Urinary excretion measurement of cysteine and homocysteine in the form of their S-pyridinium derivatives by high-performance liquid chromatography with ultraviolet detection.
Several human diseases, in particular metabolic disorders, often lead to the accumulation of characteristic metabolites in plasma, urine and cells. The selected diseases of this type include cystinuria and homocystinuria. In the typical laboratory diagnosis of these two diseases, a positive nitroprusside test is followed by quantitative analysis of urine cysteine and homocysteine in order to differentiate between cystinuria and homocystinuria. A sensitive and reproducible assay for total urine cysteine and homocysteine has been developed. The essential steps in the assay include conversion of disulphides to free thiols with tributylphosphine, conjugation of the thiols with 2-chloro-1-methyl pyridinium iodide, separation of S-pyridinium derivatives of cysteine and homocysteine from other endogenous urine thiol derivatives by reversed-phase high-performance liquid chromatography, and detection and quantitation by spectrophotometry. The method has a sensitivity of 4 pmol and is reproducible, intra- and inter-day coefficients of variation are from 1.37 to 4.14% and from 2.38 to 5.01%, respectively. The mean concentration of total urine cysteine and homocysteine in healthy donors (7 men and 7 women) were for women. 92.0 +/- 45.8 and 16.4 +/- 4.8 respectively, and for men 120.9 +/- 46.6 and 21.5 +/- 7.4 nmol/ml, respectively. Total urine homocysteine represents approximately 17.7% of cysteine in the urine of normal individuals.
Kaniowska
E
E
Department of Environmental Chemistry, University of Lodz, Poland.
Chwatko
G
G
Głowacki
R
R
Kubalczyk
P
P
Bald
E
E
D000328:Adult; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000652:urine*; D004220:Disulfides / Q000737:chemistry; D005260:Female; D006710:Homocysteine / Q000652:urine*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008297:Male; D008875:Middle Aged; D010720:Phosphines / Q000737:chemistry; D011726:Pyridinium Compounds / Q000737:chemistry*; D011786:Quality Control; D012016:Reference Values; D012680:Sensitivity and Specificity; D013438:Sulfhydryl Compounds / Q000737:chemistry
D004220:Disulfides; D010720:Phosphines; D011726:Pyridinium Compounds; D013438:Sulfhydryl Compounds; D006710:Homocysteine; C043255:tri-n-butylphosphine; D003545:Cysteine