Liquid chromatographic determination of total homocysteine in blood plasma with photometric detection.
A rapid and sensitive method for quantification of homocysteine total forms and glutathione levels in blood plasma via HPLC was developed. Dithiotreitol as a water soluble agent has been used as a reductant for both protein and nonprotein disulphides. Dithiotreitol reacts with the mixed disulphides under 60 degrees C treatment within 10 min. Reduced aminothiols and homocystein were easily derivated with 5,5'-dithiobis-(2-nitrobenzoic acid) and the resultant ultraviolet absorbance within 330 nm was detected by the HPLC method. The concentration of total plasma homocysteine was significantly higher in groups of patients: with the end stage of renal disease: 45.5+/-40.9 micromol/l (n=79), with cerebral vascular disorders 12.3+/-7.0 micromol/l (n=65), and with coronary atherosclerosis 15.4+/-10.9 micromol/l (n=15) than that in healthy subjects (6.2+/-1.74 micromol/l, n=20). Some major advantages of the method include: simultaneous measurement of both total homocysteine and total glutathione, no loss of oxidized form during processing of blood plasma for aminothiols measurement, use of protein-bound aminothiols solution as a calibrator.
Zhloba
Alexander A
AA
SC Department of Biochemistry, St.-Petersburg State I.P. Pavlov Medical University, L. Tolstoy Street 6/9, St. Petersburg 197089, Russia. zhloba@mail.spbnit.ru
Blashko
Eduard L
EL
D000328:Adult; D001800:Blood Specimen Collection; D002138:Calibration; D002851:Chromatography, High Pressure Liquid; D004229:Dithiothreitol / Q000737:chemistry; D005260:Female; D005978:Glutathione / Q000097:blood; D006710:Homocysteine / Q000097:blood*; D006801:Humans; D008297:Male; D008875:Middle Aged; D010084:Oxidation-Reduction; D010783:Photometry; D011485:Protein Binding; D019163:Reducing Agents / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet; D013439:Sulfhydryl Reagents / Q000737:chemistry; D013696:Temperature
D019163:Reducing Agents; D013439:Sulfhydryl Reagents; D006710:Homocysteine; D005978:Glutathione; D004229:Dithiothreitol