title: A simple and automated HPLC method for determination of total hydroxyproline in urine. Comparison with excretion of pyridinolines.
abstract: An HPLC method for measuring total hydroxyproline in human urine was validated. Hydroxyproline derivatization was achieved with 9-fluorenylmethyl chloroformate after blocking primary amino acids with orthophthaldialdehyde. The derivatives (hydroxyproline and internal standard) were separated by reversed phase high-performance liquid chromatography and detected by absorbance at 254 nm. Duplicate measurements of hydroxyproline have a coefficient of variation of 3.9% and the recovery in spiked urine samples is between 99.5 and 100.8%. We have compared the HPLC procedure with a commercial colorimetric assay. Analytical criteria of these methods are identical. Regression analysis, involving 50 samples, shows an excellent correlation between hydroxyproline chromatographic (y) and colorimetric (x) procedures: y = 0.989x + 2.99 (r = 0.976). Hydroxyproline excretion was determined in urine samples from 76 women more than 5 years post-menopause. The mean hydroxyproline/creatinine ratio in this group was 19.3 +/- 5.6 mumol/mmol (range 10.6-34.7). Finally, we compared in the same urinary samples hydroxyproline excretion with pyridinoline excretion (hydroxylysylpyridinoline and lysylpyridinoline), a new marker of bone resorption. The values show a significant correlation, with r = 0.417 for hydroxylysylpyridinoline and r = 0.443 for lysylpyridinoline.
authors.affiliation: Laboratoire d'Hormonologie, Hôpital Sud, Amiens, France.
authors.forename: S
authors.initials: S
authors.lastname: Castelain
authors.forename: S
authors.initials: S
authors.lastname: Kamel
authors.forename: C
authors.initials: C
authors.lastname: Picard
authors.forename: G
authors.initials: G
authors.lastname: Desmet
authors.forename: J L
authors.initials: JL
authors.lastname: Sebert
authors.forename: M
authors.initials: M
authors.lastname: Brazier
mesh_terms: D000368:Aged; D000596:Amino Acids / Q000652:urine*; D001862:Bone Resorption / Q000652:urine; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003124:Colorimetry; D005260:Female; D005449:Fluorenes / Q000378:metabolism; D006801:Humans; D006909:Hydroxyproline / Q000652:urine*; D008875:Middle Aged; D017698:Postmenopause; D012044:Regression Analysis; D015203:Reproducibility of Results
chemical_list: D000596:Amino Acids; D005449:Fluorenes; C015484:pyridinoline; C036020:deoxypyridinoline; C054007:1-(9-fluorenyl)methyl chloroformate; D006909:Hydroxyproline