1,721,031 research outputs found
Total plasma homocysteine analysis by HPLC with SBD-F precolumn derivatization
No full textOver the last two decades, various studies have shown that moderate and persistent hyperhomocysteinemia is implicated in the development of atherosclerosis, which is responsible for 50% of all mortality and morbidity in Western countries. Considering that the most traditional risk factors for heart disease and stroke, such as plasma lipids, cigarette smoking, hypertension, obesity, and diabetes, only account for 50% of cardiovascular disease (1,2), one can understand the reason why homocysteine (Hcy) measurement is included in the list of tests for investigating the causes of atherosclerosis and thrombosis. One of the major problems encountered in studies on the potential atherogenic role of Hcy was the development of an accurate and simple assay, capable of screening, in a normal population, subjects having a congenital predisposition to occlusive vascular disease. Several approaches have been described in literature for measuring total plasma homocysteine (tHcy), which is defined as the sum of free and protein-bound homocysteine, homocystine, and homocysteine-cysteine mixed disulfide. These procedures involve, after a reduction step, the use of gas-chromatography-mass spectrometry (GC-MS) (3), radioenzymic assay (4), and high-performance liquid chromatography (HPLC)
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
High-performance liquid chromatographic method to quantify total cysteine excretion in urine
No full textDetermination of asymmetric and symmetric dimethylarginines in plasma of hyperhomocysteinemic subjects
No full textThe aim of this study was to investigate the possible relationship among dimethylarginines (asymmetric, ADMA; symmetric, SDMA) and homocysteine (Hcy) levels in subjects affected by chronic, mild to intermediate, hyperhomocysteinemia. ADMA and SDMA were assayed by an optimised HPLC method in 75 patients (Hcy = 20.8 (mu)mol/L, 17.1-30.2; median and percentile range) and, for comparison, in 85 healthy subjects (Hcy = 8.0 (mu)mol/L, 7.0-9.1). In controls, the cut-off values were set at 0.61 (mu)mol/L for ADMA and 0.56 or 0.48 (mu)mol/L for male and female SDMA, respectively. In patients, ADMA and SDMA levels were increased (p<0.001) with respect to controls, but no correlation with Hcy was observed. Hyperhomocysteinemic subjects showed a different behaviour in respect to ADMA and SDMA levels and this allowed their stratification in 3 subgroups characterized by ADMA and SDMA in the normal range, only SDMA, or both ADMA and SDMA over the cut-off values. A lack of correlation with Hcy was again observed, thus minimizing the direct role of Hcy on ADMA and SDMA metabolism and suggesting the need for further studies on this issue. (copyright) Springer-Verlag 2005
SEPARATION OF THE VALENCE INTERMEDIATES OF HUMAN-HEMOGLOBIN BY HIGH-PERFORMANCE CHROMATOFOCUSING
Full text linkInvestigations into the properties of haemoglobin often require the isolation of the valence intermediates (αo)2β+)2 and (α+ βo2)2. Chromatofocusing with an anion-exchange gel (Mono PTM; Pharmacia, particle size 10 μm) in an HR5/20 column at various temperatures (10-25°C) provides an excellent method for this task. A linearly decreasing pH gradient (8 to 7, generated by Polybuffer 96, Pharmacia) eluted sequentially the species methaemoglobin, (αo2β+)2, (α+ βo2)2 and oxygenated haemoglobin. Calibration graphs help in quantitative analyses. This method is simpler and less time consuming and provides a similar or even better resolution than the traditional ion-exchange or isoelectric focusing methods
CHROMATOFOCUSING AND ISOELECTRIC-FOCUSING IN IMMOBILIZED PH GRADIENTS COMPARED FOR CHARACTERIZATION OF HUMAN-HEMOGLOBIN VARIANTS
No full textWe compared the performance of two highly resolving methods,
chromatofocusing (CRF) and isoelectrifcocusingin
immobilized pH gradients (IPGF), for the separation of human
hemoglobin vanants. Lysates containing 13 different
hemoglobins, including variants of clinical and geographical
importance, and four electrophoretically “silent” variants (Hb
Brockton,Hb Cheverly,Hb KOIn,and Hb Waco) were analyzed.
Both techniques showed a good intrarun precision(CV
= 0.87% for CRF, 0.27% for IPGF) and high and similar
resolving power (0.010 pH units, with the pH gradients used
in this work). The use of an ultranarrow IPGF range (pH
7.15-7.35; pH gradient = 0.019 pH/cm) allowed the resolution
between Hb Brockton,Hb KOIn,and Hb A. Insome cases
(Hb D-Los Angeles, Hb F, Hb Waco), the variants were
separated from Hb A in different orders, depending on which
technique was used, probably because of the different analytical
principles of the two methods. As a second-level test,
both procedures are informative for characterization of human
hemoglobin variants
DETERMINATION OF CREATININE IN SERUM AND URINE BY A RAPID LIQUID-CHROMATOGRAPHIC METHOD
No full textWe describe an HPLC ion-pair procedure for rapid and
specific evaluationof creatinineinserum and urine. We used
a 15 cm x 4.6 mmODS column with a 50/50 (by vol) mixture
of sodium decanesulfonic acid (10 mmol/L, pH 3.2) and
methanoland measuredabsorbance at 236 nm. Serum (100
tL) or 30-fold-diluted urine (100 L) was added to 400 pL of
acetone. After centrifugation,the supemates (300 L) were
dried, reconstituted with the mobile phase, and injected into
the HPLC. Assay precision was tested for concentrations of
10, 29, and 130mg/Land yielded, respectively, 3.1%, 2.1%,
and 1.1% for within-dayCV and 2.8%, 2.1%, and 2.2% for
totalCV. Analytical recovery was 102 (±6.7)%. Unearitywas
demonstrated in the 0-200 mg/L range for serum and 0-3.5
g/L range for urine (r 0.999). The detection limit for creatmine
(signal-to-noiseratio = 3) was 0.5 mg/L. We used
cimetidinefor internalstandardization.Correlationwasgood
between this procedureand the Jaff#{k2i3n3e}tic,the enzymatic
(creatinineamidohydrolase),and the Fuller’searth alkaline
picrate methods
Are genetic variants of the methyl group metabolism enzymes risk factors predisposing to obesity?
No full textObesity, due to the combination of inherited genes and environmental factors, is continually increasing. We evaluated the relationship between polymorphisms of methylene-tetrahydrofolate reductase (MTHFR C677T and A1298C), methionine synthase (MTR A2756G), methionine synthase reductase (MTRR A66G), betaine:homocysteine methyltransferase (BHMT G742A) and cystathionine beta-synthase (CBS 68-bp ins) genes and the risk of obesity. We studied these polymorphic variants in 54 normal and 82 obese subjects [body mass index (BMI)=22.4+/-1.8, 34.1+/-7.1; ages 35.2+/-10.7, 43.3+/-10.6 respectively]. Levels of total plasma homocysteine (t-Hcy), folates, and vitamins B6 and B12 were not significantly different, while leptin concentration was significantly higher (p=0.005) in the obese patients compared to the lean controls. The frequency of only (a) MTHFR (AC), (b) MTR (AG), and (c) MTRR (AG) heterozygous genotypes was statistically different in the obese compared to the control group (p=0.03, p=0.007, and p=0.01). Single (a), (b), and (c) heterozygous genotypes had a significant risk of developing obesity [p=0.02, 0.01, and 0.03; odds ratio (OR)=2.5, 3.0, and 2.4; 95% confidence interval (CI)=1.2-5.3, 1.3-7.1, and 1.2-5.1 respectively] and the risk remarkably increased for combined genotypes a+b, a+c, b+c, and a+b+c (p=0.002, 0.002, 0.016, 0.006; OR=7.7, 5.4, 5.8, 15.4; 95% CI=1.9-30.4, 1.7-16.8, 1.4-23.2, 1.6- 152.3). These findings suggest that in obese subjects, Hcy cycle efficiency is impaired by MTHFR, MTR, and MTRR inability to supply methyl-group donors, providing evidence that MTHFR, MTR, and MTRR gene polymorphisms are genetic risk factors for obesity
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