N- and S-homocysteinylation reduce the binding of human serum albumin to catechins.

PURPOSE: The dietary flavonoids epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECG) and epigallocatechin gallate (EGCG) have been shown to interact with circulating albumin for transport in blood to different body tissues. This interaction may modulate their bioavailability and effectiveness. METHODS: Using affinity capillary electrophoresis to assess binding constants (K b), we investigated whether posttranslational modification of human serum albumin (HSA) through N- and S-homocysteinylation, commonly observed in hyperhomocysteinemia, may modify its interaction with catechins. RESULTS: S-Hcy HSA had lower Kb values toward EC (14 %), EGC (18 %), ECG (24 %) and EGCG (30 %). Similarly, N-Hcy HSA had lower Kb values toward EC (17 %), EGC (22 %), ECG (23 %) and EGCG (32 %). No differences were observed in the affinity between catechins, albumin and mercaptalbumin. CONCLUSION: Therefore, HSA posttranslational modifications typical of hyperhomocysteinemia reduce its affinity to catechins, potentially affecting their pharmacokinetics and availability at the active sites.

Human Serum Albumin Increases the Stability of Green Tea Catechins in Aqueous Physiological Conditions.

Epicatechin (EC), epigallocatechin (EGC), epicatechingallate (ECG) and epigallocatechingallate (EGCG) are antioxidants present in the green tea, a widely used beverage whose health benefits are largely recognized. Nevertheless, major physicochemical limitations, such as the high instability of catechins, pose important questions concerning their potential pharmacological use. Recent studies indicate that binding of catechins with plasmatic proteins may modulate their plasma concentration, tissue delivery and biological activity. After 5 minutes of incubation with HSA both ECG and EGCG were fully bound to HSA, while after 48h incubation only 41% of EC and 70% of EGC resulted linked. HSA had a strong stabilizing effect on all catechins, which could be found in solution between 29 and 85% even after 48h of incubation. In the absence of HSA, EGC and EGCG disappeared in less than 24h, while ECG and EC were found after 48h at 5 and 50%, respectively. The stabilizing effect of HSA toward EGCG, obtained in aqueous physiological conditions, resulted stronger in comparison to cysteine and HCl, previously reported to stabilize this polyphenol. Because of the multitude of contradictory data concerning in vivo and in vitro antioxidant-based experimentations, we believe our work may shed some light on this debated field of research.

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

Low density lipoprotein S-homocysteinylation is increased in acute myocardial infarction patients.

OBJECTIVES: We have investigated on the levels of homocysteine linked to LDL in acute myocardial infarction patients. DESIGN AND METHODS: We used capillary electrophoresis to measure LDL-bound thiols in 16 AMI individuals and 32 healthy volunteers. RESULTS: We found a significant increase of apo-B homocysteine and cysteine levels in LDL fraction of AMI subjects. CONCLUSIONS: Our findings suggest that also LDL S-homocysteinylation may be considered among the markers for the CVD risk evaluation.

Quantification of histidine, 1-methylhistidine and 3-methylhistidine in plasma and urine by capillary electrophoresis UV-detection.

We describe a new CE method with UV-detection for the quantification of histidine (His) and its methylated forms 1-methylhistidine and 3-methylhistidine, both in plasma and urine. Analytes were basically resolved using a 60 mmol/L Tris-phosphate run buffer pH 2.2 in less than 12 min. The use of a mixture of ACN/ammonia (80:20) for protein precipitation allows the quantitative recovery of all His from plasma. The optimization of the sample volume injection permits to reach an LOD of 20 nmol/L, thus improving the sensitivity of about hundred times in comparison to the previous described assays. Moreover, the opportunity to also measure creatinine in the same run makes it possible to evaluate the renal function contemporarily, thus avoiding further dosages with significant time saving. The application method has been proved by measuring His, 1-methylhistidine and 3-methylhistidine in 44 healthy subjects. In conclusion, our new method seems to be an inexpensive, fast and specific tool to assess large numbers of patients for routine analysis both in clinical and research laboratories.

Capillary electrophoresis with laser-induced fluorescence detection for ATP quantification in spermatozoa and oocytes.

We describe a new capillary electrophoresis laser-induced fluorescence (CE-LIF) method for the quantification of adenosine 5'-triphosphate (ATP) in spermatozoa and oocytes. The optimization of the precapillary derivatization reaction between ATP and 4,4-difluoro-5,7-dimethyl-4-bora-3a,4adiaza-s-indacene-3-propionyl ethylene diamine hydrochloride (BODIPY FL EDA) has been described. BODIPY-ATP conjugate was analysed in an uncoated fused silica capillary of 75 µm ID and 50 cm effective length using a 10 mmol/L tribasic sodium phosphate buffer, pH 11.5, at 22 kV in <5 min. A good reproducibility of intra- and inter-assay tests was obtained (CV = 4.55% and 7.14%, respectively). With respect to our previous CE-UV assay, the new method showed an improvement in sensitivity that was about 120-fold (limit of quantification, 0.15 vs 18 µmol/L). Method applicability was proven on the reproductive cells of several animal species (roosters, horses, sheep and goats). Due to the elevated sensitivity, the new assay allows the measurement of adenosine 5'-triphosphate levels from just 20 oocytes. Considering that ATP concentration in reproductive cells is related to the mitochondrial integrity after cryopreservation, the proposed method could be a useful tool in assisted reproductive technologies.

Increased low-density lipoprotein S-homocysteinylation in chronic kidney disease.

BACKGROUND: Since low-density lipoprotein (LDL) S-homocysteinylation has been recently reported to enhance atherogenicity of lipoprotein, we have investigated the levels of homocysteine (Hcy) linked to LDL in chronic proteinuric patients in which lipid abnormalities highly contribute to the excess of morbidity and mortality. METHODS: We used capillary electrophoresis to measure LDL-bound thiol Hcy, cysteine (Cys), cysteinylglycine (Cys-Gly), glutathione (GSH), and glutamylcysteine (Glu-Cys) in 30 chronic kidney disease (CKD) individuals and 60 healthy volunteers. RESULTS: We found more elevated levels of total plasma Hcy, Cys, GSH and Glu-Cys in patients than in controls and also found that Hcy and Cys bound to LDL were significantly increased in nephropathic subjects. By multiple linear regression, we found that in healthy people, total Hcy was the most important determinant of LDL-bound Hcy and Cys-Gly was negatively associated with apoB-Hcy concentrations. In CKD the most important determinant of homocysteinylation was creatinine while total plasma Hcy is weakly associated with apoB-Hcy. CONCLUSIONS: The increased levels in Hcy-LDL observed in CKD patients might account, at least in part, for the excess of cardiovascular risk; thus LDL S-homocysteinylation can be considered a key marker of risk for cardiovascular disease in these individuals.

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.

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.

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.

Improved rapid assay of plasma uric acid by short-end injection capillary zone electrophoresis.

A rapid and simple short-end injection capillary zone electrophoresis method was developed for the quantification of plasma uric acid. The separation was performed in an uncoated fused-silica capillary (50 microm ID, 60 cm total length, 10.2 cm effective length) by using as a background electrolyte a 75 mmol/L glycylglycine solution titrated with NaOH 5 mol/L to pH 9.0, a voltage of 28 kV, a cartridge temperature of 15 degrees C, and direct UV detection at 292 nm. Under optimized conditions, uric acid was determinate in little more than 1 min (1.076 minutes). In order to verify the accuracy of the analysis, urate levels were measured in 543 apparently healthy volunteers by the new assay and our previous method, and the obtained data were compared by Passing-Bablock regression, Bland-Altman test, and a new regression-based approach, which showed a good agreement between two methods.

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.

Evaluation of low molecular mass thiols content in carotid atherosclerotic plaques.

OBJECTIVES: Despite the evidence that both homocysteine and cysteine are important risk factors for vascular disease and atherosclerosis no information are reported about their effective amount in plaque and on the relationship with the other low molecular weight thiols. DESIGN AND METHODS: We used capillary electrophoresis to measure thiols in both carotid plaque specimens and plasma samples from 37 patients undergoing carotid endarterectomy. RESULTS: Pearson's correlation shows that intraplaque homocysteine, cysteine and cysteinylglycine levels are related to their plasma concentrations. The distribution of intraplaque GSH and Glu-Cys was higher than that of the same thiols in plasma, whereas the other thiols were significantly less prevalent in plaque than in plasma. Intraplaque haemoglobin and GSH levels were correlated, thus suggesting their common origin from erythrocytes lysis. CONCLUSION: Data suggest that increased levels of intraplaque glutathione may induce important effects on plaque fate by perturbing the normal LMW thiol redox state.

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.

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.

Determination of protein-incorporated methylated arginine reference values in healthy subjects whole blood and evaluation of factors affecting protein methylation.

OBJECTIVES: Protein arginine methylation is a post-translational modification involved in the regulation of signal transduction, RNA export, and cell proliferation. Reference values of arginine methylation of whole blood proteome remain to be determined. DESIGN AND METHODS: Asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA) and monomethylarginine (MMA) incorporated in whole blood protein and methionine, cysteine and homocysteine plasma levels from 134 healthy subjects were measured by capillary electrophoresis. RESULTS: The mean protein-incorporated concentration of the selected population was 4.11+/-0.77 nmol/mg protein for ADMA; 1.66+/-0.42 nmol/mg protein for SDMA and 4.31+/-1.17 nmol/mg protein for MMA. Multiple correlation analysis showed that plasma Hcy was positively related to incorporated protein ADMA (P=0.002) and MMA (P=0.049). CONCLUSIONS: The study was able to define a reference value for protein-incorporated ADMA, SDMA and MMA levels and found a positive association between protein-incorporated ADMA and plasma homocysteine.

Assessment of protein-incorporated arginine methylation in biological specimens by CZE UV-detection.

Protein arginine methyltransferases methylate post-translationally arginine residues in proteins to synthesize monomethylarginine (MMA), asymmetric dimethylarginine (ADMA), or symmetric dimethylarginine. Protein arginine methylation is involved in the regulation of signal transduction, RNA export, and cell proliferation. Moreover, upon proteolysis, arginines are released into the cytosol in which they exert important biological effects. Both MMA and ADMA are inhibitors of nitric oxide synthase and especially elevated levels of ADMA are associated with endothelial dysfunction and cardiovascular disease. Quantification of these analytes is commonly performed by HPLC after sample cleanup and derivatization. We propose a CE method in which these steps have been avoided and the procedure for sample preparation has been simplified. After acidic hydrolysis of proteins, samples were dried, resuspended in water, and directly injected in CE. A baseline separation of analytes was reached in a 60 cm x 75 microm id uncoated silica capillary, by using a Tris-phosphate run buffer at pH 2.15. This method allows an accurate assessment of protein arginine methylation degree in different biological samples such as whole blood, plasma, red blood cells, cultured cells, and tissue. Moreover, its good sensitivity permits to evaluate the methylation of a single protein type after the opportune purification steps. A method applicability concerns both clinical laboratories, where the evaluation of blood protein from numerous samples could be rapidly performed, and research laboratories where the factors affecting the arginine protein methylation degree could be easily studied.

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.