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

Abstract

The measurement of the 3-OH pyridinium compounds, pyridinoline (Pyr) and deoxypyridinoline (Dpyr), in urine by high performance liquid chromatography 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 CF1 cellulose, produced uncontaminated 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.

A population-based study of the relationship between salt intake, bone resorption and bone mass

“…Day to day variation in the sodium excretion of individuals is so great that`regression dilution bias' may reduce the slope of the regression line of sodium on a dependent variable such as blood pressure to a quarter of its true value (Frost et al, 1991) and this may well apply to bone mass also. This short-term variability in sodium excretion may also explain in part the well demonstrated short-term variability in pyridium cross-link assays (Colwell et al, 1993). Indeed, our data show bone resorption markers vary rapidly with sodium intake suggesting this is the case.…”

No effect of copper supplementation on biochemical markers of bone metabolism in healthy young adult females despite apparently improved copper status

“…Each of the separate daily urine samples from each individual collected over three consecutive days was analysed in triplicate using a three-step procedure. Urine was ®rst hydrolysed with an equal volume of 12 M HCl at 110 C for 18 h, the crosslinks were then extracted by CF1 cellulose chromatography with the use of an internal standard (acetylated pyridinoline, MetraBiosystems Ltd, Wheatley, Oxon, UK) and were measured using a reversed-phase HPLC method with¯uor-escence detection (Colwell et al, 1993). The acetylated pyridinoline was used in accordance with the method as described by Calabresi et al (1994) and Robins et al (1994).…”

“…The mean content of the pyridinium crosslinks in the three separate urine collections was used to represent the individual's excretion in that dietary period. The intra-assay coef®cient of variation (CV) for pyridinoline (Pyr) and deoxypyridinoline (Dpyr), measured as the variation between 10 chromatograms obtained between column regenerations as described by Colwell et al (1993), was 6% and 7%, respectively. Inter-assay variation was avoided by analysing all samples from an individual in the same run.…”

Measurement of urinary collagen cross-links indicate response to therapy in patients with breast cancer and bone metastases

“…All samples were coded, and all samples from individual patients were assayed in the same batch. Following acid hydrolysis of urine at 110°C overnight, and partial purification on a CF1 cellulose column, Pyr and Dpyr were separated by reverse phase high-performance liquid chromatography (HPLC) and detected by fluorescence (Colwell et al, 1993). The intra-assay coefficients of variation for Pyr and Dpyr were 7% and 10% respectively, and for inter-assay variation were <10%.…”