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Automation and Analytical Techniques |
1
Laboratory of Biochemistry, Children Hospital "Bambino Gesù" IRCCS, Piazza S. Onofrio, 4-00165 Rome, Italy.
2
University of Rome "Tor Vergata", Department of
Internal Medicine, Via di Tor Vergata, 135-00133 Rome, Italy.
a Address correspondence to this author at: Fax 39668592210; e-mail Federici@risc1.obg-irccs.rm.it or Federici{at}utorvrm.it.
We describe a 6-min HPLC method to measure the total concentrations of the most important thiols in plasma and urine–cysteine, homocysteine, cysteinylglycine, and glutathione–as well as the concentrations in plasma and urine, respectively, of cysteamine and 2-mercaptopropionylglycine, two compounds used to treat disorders of cysteine metabolism. Precolumn derivatization with bromobimane and reversed-phase HPLC were performed automatically by a sample processor. Throughput was up to 100 samples in 24 h. The within-run CV ranged from 0.9% to 3.4% and the between-run CV ranged from 1.5% to 6.1%. Analytical recovery was 97–107%, with little difference between plasma and urine samples. The detection limit was ~50 nmol/L for all the analytes studied. Thiol concentrations were determined in the plasma of 206 healthy donors and in the urine of 318 healthy donors distributed for age and sex. Mean values of plasma cysteine and homocysteine were significantly lower in infants (ages, <1 y) compared with other age groups (P <0.005). In adults, mean plasma homocysteine values were higher in males than in females (9.2 vs 6.7 µmol/L, P <0.0001) and in the 6- to 10-year-old group (P <0.05). Mean values for glutathione and cysteinylglycine were not sex- and age-dependent. In urine, both cysteine and homocysteine showed a wide range of variation.
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