Carotid restenosis is associated with plasma ADMA concentrations in carotid endarterectomy patients.

BACKGROUND: The aim of this work has been to study the association between plasma asymmetric dimethylarginine (ADMA) concentrations and carotid stenosis in a group of 64 patients undergoing carotid endarterectomy (CEA). METHODS: Arginine, ADMA and symmetric dimethylarginine (SDMA) were measured using capillary electrophoresis with UV detection. An evaluation of plasma concentrations of total cysteine (tCys) and total homocysteine (tHcy) was also performed. RESULTS: Pearson's analysis show a positive correlation between ADMA and carotid stenosis (r=0.37, p=0.003), which is also confirmed after stepwise multiple linear regression analysis. ADMA plasma concentrations were significantly associated with tHcy (r=0.40, p=0.001) and to a lesser extent, even if not significantly, with tCys (r=0.23, p=0.07). CONCLUSIONS: Our data suggest that plasma ADMA is involved in carotid narrowing after CEA intervention. This suggests that this molecule may have an important role in the events that lead to stenosis.

Quantification of neurotransmitter amino acids by capillary electrophoresis laser-induced fluorescence detection in biological fluids.

The role of neurotransmitter amino acids (NAAs) in the functioning of the nervous system has been the focus of increasingly intense research over the past several years. Among the various amino acids that have important roles as neurotransmitters, there are alanine (Ala), glutamic acid (Glu), aspartic acid (Asp), serine (Ser), taurine (Tau) and glycine (Gly). NAAs are present in plasma, cells and--at trace levels--in all biological fluids, but complex components in biological matrices make it difficult to determine them in biological samples. We describe a new capillary electrophoresis (CE) method with laser-induced fluorescence detection by which analytes are resolved in less than 12 minutes in a 18 mmol/L phosphate run buffer at pH 11.6. The use of elevated temperatures during sample derivatization leads to a drastic reduction in the reaction time, down to 20 min, compared to the 6-14 h usually described for reactions between FITC and amino acids at room temperature. In order to demonstrate its wide range of applications, the method was applied to the analysis of NAA in human plasma and in other sample types, such as red blood cells, urine, cultured cells, cerebrospinal fluid, saliva and vitreous humor, thus avoiding the typical limitations of other methods, which are normally suitable for use with only one or two matrix types.

Taurine determination by capillary electrophoresis with laser-induced fluorescence detection: from clinical field to quality food applications.

In this work we describe a new method for taurine quantification in plasma by capillary electrophoresis laser-induced fluorescence detection. Taurine is derivatized with fluorescein isothiocyanate at 100 degrees C in 20 min. These conditions allow to reduce the pre-analytical times and to derivatize quantitatively the taurine contained in the reaction mixture, contrary to the room temperature derivatization commonly adopted. FITC-taurine adduct is analyzed in an uncoated fused-silica capillary, 75 mum ID and 40 cm effective length using a 20 mmol/L tribasic sodium phosphate buffer pH 11.8, at 22 kV. To avoid the typical problems due to instability of FITC-adduct, we use the homocysteic acid as internal standard. The loss of FITC-taurine signal during the sequence analysis is compensated by the same loss of FITC-internal standard adduct, thus giving a noteworthy improvement in the assay precision. The method shows a good reproducibility of the migration times (coefficient of variation, CV%, 1.93) and the peak areas (CV%, 3.65). Intra- and interassay CV were 4.63 and 6.44%, respectively, and analytical recovery was between 98.1 and 102.3%. Assay application was tested measuring taurine plasma levels in 50 healthy volunteers in which a mean value of 60.2 +/- 17.9 micromol/L was found. Moreover, the applicability of the method was also checked on energy drinks and milk.

Protein-bound glutathione measurement in cultured cells by CZE with LIF detection.

Protein modification due to S-glutathio(ny)lation, usually a reversible process in intact cells, arises interest as a possible mode of regulatory events that may potentially modify a large number of cellular processes. However, since less than 1% of the total protein is S-thiolated in resting cells, high sensitivity methods are required for its evaluation. We set up a new method by CE with LIF detection that allows to measure all forms of intracellular GSH involved in the process. For total and reduced glutathione, cell lysates were rapidly derivatized by 5-iodoacetoamidofluorescein (5-IAF), a selective reagent which traps thiol groups, thus minimizing auto-oxidation. Derivatized samples were separated in a 47 cmx75 microm id capillary by using 7 mmol/L sodium phosphate at pH 11.6. For the evaluation of S-glutathio(ny)lation, intracellular proteins from cell lysates were precipitated and washed to eliminate free GSH. After protein resuspension with NaOH and reduction treatment with tri-n-butylphosphine (TBP), the freed GSH was dried in a vacuum concentrator and directly dissolved in the derivatization mixture. GSH-IAF adduct was detected in a 6 mmol/L sodium phosphate, 3 mmol/L boric acid, and 75 mmol/L N-methylglucamine run buffer in less than 5 min. The high sensitivity ensured by 5-IAF use and sample concentration, allowed to quantify GSH at levels as low as 5 nmol/L, value suitable for the evaluation of protein S-glutathio(ny)lation. The method suitability was checked both in HUVEC and ECV304 cultured cells.

Plasma methionine determination by capillary electrophoresis-UV assay: application on patients affected by retinal venous occlusive disease.

Methionine is an important amino acid involved in protein synthesis and transmethylation reactions. It is also the precursor of homocysteine and cysteine, two important risk factors for cardiovascular diseases. As homocysteine research has gained impulsion, the evaluation of plasma methionine concentrations has acquired importance. Methionine measurement generally has been performed by HPLC after o-phthalaldehyde derivatization. Its separation from other amino acids is time-consuming. We set up a new specific capillary electrophoresis method in which analyte derivatization was avoided by sample concentration before analysis. Methionine was detected by UV absorbance at 204 nm with a detection limit of 0.5 micromol/L. By a capillary with an effective length of 50 cm filled with 125 mmol/L Tris phosphate buffer at pH 2.3, the separation occurred in less than 14 min. Precision tests indicated a good test repeatability for both migration times (coefficient of variation [CV]<0.3%) and areas (CV<2.0%). Moreover, a good reproducibility of intraassay and interassay tests was obtained (CV<2.9% and CV<3.5%, respectively). The Passing-Bablok regression and the Bland-Altman test for methods comparison suggest that the data obtained by our method and by a reference HPLC assay are similar. Assay performance was evaluated measuring methionine concentrations in retinal venous occlusive disease.

Assay for the simultaneous determination of guanidinoacetic acid, creatinine and creatine in plasma and urine by capillary electrophoresis UV-detection.

Guanidinoacetic acid (GAA) measurement has recently become of great interest for the diagnosis of creatine (Cn) metabolism disorders, and research calls for rapid and inexpensive methods for its detection in plasma and urine in order to assess a large number of patients. We propose a new assay for the measurement of GAA by a simple CZE UV-detection without previous sample derivatization. Plasma samples were filtered by Microcon-10 microconcentrators and directly injected into the capillary, while for urine specimens a simple water dilution before injection was needed. A baseline separation was obtained in less than 8 min using a 60.2 cm x 75 microm uncoated silica capillary, 75 mmol/L Tris-phosphate buffer pH 2.25 at 15 degrees C. The performance of the developed method was assessed by measuring plasma creatinine and Cn in 32 normal subjects and comparing the data obtained by the new method with those found with the previous CE assay. Our new method seems to be an inexpensive, fast and specific tool to assess a large number of patients both in clinical and in research laboratories.

Thiol redox status evaluation in red blood cells by capillary electrophoresis-laser induced fluorescence detection.

Thiols and in particular glutathione (GSH) play a central role in human metabolism, including the detoxification of xenobiotics, cell homeostasis, radioprotection, and antioxidant defence. Here, a new method is provided for the measurement of reduced and total forms of thiols in red blood cells. In order to minimize oxidation of reduced thiols, a water erythrocyte lysis (15 min at 4 degrees C) was performed followed by a protein precipitation step with acetonitrile. The supernatant was rapidly derivatized with 5-iodoacetoamidefluorescein that trapped thiol groups, thus minimizing auto-oxidation. Derivatized samples were separated in a 57 cm x 75 microm ID capillary by using 5 mmol/L sodium phosphate, 4 mmol/L boric acid as electrolyte solution with 75 mmol/L N-methyl-D-glucamine at pH 11.0. Under these conditions, cysteinylglycine (CysGly), cysteine (Cys), glutathione, and gamma-glutamylcysteine (GluCys) were baseline-resolved in approximately 4 min. Precision tests showed a good repeatability of our method both for migration times (coefficient of variation CV < 0.8%) and areas (CV < 3.3%). Furthermore, a good reproducibility of intrassay and interassay tests was obtained (CV < 5% and CV < 8%, respectively). The method was employed to investigate the effect of acidic precipitation on intracellular thiol concentration. Our data suggest that sample acidification causes a modification of the measured redox thiol status due to the development of a pro-oxidant environment; moreover, the thiol redox status of red blood cells was evaluated in 22 healthy volunteers.

Plasma creatinine and creatine quantification by capillary electrophoresis diode array detector.

Traditional clinical assays for nonprotein nitrogen compounds, such as creatine and creatinine, have focused on the use of enzymes or chemical reactions that allow measurement of each analyte separately. Most of these assays are mainly directed to urine quantification, so that their applicability on plasma samples is frequently hard to perform. This work describes a simple free zone capillary electrophoresis method for the simultaneous measurement of creatinine and creatine in human plasma. The effect of analytical parameters such as concentration and pH of Tris-phosphate running buffer and cartridge temperature on resolution, migration times, peak areas, and efficiency was investigated. Good separation was achieved using a 60.2-cm x 75-microm uncoated silica capillary, 75 mmol/L Tris-phosphate buffer, pH 2.25, at 15 degrees C, in less than 8 min. We compared the present method to a validated capillary electrophoresis assay, by measuring plasma creatinine in 120 normal subjects. The obtained data were compared by the Passing-Bablok regression and the Bland-Altman test. Moreover the performance of the developed method was assessed by measuring creatine and creatinine in 16 volunteers prior to and after a moderate physical exercise.

Carotid narrowing degree and plasma thiol levels in carotid endarterectomy patients.

The aim of this work was to study the association between plasma thiol levels and percentage carotid narrowing in a group of 68 patients who underwent a carotid endarterectomy, pertained as a risk factor for vascular and cardiovascular disease. Total plasma thiols were measured by capillary electrophoresis laser-induced fluorescence. The mean values of the hematological parameters studied were within normal limits and 25% of the patients were hyperhomocysteinemic (homocysteine >15 micromol/L). Pearson's correlation between carotid narrowing degree and the most common risk factors for atherosclerosis showed a positive relationship only between carotid narrowing degree and cysteine levels (r=0.252; p<0.05). Stepwise multiple linear regression with carotid narrowing degree as the dependent variable, and cysteine, homocysteine, age, triglyceride and low-density lipoprotein-cholesterol as independent variables confirmed that cysteine was significantly associated with these variables. By regrouping the population according to cysteine and homocysteine concentration percentiles, we found positive correlation between these parameters and median values of carotid narrowing degree. Our study provides experimental evidence to confirm that plasma homocysteine and cysteine are involved in carotid narrowing after carotid endarterectomy intervention, suggesting that cysteine may be involved in the deleterious molecular mechanisms active in carotid stenosis.