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 μm 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 °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.     

A HIGH RESOLUTION FLUORESCENCE DECAY AND DEPOLARIZATION STUDY OF HUMAN PLASMA APOLIPOPROTEINS

Abstract— Human plasma apolipoprotein A-I (apoA-I) and apolipoprotein C-I (apoC-I) were investigated by time-resolved fluorescence decay and depolarization. The tryptophyl fluorescence of apoA-I undergoes a double-exponential decay with lifetimes of 1.07 and 3.43 ns which remain unchanged over the range of apoA-I concentration studied.
The time-resolved fluorescence of both native and denatured forms of apoC-I exhibits an unusual tryptophyl fluorescence decay that was best fit to a triexponential function with lifetimes at 3.7 ± 0.2, 1.1 ± 0.1 and 0.1 ns at 2°C. The native and denatured forms of apoC-I had rotational correlation times of 1.42 and 1.19 ns at 20°C respectively. A shorter rotational correlation time associated with the internal tryptophan motions was not observed or resolved.
The decay of tryptophyl fluorescence in apoC-I/DPPC/cholesterol complex at 20°C is also triexponential with lifetimes at 4.94, 1.28 and 0.21 ns, which are longer than those of the uncomplexed forms. Two rotational correlation times of 28.32 and 0.59 ns at 20°C were resolved by fluorescence depolarization measurements. The long rotational time remained constant with temperatures above 30°C. Also, the temperature dependence of the order parameter, S², resembled a lipid phase transition curve with a transition midpoint at 38°C. The tryptophan and thus apoC-I are found to be affected by the bulk changes in the lipid.     

High-resolution characterization of liquid-crystalline [60]fullerenes using solid-state nuclear magnetic resonance spectroscopy

Liquid-crystalline materials containing fullerenes are valuable in the development of supramolecular switches and in solar cell technology. In this study, we characterize the liquid-crystalline and dynamic properties of fullerene-containing thermotropic compounds using solid-state natural abundance (13)C NMR experiments under stationary and magic angle spinning sample conditions. Chemical shifts spectra were measured in isotropic, liquid-crystalline nematic and smectic A and crystalline phases using one-dimensional (13)C experiments, while two-dimensional separated local-field experiments were used to measure the (1)H- (13)C dipolar couplings in mesophases. Chemical shift and dipolar coupling parameters were used to characterize the structure and dynamics of the liquid-crystalline dyads. NMR data of fullerene-containing thermotropic liquid crystals are compared to that of basic mesogenic unit and mesomorphic promoter compounds. Our NMR results suggest that the fullerene-ferrocene dyads form highly dynamic liquid-crystalline phases in which molecules rotate fast around the symmetry axis on the characteristic NMR time scale of approximately 10 (-4) s.     

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.     

Influence of binary surfactant mixtures on the rheology of associative polymer solutions

Hydrophobically modified alkali-soluble emulsion polymers (HASE) are a class of comblike associative polymers that can impart high viscosities to aqueous solutions. The rheology of HASE solutions can be tuned by the addition of surfactants, such as nonylphenol ethoxylates (NP e), where e is the length of the hydrophilic (ethoxylate) chain. While previous studies have considered individual surfactants, our focus here is on binary surfactant mixtures. We find that equimolar NP4-NP12 mixtures significantly enhance the zero-shear viscosities of HASE solutions as compared to equivalent amounts of NP8, especially at high overall surfactant concentrations. Dynamic rheological measurements suggest that the higher viscosities are due to increases in the lifetime of hydrophobic junctions in the polymer-surfactant network. In contrast to the above results, equimolar NP4-NP8 mixtures are rheologically identical to equivalent solutions of NP6. The differences between the two sets of mixtures are further correlated with cloud point measurements and thereby with the overall hydrophilic-lipophilic balance (HLB) of the surfactant system.     

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.     

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.     

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.     

Direct chromatographic methods for the rapid determination of homogentisic acid in strawberry tree (Arbutus unedo L.) honey

Two rapid and direct chromatographic methods based on reverse phase-high performance liquid chromatography (RP-HPLC) and ion chromatography (IC) were developed for the determination of homogentisic acid (HA) in honey. This is the marker of the botanic origin of strawberry tree honey. The methods were validated and tested using 22 samples from Sardinia, Italy. The IC method is faster than the RP-HPLC one (6 min versus 13 min of total run), but it is slightly less sensitive (the limit of detection (LOD), is 26 mg kg(-1) versus 15 mg kg(-1)) and reproducible (relative standard deviation, RSD, of 10.4 and 4.4%, respectively). The whole dataset of validation parameters allows both the proposed methods to be considered as bias-free (by recovery tests, comparison of analytical results of the two independent methods and analysis of a synthetic sample) and precise (both the techniques show a repeatability better than 2% repeatability in the range between 70 and 600 mg kg(-1)).