Maria Franca Usai

Simultaneous detection of N-acetyl-l-cysteine and physiological low molecular mass thiols in plasma by capillary electrophoresis

N-acetyl-l-cysteine (NAC) is a therapeutic drug widely used as mucolytic agent in the treatment of respiratory diseases. Recently it has been proposed that NAC administration may modify the plasma levels of low molecular weight thiols (LMW) like cysteine, homocysteine and glutathione, though it has been still debated if their plasma concentration increases or decreases during the therapy. Therefore research calls for methods able to analyze simultaneously NAC and the other plasma LMW thiols in order to evaluate if NAC is able to modify plasma thiols concentration and in particular to reduce homocysteine levels in hyperhomocysteinemia. In this paper we present a new capillary electrophoresis method that allows a baseline separation of plasma NAC from the physiological thiols. The proposed method has been utilized to measure the drug and the physiological LMW thiols in NAC administered chronic obstructive broncho-pneumopathy (COPB) disease patients.

Albumin-bound low molecular weight thiols analysis in plasma and carotid plaques by CE

We describe a new method for the quantification of low molecular weight thiols, as homocysteine, cysteine, cysteinylglycine, glutamylcysteine and glutathione bound to human plasma albumin. After albumin isolation and purification by SDS-PAGE, thiols were freed from protein with tri-n-butylphosphine and successively derivatized with 5-iodoacetamidofluorescein. Samples were then injected and quantified in about 18 min by CE with laser induced fluorescence detection. Precision tests indicate a good repeatability of the method both for migration times (RSD<0.63%) and areas (RSD<2.98%). The method allows to measure all five low molecular weight thiols released from just 3 microg of albumin thus improving the other described methods in which only three or four thiols were detected. Due to the elevated sensitivity (LOD of 0.3 pM for all thiols), also low molecular weight thiols bound to albumin filtered in tissues could be quantified.

Ultra-fast adenosine 5′-triphosphate, adenosine 5′-diphosphate and adenosine 5′-monophosphate detection by pressure-assisted capillary electrophoresis UV detection

Herein, we report a new CE method to measure adenine nucleotides adenosine 5′‐triphosphate, adenosine 5′‐diphosphate, and adenosine 5′‐monophosphate in red blood cells. For this purpose, 20 mmol/L sodium acetate buffer at pH 3.80 was used as running electrolyte, and the separation was performed by the simultaneous application of a CE voltage of 25 kV and an overimposed pressure of 0.2 psi from inlet to outlet. A rapid separation of these analytes in less than 1.5 min was obtained with a good reproducibility for intra‐ and inter‐assay (CV<4 and 8%, respectively) and an excellent analytical recovery (from 98.3 to 99%). The applicability of our method was proved by measuring adenine nucleotides in red blood cells.

Increased plasma asymmetric dimethylarginine (ADMA) levels in retinal venous occlusive disease

Abstract Background: We investigated the levels of the endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA), as well as homocysteine and cysteine thiols, in a cohort of subjects affected by retinal vein occlusion (RVO) disease. Methods: Capillary electrophoresis analysis was performed in both RVO subjects (n=54) and in a control group (n=32). Results: No differences were found between controls and patients; however, after categorisation for RVO type, central RVO (CRVO) patients showed higher levels of ADMA (0.710±0.139 μmol/L) than controls (0.635±0.117 μmol/L) and branch RVO patients (0.642±0.096 μmol/L). Moreover, cysteine plasma levels were also significantly higher in CRVO patients than in controls (265.8±46.9 vs. 226.7±51.9 μmol/L, p<0.01), while homocysteine plasma concentration was more or less identical in all groups. Conclusions: We hypothesise that the elevated levels of cysteine in CRVO patients may post-translationally inhibit dimethylarginine dimethylaminohydrolase enzyme activity, as already described for homocysteine, thus contributing to the accumulation of ADMA in this patient group. Clin Chem Lab Med 2008;46:387–92.