Application of the capillary electrophoresis method for the study of plasma proteins homocysteinylation.

Purpose: Purpose. This article describes the use of capillary electrophoresis with UV detection to determine the ratio of protein-bound homocysteine and cysteine concentrations in human plasma. Methods: Plasma samples were reduced with dithiothreitol and derivatized by thiocarbonyldiimidazole before being filtered again for purification of proteins. The pre-concentration of analytes was carried out directly in the capillary (48.5 cm in length and an inner diameter of 50 mkm) by NaOH post-injection. The eletrophoretic separation of analytes was carried out using 0.2 M ammonium acetate with 25 mM hexadecyltrimethylammonium bromide. Results: Limit of quantitation for homocysteine was 0.8 mkM, reproducible ratio of cysteine/homocysteine <5%, full analysis time 15 min. Conclusion: The ratio of bound cysteine to homocysteine is characterized by the same regularity as the ratio of their total content. It has a fairly high degree of correlation with the level of bound homocysteine and it is characterized by less variability than the level of total homocysteine. This has the advantage of use the bound cysteine/homocysteine ratio for assessing the risk of cardiovascular disease complications.

S-Adenosylhomocysteine Assay in the Urine by Capillary Electrophoresis.

We present a simple and effective method for measuring urine S-adenosylhomocysteine by capillary electrophoresis without using modifiers. The detection threshold of the method is 0.1 µM S-adenosylhomocysteine, the time of analysis 13 min, reproducibility at physiological concentrations within 4%.

Effect of cerebral ischemia on redox status of plasma aminothiols.

We measured the content of reduced aminothiols (cysteine, homocysteine, and glutathione) after mechanical occlusion of the middle cerebral artery in rats. During acute period of ischemia (3 h), these parameters decreased by ~10 times. In 7 days, the content of reduced aminothiols in some animals remained low, but in the others surpassed the initial levels. These results indicate significant impairment of the redox status of the circulating aminothiol pool apparently caused by oxidative stress of the peripheral vascular endothelial occurring as a response to cerebral ischemia. Thus, the total amount of homocysteine is considered a risk factor for stroke, while its reduced fraction can reflect the post stroke state.

[Total aminothioles of rat plasma under intraperitoneal and subcutaneous introduction of homocysteine].

In the presented work a variation of total aminothiols (cysteine, glutathione, cysteinylglycine and homocysteine) in blood plasma have been shown at modelling hyperhomocysteinemia by daily intraperitoneal (0.6 mkmol/g body weight) and subcutaneous (0.12 mkmol/g body weight) introduction of homocysteine. During two weeks of the intraperitoneal introduction a significant concentration growth (from -40 to 180 mkM) of cysteine was observed. We also observed a moderate change of concentration levels for glutathione (from 10-15 to 30 mkM) and cysteinylglycine (from 1,5 to 4,5 mkM). The homocysteine level has decreased from 300 to 200-250 mkM at second week of experiments. Experimental results with subcutaneous introduction were similar. In this case a stable homocysteine level (-70 mkM) and increase of cysteine level (to 60 mkM) was observed at second week. These data reflect dose-depended processes of organism adaptation to hyperhomocysteinemia, i.e. reinforced capability for homocysteine metabolism and at the same time retention low glutathione level which correlates with hyperhomocysteinemia degree and duration.

[Mass-spectrometry in biology and medicine of XXI century].

This article for those who faces for the first time with mass spectrometry, but for obvious reasons they don't want to refer to mass spectrometers as to "black boxes". In this article all mass spectrometers which are used in analytical practice are mentioned. The huge need of analytical practice for very sensitive and selective methods of detection have stimulated the attraction of various mass analyzers by mass spectral laboratories. The concentration of analytes, requirements for limit of detection and sample content define the choice of the device.

The use of N-ethylmaleimide for mass spectrometric detection of homocysteine fractions in blood plasma.

Fraction analysis of homocysteine in biological fluids is important for the diagnosis and studies of cardiovascular, nervous, urological, and other diseases. Measurements of total, free, and reduced homocysteine by mass spectrometry with HPLC by means of its modification with N-ethylmaleimide are proposed. The linearity and detection threshold were 0.025-10 and 0.001 µM, respectively, for reduced homocysteine fraction and 0.2-100 and 0.1 µM, respectively, for the rest fractions. The accuracy and reproducibility of the method were within 12%.