantitation is carried out on total lipid extracts. In the case of cholesterol 7 alpha-hydroxylase, the procedure does not require the use of exogenous cholesterol as the substrate, so problems related to its equilibration with endogenous microsomal cholesterol are avoided
Increased free malondialdehyde concentrations in smokers normalise with a mixed fruit and vegetable juice concentrate : a pilot study
No full textBackground: Cigarette smoking, a cardiovascular risk factor leading to oxygen free radical formation, is involved in the development of serious pathological conditions. On the other hand, a healthy diet and adequate supplementation can help prevent many diseases. The aim of our study was to evaluate in healthy light smokers the effects of supplementation with mixed fruit and vegetable juice powder concentrate on homocysteine metabolism and oxidative status. Methods: In this pilot study, 32 healthy volunteers, 16 light smokers and 16 non-smokers, on twice daily supplementation were monitored at time zero and after 30 days. Plasma homocysteine, and serum vitamin B(12) and folate concentrations were measured by immunoenzymatic assays; reactive oxygen species, total antioxidant capacity and thiol groups by spectrophotometric methods; and total and free malondialdehyde concentrations by gas chromatography-mass spectrometry with isotopic dilution. Results: Baseline free malondialdehyde concentrations were significantly higher in smokers than in non-smokers and normalised after 30-day supplementation. Baseline results for all the other parameters remained unchanged after supplementation, with no significant differences between smokers and non-smokers. Conclusion: This is the first study showing a significant decrease in free malondialdehyde levels in light smokers after 1-month phytonutrient supplementatio
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
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