1,721,008 research outputs found
Total 25-Hydroxyvitamin D Determination by an Entry Level Triple Quadrupole Instrument: Comparison between Two Commercial Kits.
No full textObjective. 25-hydroxyvitamin D2/D3 (25-OHD2/D3) determination is a reliable biomarker for vitamin D status. Liquid chromatography-tandem mass spectrometry was recently proposed as a reference method for vitamin D status evaluation. The aim of this work is to compare two commercial kits (Chromsystems and PerkinElmer) for 25-OHD2/D3 determination by our entry level LC-MS/MS. Design and Methods. Chromsystems kit adds an online trap column to an HPLC column and provides atmospheric pressure chemical ionization, isotopically labeled internal standard, and 4 calibrator points. PerkinElmer kit uses a solvent extraction and protein precipitation method. This kit can be used with or without derivatization with, respectively, electrospray and atmospheric pressure chemical ionization. For each analyte, there are isotopically labeled internal standards and 7 deuterated calibrator points. Results. Performance characteristics are acceptable for both methods. Mean bias between methods calculated on 70 samples was 1.9 ng/mL. Linear regression analysis gave an R (2) of 0.94. 25-OHD2 is detectable only with PerkinElmer kit in derivatized assay option. Conclusion. Both methods are suitable for routine. Chromsystems kit minimizes manual sample preparation, requiring only protein precipitation, but, with our system, 25-OHD2 is not detectable. PerkinElmer kit without derivatization does not guarantee acceptable performance with our LC-MS/MS system, as sample is not purified online. Derivatization provides sufficient sensitivity for 25-OHD2 detectio
Plasma thiols levels in Alzheimer's disease mice under diet-induced hyperhomocysteinemia: effect of S-adenosylmethionine and superoxide-dismutase supplementation.
No full textWidely confirmed reports were published on association between hyperhomocysteinemia, B vitamin deficiency, oxidative stress, and amyloid-β in Alzheimer's disease (AD). Homocysteine, cysteine, cysteinylglycine and glutathione are metabolically interrelated thiols that may be potential indicators of health status and disease risk; they all participate in the metabolic pathway of homocysteine. Previous data obtained in one of our laboratories showed that B vitamin deficiency induced exacerbation of AD-like features in TgCRND8 AD mice; these effects were counteracted by S-adenosylmethionine (SAM) supplementation, through the modulation of DNA methylation and antioxidant pathways. Since the cellular response to oxidative stress typically involves alteration in thiols content, a rapid and sensitive HPLC method with fluorescence detection was here used to evaluate the effect of SAM and superoxide-dismutase (SOD) supplementation on thiols level in plasma, in TgCRND8 mice. The quantitative data obtained from HPLC analysis of mice plasma samples showed significant decrease of thiols level when the B vitamin deficient diet was supplemented with SAM + SOD and SOD alone, the latter showing the greatest effect. All these considerations point out the measurement of plasma thiols concentration as a powerful tool of relevance for all clinical purposes involving the evaluation of oxidative stress. The coupling of HPLC with fluorimetric detection, here used, provided a strong method sensitivity allowing thiols determination at very low levels
A Reversed-Phase HPLC Fluorimetric Method for Simultaneous Determination of Homocysteine-Related Thiols in Different Body Fluids
No full textBackground: Low molecular weight thiols (cysteine, cysteinylglycine,
glutathione {[GSH] and homocysteine) are important intermediates in
different metabolic pathways. Glutathione has a relevant role as an
antioxidant in detoxification of toxic compounds and xenobiotics, and
homocysteine represents a risk factor for cardiovascular, neurological,
and congenital diseases. Homocysteine and GSH are metabolically related
in a pathway including cysteine and cysteinylglycine as intermediates.
For these reasons, determination of homocysteine and related thiols is
of great importance in the diagnosis of several diseases.
Methods: The measurement of these sulphur compounds can be performed by
using different methods, such as liquid or gas chromatography/mass
spectrometry, high-performance liquid chromatography (HPLC), or
capillary electrophoresis.
Results: This study describes an HPLC method coupled with fluorimetric
detection for the simultaneous determination of cysteine,
cysteinylglycine, GSH, and homocysteine in different biological fluids
(blood, saliva, urine, and cerebrospinal fluid [CSF]).
Conclusion: The comparison of the results obtained by other authors, as
well as the method validation and the analytical costs, indicate that
this HPLC method is particularly suitable for routine measurement of
thiols in different body fluids.
Simultaneous quantification of 17-hydroxyprogesterone, androstenedione, testosterone and cortisol in human serum by LC-MS/MS using TurboFlow online sample extraction
No full textBackground: The simultaneous quantification of a steroid hormones panel provides more clinical information than a single steroid assay. Traditionally, steroids have been quantified with immunoassays which are characterized by high rate of positive results. Aim of this work, was to develop a TurboFlow-LC-MS/MS method for the simultaneous quantification of 17-hydroxyprogesterone, androstenedione, cortisol and testosterone in serum.
Methods: To 100 mu L of serum, 100 mu L of internal standard solution in methanol were added; after centrifugation the supernatant was injected in the TurboFlow for further purification. Steroids were determined using a TSQ Vantage operating with an atmospheric pressure chemical ionization source. Method was fully validated and results compared with immunoassay methods.
Results: Limit of quantification ranged from 0.02 ng/mL to 1 ng/mL. The precision was lower than 11% and accuracy ranged from 93.5 to 121.6%. The correlation was acceptable for all analytes except for low levels of testosterone. However, the Bland-Altman plots display a positive bias for androstenedione and 17-hydroxyprogesterone, and a negative bias for cortisol and testosterone.
Conclusions: TurboFlow analysis provides a simple and effective clean-up procedure minimizing the interference of the matrix. The presented method is selective, precise, and sensitive being suitable in a clinical laboratory
A simple liquid chromatography-tandem mass spectrometry method for urinary free cortisol analysis: suitable for routine purpose
No full textBackground: The best index of adrenal dysfunction is urinary free
cortisol (UFC) measurements performed using a 24-h urine collection.
This measurement is also useful in the investigation of Cushing's
syndrome. In this paper, we report a simple and selective method for the
analysis of UFC by liquid chromatography-tandem mass spectrometry
(LC-MS/MS) suitable for use in a high-volume clinical laboratory
routine. The results were compared to those obtained using a commercial
immunoassay method used in our laboratory.
Methods: Urine samples containing 50 ng of internal standard
(Cortisol-9,11,12,12-d(4)) were deproteinized using centrifugal filters
with a molecular weight 10,000 Da cut-off and injected on a reversed
phase column. Cortisol was analyzed in highly selective reaction
monitoring in positive atmospheric pressure chemical ionization mode, at
a resolution of 0.4 amu full width half maximum, and following the
transitions related to the precursor 363.2 for cortisol and 367.2 for
deuterated cortisol. The method validation included analysis of
precision, linearity, sensitivity, recovery and interference from
structurally similar steroids. UFC from 230 subjects was measured using
LC-MS/MS and electrochemiluminescence immunoassay (ECLIA) methods.
Results: The calibration curves exhibited linearity and reproducibility
in the range 7-10,000 nmol/L. Total imprecision was lower than 10\%. The
limit of detection and limit of quantification were 2 and 7 nmol/L,
respectively. Mean recovery was higher than 90\%. Structurally similar
steroids do not interfere in the proposed method, but cause a
significant change in the ECLIA results. Cortisol values obtained using
the ECLIA method were always higher than those obtained using the
LC-MS/MS method, with the bias directly proportional to cortisol
concentrations. The reference values calculated using 180 normal
subjects were 11-70 mu g/day.
Conclusions: The proposed method is sensitive, simple, free from
interferences and reliable for routine use. Clin Chem Lab Med
2010;48:1433-7
Effect of smoking one cigarette on antioxidant metabolites in the saliva of healthy smokers
No full textConcentrations of glutathione, uric acid and total antioxidant activity, expressed as Trolox (a water-soluble vitamin E analogue) equivalent, were measured in the saliva of healthy non-smokers and smokers before and just after smoking a single cigarette. There was no statistically significant difference between smokers and non-smokers in uric acid concentrations and total radical-trapping antioxidant capacity, but glutathione concentrations were significantly (p < 0.05) higher in smokers. Smoking of a single cigarette induced a significant reduction in glutathione concentration (p < 0.05). Salivary antioxidant power may affect individual sensitivity toward tobacco stress
Comparing different methods for homocysteine determination
No full textSlightly elevated values of homocysteine are commonly associated with thromboembolic diseases, while high values can be found in patients with congenital metabolic defects or nutritional problems. The clinical use of homocysteine as an independent marker of cardiovascular disease was limited in the past by technical problems with its measurement, the instrumentation (HPLC, radioenzymatic assays, gas chromatography-mass spectrometry, etc.) and the necessary skills required. Commercially available immunoassays now permit a simpler and more rapid measurement of homocysteine, that is more suitable for routine clinical laboratories; in this paper we analyze the results obtained by using three fully automated methods for homocysteine determination (Abbott IMx immunoassay, Abbott AxSYM immunoassay and Immulite 2000 homocysteine immunoassay) and their correlation with the widely used HPLC method. The results clearly indicate that all three automated immunochemical methods correlate well with the HPLC method (slope 0.97-1.03; intercept 0.95-1.91 with a recovery above 95% for all three methods)
A simplified method for the determination of total homocysteine in plasma by electrospray tandem mass spectrometry.
No full textHyperhomocysteinemia is a risk factor for different diseases. Several methods have been developed to analyze homocysteine and the immunometric ones, although expensive, they are in widespread use. A rapid LC-MS/MS method for homocysteine assay has been developed for the application of large clinical chemistry routines. Selected reaction monitoring was performed through the transitions m/z 136.0→90.1 for homocysteine and m/z 140.0→94.0 for the internal standard. ESI was used to generate [H+] adduct ions. Chromatographic isocratic separation was achieved using a strong cation exchange column. The mobile phase was methanol/water (20:80 v/v, containing 0.1% formic acid and 1.5 mmol/L ammonium formate in the water phase) at a flow rate of 0.250 mL/min (35°C). Samples treatment consisted in the reduction with DTT and deproteinization with methanol. Recovery, linearity, LOD, LOQ and total imprecision were evaluated to validate the method. Homocysteine values on 100 serum samples were compared with those obtained by HPLC and immunometric methods. The method is robust, selective and precise in the whole range of values studied. Moreover, low reagent cost and easiness of sample treatment make this method useful, not only for research, but also for routine work
Is Homocysteine a pro-oxidant ?
No full textHigh plasma homocysteine concentrations have been found to be associated with atherosclerosis and thrombosis of arteries and deep veins. The oxidative damage mediated by hydrogen peroxide production during the metal-catalyzed oxidation of homocysteine is to date considered to be one of the major pathophysiological mechanisms for this association. In this work, a very sensitive and accurate method was employed to measure the effective production of H2O2 during homocysteine oxidation. Furthermore, the interaction of homocysteine with powerful oxidizing species (hypochlorite, peroxynitrite, ferrylmyoglobin) was evaluated in order to ascertain the putative pro-oxidant role of homocysteine. Our findings indicate that homocysteine does not produce H2O2 in a significant amount (1/4000 mole/mole ratio of H2O2 to homocysteine). Moreover, homocysteine strongly inhibits the oxidation of luminol and dihydrorhodamine by hypochlorite or peroxynitrite and rapidly reduces back ferrylmyoglobin, the oxidizing species, to metmyoglobin. All these results should, in our opinion, lead to a rethinking of the commonly held view that homocysteine oxidation is one of the main causative mechanisms of cardiovascular damage
A fast chemiluminescent method for H2O2 measurement in exhaled breath condensate.
No full textAbstract
BACKGROUND:
Breath condensate can give useful information on volatile compounds produced at alveolar level. Actual concentration of H(2)O(2) in breath condensate is dependent on its production at alveolar level and on the efficacy of the detoxifying systems, catalase, glutathione peroxidase, etc.
METHODS:
In the present paper, a simple chemiluminescent method for the determination of the H(2)O(2) collected in exhaled breath is shown and data of both smokers and nonsmokers volunteers are presented.
RESULTS:
The chemiluminescent response is linear up to 100 micromol/l H(2)O(2). The analytical sensitivity is about 0.01 micromol/l. Most of the nonsmokers have a H(2)O(2) content lower than 0.05 micromol/l, while smokers have a content ranging from 0.1 to 0.6 micromol/l
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