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KMID : 0371020020350040282
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Journal of Preventive Medicine and Public Health
2002 Volume.35 No. 4 p.282 ~ p.286
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Determination of Free-4-hydroxyproline with Dansylchloride by HPLC in Human Urine
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Lee Keou-Weon
Lee Kyung-Jong
Cho Young-Bong
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Abstract
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Objectives: The level of 4-hydroxyproline (4Hyp) in human urine was measured using high performance liquid chromatography (HPLC) with a fluorescence detector. This method is useful for medical examinations and investigating the radicals induced by physical, chemical, mental stresses. This method is superior to many published several methods in terms of its low cost and ability to analyze many samples.
Methods: The urine from workers in a tire manufacturing company (22 male pre- and post-shift workers) and 18 office-workers as controls were analyzed. Date concerning age, the cumulative drinking amount and the cumulative smoking amount was collected with a questionnaire. The optimum applied amount of dansyl-Cl, the optimum reaction temperature and time, the recoveries and the optimum pH of the eluent and buffer were determined. 4-Hyp from human urine was derivatized with dansyl-Cl diamethylamino-naphthalene-1-sulfonyl chloride) after removing the α-amino acid by a treatment with phthalic dicarboxaldehyde (OPA) and cleaned with Bond Elut C 18 column. The 4-Hypderivatives were separated on a reversed phase column by gradient elution with a phoshate buffer ( 5 m㏖, pH 8.0) and acetonitrile, and detected by fluorescence measurements at 340 ㎚ (excitation) and 538 ㎚ (emission).
Results: The detection limit for the urinary free 4-Hpy was 0.364 μ㏖/ℓ. The recovery rate of 4-Hpy was 99.7%, and the effective pH of the phosphate buffer and borate buffer were 3.0 and 8.0, respectively. From statistical analysis, age, drinking and smoking did not affect the urinary free 4-Hpy in both the controls and workers. The range of urinary 4-Hpy in the controls, pre-shift, and post-shift workers were 0.33-16.44, N.D-49.06, and 0.32-56.27 μ㏖/ℓ. From the pared-sample t-test, the urinary 4-Hpy levels in post shift workers (11.82±16.73 n㏖/㎎ Cre) were 2-fold higher than in pre-shift workers (5.36±5.53 n㏖/㎎ Cre) and controls (4.91±4.89 n㏖/㎎ Cre).
Conclusions: This method was developed with high sensitivity, accuracy, and precision. The present method was effectively applied to analyzed the urinary free 4-Hpy in both controls and workers.
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KEYWORD
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hydroxyproline, Dimethylamino naphthalene-1-sulfonyl chloride, Dansyl chloride, HPLC, Stress
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