Rapid screening of urinary proline–hydroxyproline dipeptide in bone turnover studies

In a recent report [J. Chromatogr. B 678 (1996) 165] a urinary hydroxyproline-containing peptide has been preliminarily suggested as a possible alternative to hydroxyproline (HP) determination in bone resorption studies. For this purpose a simple and practical procedure was developed for a rapid high-performance liquid chromatographic (HPLC) assay of the peptide in non-hydrolyzed urine samples. Hundreds of randomly selected urine samples were assayed for both the peptide and HP, the latter in hydrolyzed urine, and a high correlation between them was found. The promising results prompted us to search for the postulated biomarker of bone resorption in urine samples of postmenopausal women examined as osteoporosis suspects. As an alternative to the HPLC determination, an equally rapid procedure has been developed for the peptide assay using capillary gas chromatography (GC) with flame ionization detection (FID). By means of a solid-phase and a liquid–liquid phase extraction, involving ethyl chloroformate (ECF) as the derivatizing agent, two dipeptides and some urinary amino acids could be analyzed within 5 min. A high correlation between both HPLC and GC peptide assay was confirmed (r=0.944) and the compound was identified as proline–hydroxyproline (PHP) dipeptide.