Factors affecting the assay of urinary 3–hydroxy pyridiniurn crosslinks of collagen as markers of bone resorption

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Abstract
The measurement of the 3–OH pyridinium compounds, pyridinoline (Pyr) and deoxypyridinoline (Dpyr), in urine by high performance liquid chromato-graphy is potentially useful in clinical studies, since they are specific biochemical markers of bone resorption. The aims of the present study were to improve assay performance and optimize sample collection. An isocratic high performance liquid chromatogram (HPLC) separation with baseline resolution was accomplished within 4 min using heptafluorobutyric acid as an ion-pair. The sample preparation for HPLC, using CFl cellulose, produced uncontami-nated samples with a recovery higher than 90% for both crosslinks. An elastin-derived material, tentatively identified as isodesmosine (Ides), was also tested and proved to be a suitable internal standard. Use of this standard improved assay precision. The effect of an oral gelatin load on the excretion of Pyr and Dyr was investigated. The creatinine corrected excretion of Pyr and Dpyr was unchanged over a 6 h period, in contrast to the 10–fold increase in the excretion of urinary hydroxyproline with a peak 2–4 h after ingestion. In 20 postmenopausal women, 2 h fasting morning urine results correlated with results from 24–h urine collections Dpyr/Cr (r = 0.70, n= 20). There was a day-to-day variation of 26% in adults studied for 10 days.