Salvatore Sotgia

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.

Rapid quantification of total genomic DNA methylation degree by short-end injection capillary zone electrophoresis

We propose a new capillary zone electrophoresis method applying short-end injection technique for the fast evaluation of methylcystosine/total cytosine ratio after acidic DNA hydrolysis. By short-end injection and by using a 100 mmol/l Tris solution titrated with 1 mol/l phosphoric acid to pH 3.75 as background electrolyte, cytosine and methylcytosine were separated with a good resolution in less than 1.5 min. Stepwise multiple linear regression with DNA methylation degree as the dependent variable and age, cysteine, homocysteine and methionine as independent variables, showed a negative association with age and that total cysteine is the most important determinant of DNA methylation.

S-homocysteinylated LDL apolipoprotein B adversely affects human endothelial cells in vitro.

OBJECTIVE In recent years elevated homocysteine (Hcy) levels have been widely recognized as a risk factor for cardiovascular diseases (CVDs) and a connection between hyperhomocysteinemia and lipid metabolism has been suggested to have a possible role in endothelial vascular damage as lipoprotein fractions contain higher Hcy levels in hypercholesterolemia, compared to normolipidemic individuals. However, the biochemical events underlying the interaction between Hcy and LDL are still poorly understood. METHODS AND RESULTS Herein we have investigated the interaction of LDL with Hcy by measuring thiols S-linked to apoprotein using capillary electrophoresis and have evaluated the effect of S-homocysteinylated LDL on human endothelial cells (HECs). We found that Hcy binds to LDL in a dose dependent manner and the saturation binding is achieved at 100 micromol/L Hcy in about 5h. Addition of Hcy resulted in a rapid displacement of other thiols bound to apoprotein and this was dependent on the concentration of Hcy added. For the first time we also demonstrated that treatment of HECs with homocysteine-S-LDL (Hcy-S-LDL) resulted in the induction of significantly higher levels of reactive oxygen species (ROS) compared to N-LDL (native LDL). Furthermore, the Hcy-S-LDL-induced a rise in intracellular ROS production was followed by a marked reduction of HECs proliferation and viability. CONCLUSIONS Although the mechanism by which Hcy-S-LDL elicits the current cellular effects needs further investigation, our data suggest that intracellular ROS production induced by Hcy-S-LDL might be responsible for the observed HECs damage and indicate that Hcy-S-LDL may have some role in CVD.

Enantiomeric reversed-phase high-performance liquid chromatography resolution of D-/L-penicillamine after spirocyclization with ninhydrin and by using copper(II)-L-proline complex as a chiral selector in the mobile phase.

A new HPLC method by fluorescence or UV/vis absorbance detection has been developed for the separation and quantification of penicillamine stereoisomers after their spirocyclization with ninhydrin. The separation was performed on an achiral C18 column by isocratic elution with a copper(II)-l-proline complex as a chiral selector in the mobile phase. The method was able to detect traces of l-penicillamine in samples of d-penicillamine below 0.1% in fairly short times (about 16 min) with a good resolution (R(s)=1.31). On the whole, the method was found to be stable and useful in the quality control of the bulk material and formulations.

Evaluation of non-covalent interactions between serum albumin and green tea catechins by affinity capillary electrophoresis

The natural antioxidant-associated biological responses appear contradictory since biologically active dosages registered in vitro experiments are considerably higher if compared to concentrations found in vivo. The recent research indicates that natural antioxidants, including the major catechins of green tea epicatechin (EC), epigallocatechin (EGC), epicatechingallate (ECG) and epigallocatechingallate (EGCG) form non-covalent complexes with albumin, a crucial aspect that may modulate their plasma concentration, tissue delivery and biological activity. Affinity capillary electrophoresis (ACE) was used to characterize the binding of the four catechins to human serum albumin (HSA) and bovine serum albumin (BSA) at near-physiological conditions: 10 mmol/L phosphate buffer, HEPES 50 mmol/L (pH 7.5), temperature 37°C. The studied flavonoids displayed affinities toward the albumin with binding constants in the range 10(3)-10(5)M(-1), with a greater affinity of catechins toward HSA than BSA (between 3 and 3.5 fold higher). We also confirmed that catechins having a galloyl moiety (ECG and EGCG) have a higher binding affinity toward albumin than the catechins lacking the galloyl moiety (EC and EGC), and that for both albumins the order of affinity is EC<EGC<ECG<EGCG. We believe that our work can provide useful information for better understanding the intercurrent relationships between cathechins bioavailability and their elicited biological effects.

Pre-analytical stability of the plasma proteomes based on the storage temperature

BackgroundThis study examined the effect of storage temperature on the protein profile of human plasma. Plasma samples were stored for 13 days at -80°C, -20°C, +4°C and room temperature (20-25°C) prior to proteomic analysis. The proteomic comparisons were based on the differences of mean intensity values of protein spots between fresh plasma samples (named “time zero”) and plasma samples stored at different temperatures. To better understand the thermally induced biochemical changes that may affect plasma proteins during storage we identified proteins with different expressions with respect to the time zero sample.ResultsUsing two-dimensional electrophoresis followed by MALDI-TOF MS and /or LC-MS/MS 20 protein spots representing 10 proteins were identified with significant differences in abundance when stored at different temperatures. Our results, in agreement with various authors, indicate that during storage for a short period (13 days) at four different temperatures plasma proteins were more affected by degradation processes at +4°C compared to the other temperatures analysed. However, we founded that numerous protein spots (vitamin D binding protein, alpha-1-antitrypsin, serotransferrin, apoplipoprotein A-I, apolipoprotein E, haptoglobin and complement factor B) decrease in abundance with increasing temperature up to 4°C, but at room temperature their intensity mean values are similar to those of time zero and -80°C. We hypothesize that these proteins are labile at 4°C, but at the same time they are stable at room temperature (20-25°C). Furthermore we have grouped the proteins based on their different sensitivity to the storage temperature. Spots of serum albumin, fibrinogen gamma chain and haptoglobin are more resistant to the higher temperatures tested, as they have undergone changes in abundance only at room temperature; conversely, other spots of serum albumin, fibrinogen beta chain and serotransferrin are more labile as they have undergone changes in abundance at all temperatures except at -80°C.ConclusionsAlthough there are many studies concerning protein stability of clinical samples during storage these findings may help to provide a better understanding of the changes of proteins induced by storage temperature.

Resveratrol alters human endothelial cells redox state and causes mitochondrial-dependent cell death

Studies analyzing the impact of natural antioxidants (NA) on Endothelial Cells (ECs) have dramatically increased during the last years, since a deregulated ECs redox state is at the base of the onset and progression of several cardiovascular diseases. However, whether NA can provide cardiovascular benefits is still a controversial area of debate. Resveratrol (RES), a natural polyphenol found in grapes, is believed to provide cardiovascular benefits by virtue of its antioxidant effect on the endothelium. Here, we report that tissue-attainable doses of resveratrol increased the intracellular oxidative state, thus affecting mitochondrial membrane depolarization and inducing EC death. Cyclosporine A, a mitochondrial permeability transition pore inhibitor, prevented oxidative-mediated cell death, thus implicating mitochondria in resveratrol-induced EC impairment. The specific cytochrome P450 (CYP) 2C9 inhibitor, sulfaphenazole, counteracted both oxidative stress and mitochondrial membrane depolarization, providing EC protection against resveratrol-elicited pro-oxidant effects. Our findings strongly suggest that CYP2C9 mediates resveratrol-induced oxidative stress leading to mitochondria impairment and EC death.

Field-amplified online sample stacking capillary electrophoresis UV detection for plasma malondialdehyde measurement

Malondialdehyde (MDA) determination is the most widely used method for monitoring lipid peroxidation. Here, we describe an easy field‐amplified sample injection (FASI) CE method with UV detection for the detection of free plasma MDA. MDA was detected within 8 min by using 200 mmol/L Tris phosphate pH 5.0 as running buffer. Plasma samples treated with ACN for protein elimination were directly injected on capillary without complex cleanup and/or sample derivatization procedures. Using electrokinetic injection, the detection limit in real sample was 3 nmol/L, thus improving of about 100‐fold the LOD of the previous described methods based on CE. Precision tests indicate a good repeatability of our method both for migration times (CV=1.11%) and for areas (CV=2.05%). Moreover, a good reproducibility of intra‐ and inter‐assay tests was obtained (CV=2.55% and CV=5.14%, respectively). Suitability of the method was tested by measuring MDA levels in 44 healthy volunteers.

Determination of homocysteine thiolactone, reduced homocysteine, homocystine, homocysteine-cysteine mixed disulfide, cysteine and cystine in a reaction mixture by overimposed pressure/voltage capillary electrophoresis.

An elevated level of thiol amino acid homocysteine is associated with several complex disorders. Homocysteine ability to bind proteins, thereby modulating their structure and function, is proposed to be one of the mechanisms of homocysteine induced pathogenecity. Homocysteine and homocysteine thiolactone bind to protein cysteine and lysine residues respectively. A major hurdle in studying protein homocysteinylation is the lack of suitable analytical techniques to determine simultaneously the concentrations of reduced and oxidized forms of homocysteine and cysteine (especially homocysteine-cysteine mixed disulfide) together with thiolactone formed during the reaction of homocysteine or thiolactone with proteins. Herein we report a capillary electrophoresis method to determine simultaneously the levels of these intermediates. For this 40 mmol/L Tris phosphate buffer at (pH 1.60) was used as running electrolyte, and the separation was performed by the simultaneous application of a CE voltage of 15kV and an overimposed pressure of 0.1 psi. A rapid separation of these intermediates in less than 6 min with a good reproducibility of both peak areas (CV<2%) and migration time (CV<0.2%) was obtained. The applicability of our method was validated by incubating reduced homocysteine and albumin and measuring the reaction intermediates in the solution mixture.

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.