Identification of the Main Intermediate Precursor of l-Ergothioneine Biosynthesis in Human Biological Specimens.

A capillary electrophoresis coupled to tandem mass spectrometry (CE-MS/MS) has been used to make a qualitative determination of hercynine-the main precursor of l-ergothioneine biosynthesis-in some key human biological specimens, such as urine, whole blood, plasma, and saliva. From semiquantitative analysis results, the highest concentrations of hercynine were detected in saliva and whole blood, whereas much lower concentrations were measured in urine and plasma. Whole blood was the biological matrix with the highest concentration of l-ergothioneine followed by plasma, saliva, and urine. The antioxidant effects attributed to l-ergothioneine, along with its peculiar antioxidant mechanism, offer a possible explanation for the presence of the hercynine, as well as its concentration, in the considered biological matrices.

An isotope dilution capillary electrophoresis/tandem mass spectrometry (CE-MS/MS) method for the simultaneous measurement of choline, betaine, and dimethylglycine concentrations in human plasma.

Plasma concentrations of choline, betaine, and dimethylglycine provide valuable information on the flow of methyl groups in key biological processes, particularly during folate deficiency states. We developed a new method to simultaneously measure these analytes in human plasma. Following sample deproteinization using acetonitrile, an aliquot was evaporated to dryness under vacuum to be then taken up by water. Finally, analytes were separated by capillary electrophoresis and detected by electrospray ionization triple-quadrupole mass spectrometry, in multiple reaction monitoring mode, using two stable isotope-labeled internal standards. Linearity of the calibration curves of each analyte was good (R(2) > 0.99). Average limits of detection (LODs) and limits of quantification (LOQs) for choline, betaine, and dimethylglycine were, respectively, 0.43, 0.62, and 0.31 µmol/L and 1.52, 2.11, and 0.97 µmol/L. Mean recovery of three replicates of two spiked concentrations levels was close to 100 % for all of the analytes. Repeatability and intermediate precision, expressed as %RSD of measurements, were <9 %. The method, applied to measure analytes in samples from 30 patients with chronic kidney disease and 30 age- and sex-matched healthy controls, was able to detect differences between groups and the sexes.

Simultaneous determination of the main amino thiol and thione in human whole blood by CE and LC.

BACKGROUND: Two precolumn fluorescence derivatization procedures by two different sulfhydryl-reactive iodoacetyl reagents were established to measure simultaneously glutathione and l-ergothioneine in human whole blood by means of CE and LC. MATERIALS & METHODS: Separations were achieved in <5 min on a reverse-phase column (100 mm × 4.6 mm Zorbax Eclipse Plus C18 3.5 µm) for LC analysis, and on an uncoated fused-silica capillary (60 cm × 50 µm) for CE analysis, monitoring the fluorescence of derivatives. RESULTS: Performance of the assays was good in terms of linearity, recovery, intra- and inter-day precision and LOD and LOQ. CONCLUSION: This novel approach allows rapid assessment of circulating glutathione and l-ergothioneine concentrations for clinical and research purposes.

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.

Simultaneous determination of aromatic amino acids in human blood plasma by capillary electrophoresis with UV-absorption detection.

Phenylalanine, tyrosine, and tryptophan, also known as aromatic amino acids, are involved in many physiological and pathophysiological conditions and are indicative of the liver and kidney function. In this work, we describe a simple and accurate method for their simultaneous quantification, in a single capillary electrophoresis run. This method requires minimal sample manipulation, no derivatization procedures, and methyl tryptophan as internal standard. The human blood plasma sample was precipitated using sulfosalicylic acid and the supernatant was used for the analysis. All the analytes were baseline resolved within 16 min and detected at 200 nm using Tris phosphate 80 mmol/L at pH 1.4 as the background electrolyte. The proposed method showed good linearity (r = 0.998) and repeatability (intra-assay RSD < 2.78%, interassay RSD < 5.4%) for all the analytes. The limit of quantification was 13 µmol/L for phenylalanine and 5 µmol/L for tyrosine and tryptophan. The method suitability was tested measuring aromatic amino acids level in 20 chronic kidney disease patients at basal level and after simvastatin/ezetimibe treatment.

Simultaneous determination of citrulline and arginine in human blood plasma by capillary electrophoresis with ultraviolet absorption detection.

A new capillary electrophoresis method to measure human blood plasma arginine and citrulline levels in a single run without derivatization was established. After adding homoarginine as internal standard, plasma proteins were removed by a 90:10 v/v acetonitrile/ammonia mixture. Arginine and citrulline were detected by an ultraviolet detector at 190 nm and separated in 11.65 and 20.43 min, respectively, by using a 75 mmol/L Tris phosphate solution at pH 1.2 as a background electrolyte. Limits of detection were 0.8 and 5 µmol/L for arginine and citrulline, respectively. Precision tests indicated a good repeatability of migration times and of peak area both for citrulline (CV% = 0.82 and 3.19) and arginine (CV% = 0.65 and 2.79). The CV% for intra- and interassay tests were, respectively, 1.84 and 3.23 for citrulline and 1.25 and 1.50 for arginine. Mean recovery was 101.5 and 98.5% for citrulline and arginine, respectively. The performance of the developed method was assessed by measuring plasma arginine levels in 52 subjects and the data were compared with those obtained by our previous assay. The new method was then applied to assess plasma citrulline and arginine in ten chronic kidney disease patients under hypolipidemic therapy with statin.