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Technical Briefs |
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Ist. Clin. Humanitas, Rozzano (MI), Italy;
a address for correspondence: Lab. HPLC, H. San Raffaele, Via Olgettina 60, 20132 Milano, Italy, fax 39-2-26432640
Increased understanding of bone turnover has led to the development of several biochemical tests of bone metabolism. Among the biochemical indexes of bone resorption is measurement of urinary excretion of pyridinoline (Pyr) and deoxypyridinoline (Dpyr), molecules that cross-link the collagen chains and are released into the systemic circulation after the breakdown of mature bone collagen (1)(2)(3). Because they are not metabolized in vivo, they are excreted directly into urine in free (~40%) and peptide-bound forms (~60%) (4). Methods to measure cross-links in urine involve mainly two technical approaches: HPLC analysis, which, after hydrolytic and purification steps, allows the determination of the total forms of cross-links (5)(6)(7), and monoclonal antibody immunoassay methods able to quantify the sum of free Pyr and Dpyr or only the free Dpyr form (8)(9). The quantification of total or free cross-links forms provides, in any case, similar clinical information (9)(10).
Here we report the evaluation of the Cross LinksTM HPLC kit recently introduced by Beckman Labs. to quantify the total forms of Pyr and Dpyr. We compared the performance of this procedure with the Chrom- LinksTM HPLC method from Bio-Rad Labs. In addition we investigated the determination of free Dpyr by the Cross Links method by comparing it with the PyrilinksTM-D immunoassay (Metra Biosystem) (11).
After the Beckman procedure 0.25 mL of urine was hydrolyzed with 0.25 mL of HCl (12 mol/L) (R1 reagent) at 115 °C overnight. After the addition of 0.5 mL of the internal calibrator solution and 2 mL of 1-butanol (R2 reagent), samples were loaded onto solid-phase columns and washed with 9 mL of R3 reagent (a mixture of butanol:water:acetic acid). After the addition of 0.5 mL of an organic solvent reagent (R4) to clean up the columns from the previous R3 reagent, cross-links were eluted with 0.5 mL of the extraction reagent (R5) containing heptafluorobutyric acid in water, and 50 µL was injected into the HPLC. Unlike the Bio-Rad assay, which employs gravimetric columns, purification of urine is carried out under vacuum by solid-phase extraction, with a notable time saving and minimal solvent exposure for the technical staff.
The HPLC equipment consisted of a Beckman System Gold Model 116 on line
with an autosampler Model 507 and a Shimadzu Model RF 551 fluorometric
detector with excitation at 295 nm and emission at 395 nm. The
chromatographic separation lasted 10 min (Fig. 1
) and was performed on a reversed-phase column (100 x 4.6
mm) in isocratic mode, at a flow rate of 1.0 mL/min. The
eluting solution was prepared by mixing the stock buffer
(heptafluorobutyric acid solution) purchased from Beckman with
acetonitrile and water in the proportion 0.25:0.65:9.1 (by vol). The
column was thermoregulated at 37 °C.
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The intraassay CVs (n = 10) for a urine pool with Pyr = 500 pmol/mL and Dpyr = 89 pmol/mL were 3% and 4.9%, respectively, and for Pyr = 950 pmol/mL, Dpyr = 193 pmol/mL 3.4% and 2.8%, respectively. The interassay variation was calculated from the analysis of the same urine pools for 7 consecutive days. The CVs for Pyr and Dpyr were 5.5% and 8.7% for the lower concentrations and 4.0% and 5.2% for the higher concentrations. The mean recoveries were 95% ± 4.6% for Pyr (n = 3) and 99.2% ± 2.0% for Dpyr (n = 3). The assay was linear (r = 0.999) in the tested ranges (0–3000 pmol/mL and 0–500 pmol/L for Pyr and Dpyr, respectively). A signal-to-noise ratio of 3 (detection limit) was achieved at 1 pmol injected.
Comparison of the Beckman HPLC kit vs Bio-Rad on 50 urine samples from postmenopausal women (57 ± 5.7 years) who were referred to the Orthopaedic Division of our Institute yielded the following equations: Bio-Rad = 0.970 (± 0.0175) x Beckman + 8.0 (± 14.59) (±SE), r = 0.994, Sx|y = 45.7 for Pyr, and Bio-Rad = 0.870 (± 0.0274) x Beckman + 17.4 (± 5.56), r = 0.983, Sx|y = 17.5 for Dpyr. The slight underestimation of the Bio-Rad kit may be imputable to the lack of an internal calibrator to minimize handling errors.
Comparison of the concentrations of free forms of cross-links measured with the Cross Links with the mean of duplicate results of the Pyrilinks-D kit in 35 urine samples yielded: ELISA = 0.865 (± 0.0519) x Beckman + 3.71 (4.489), Sx|y = 10.3 (r = 0.947). Free Dpyr concentrations found with both kits represented ~40% of the corresponding total form, in agreement with data reported in literature (11).
We conclude that the Cross Links HPLC kit provides accurate results with a short and automated analysis (10 min) that can be performed overnight, thus reducing the technician time. We proved also that the detection limit of the Beckman kit allows the quantification of the free forms of cross-links (avoiding the time-consuming hydrolysis step) with a simple open monopump HPLC system. Despite the comparable costs (HPLC ~$29/sample vs the EIA ~$35/sample), the possibility of achieving in a single run the quantification of Pyr, Dpyr, and their ratio contributed to making this procedure an attractive alternative to the easily performed immunoassay.
Acknowledgments
We thank G. Mazzitelli and Beckman Analytical S.P.A. Italy for the gift of the Cross Links kit.
Footnotes
Lab. of Chromatogr. Techniques, Dept. of Lab. Med., IRCCS H. San Raffaele, Milano, Italy
References
The following articles in journals at HighWire Press have cited this article:
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  Y.-C. Chung, C.-H. Ku, T.-Y. Chao, J.-C. Yu, M. M. Chen, and S.-H. Lee Tartrate-resistant Acid phosphatase 5b activity is a useful bone marker for monitoring bone metastases in breast cancer patients after treatment. Cancer Epidemiol. Biomarkers Prev., March 1, 2006; 15(3): 424 - 428. [Abstract] [Full Text] [PDF]
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 T. G. Rosano, R. T. Peaston, H. G. Bone, H. W. Woitge, R. M. Francis, and M. J. Seibel Urinary free deoxypyridinoline by chemiluminescence immunoassay: analytical and clinical evaluation Clin. Chem., October 1, 1998; 44(10): 2126 - 2132. [Abstract] [Full Text] [PDF]
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ex = 295 nm,