| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Technical Briefs |
1
Exacta Clinical Trials Service, Vicolo Chiodo 8, 37121 Verona, Italy
a author for correspondence: fax 39-45-8010868, e-mail exacta{at}tin.it
The determination of bone metabolism markers is useful in monitoring pharmacological therapy for osteoporosis (1)(2)(3). During the course of multicenter clinical studies designed to evaluate the therapeutic efficacy of several drugs, it often is useful to carry out analysis in batches after storing the samples for differing periods, thus reducing analytical variation and cost. This requires knowledge of the behavior of the analyte in the biological matrix in terms of the length and conditions of storage. Unfortunately, these data frequently are lacking, and the information provided by a kit’s manufacturer more often than not is contradictory.
Data published relative to long-term storage in biological matrices of bone turnover markers, including the seric NH2-terminal propeptide of type I procollagen (P1NP), the urinary cross-linked N-telopeptides of type I collagen (NTx), and the urinary pyridinium cross-links pyridinoline (PYD) and deoxypyridinoline (DPD), are extremely scarce (4). Therefore, our aim was to systematically study the molecular stability of these analytes in the storage mode most frequently used. In particular, the stability of storage lengths from 1 day to 12 months at temperatures between -80 °C and 23–25 °C were studied.
The biological samples were collected from 10 healthy subjects, 7 females and 3 males, between 25 and 64 years of age (mean ± SD, 36.1 ± 13.2 years), who had fasted from midnight. The venous blood samples were collected between 0800 and 0900, and the serum was separated through centrifugation, divided into 1-mL aliquots in micro test tubes, and stored at 23–25, 2–8, -20, or -80 °C. At the same time, aliquots of the second urine (fasting) of the morning were collected in sterile containers without preservatives and stored.
The bone metabolism marker analysis was carried out on samples stored at various temperatures at established intervals: storage time, 24 h, 48 h, 7 days, and 1, 3, 6, and 12 months. All of the testing was carried out by systematically following the instructions of the manufacturer of each diagnostic kit.
P1NP was measured by radioimmunoassay (Procollagen Intact PINP; Orion Diagnostica) (5)(6), and NTx was measured with a competitive-inhibition ELISA (Osteomark; Ostex) (7)(8)(9).
The total pyridinium cross-links PYD and DPD, the end products of collagen breakdown (10)(11), were determined by HPLC (Chrom-links; Bio-Rad) (12).
The results of the study are summarized in Table 1
. Taking the result obtained at storage time as 100%, the
subsequent values are reported as a percentage of the baseline. The
mean percentages and the relative standard deviations
(mean ± SD) of the groups of data obtained from each bone marker
analyzed at different intervals and storage temperatures are shown.
|
Using one-way ANOVA, we carried out a statistical elaboration of the data with the aim of highlighting a significant difference: each group of data was compared with its respective baseline group, and the P values calculated are reported.
Only the PYD and DPD values were significantly different from the baseline data group (P <0.05) after 48 h of storage at room temperature. We can conclude that:
The instability of PYD and DPP during storage at room temperature could be caused by the lack of acidification in the sample. Indeed, Bio-Rad, the manufacturer of the kit, suggests acidifying the sample with 10 mL of 6 mol/L HCl if it is a 24-h urine collection. Nevertheless, no advice is given regarding the second urine of the morning. Additional studies are under way in our laboratory to investigate this matter. In any case, it is important to note that acidification of the urine sample is incompatible with the ELISA for the NTx, which could not, therefore, be conducted together with the pyridinium cross-links determination in 24-h urine samples.
In conclusion, this study shows that the storage of serum and urine samples at 2–8 °C for the bone metabolism markers in question can be delayed for at least 1 week; for long-term storage, freezing of the sample provides molecular stability for several months. PYD and DPD are very sensitive to the storage temperature, and therefore, immediate refrigeration of a sample determines the accuracy of their measurement.
Acknowledgments
We thank Dr. Romolo Dorizzi from the Laboratorio Analisi Ospedale Civile Maggiore (Verona, Italy) for invaluable assistance.
References
The following articles in journals at HighWire Press have cited this article:
|
|
 |
|
  T. V. Nguyen, A. E. Nelson, C. J. Howe, M. J. Seibel, R. C. Baxter, D. J. Handelsman, R. Kazlauskas, and K. K. Ho Within-Subject Variability and Analytic Imprecision of Insulinlike Growth Factor Axis and Collagen Markers: Implications for Clinical Diagnosis and Doping Tests Clin. Chem., August 1, 2008; 54(8): 1268 - 1276. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Garnero, P. Vergnaud, and N. Hoyle Evaluation of a Fully Automated Serum Assay for Total N-Terminal Propeptide of Type I Collagen in Postmenopausal Osteoporosis Clin. Chem., January 1, 2008; 54(1): 188 - 196. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Jehle, A. Zanetti, J. Muser, H. N. Hulter, and R. Krapf Partial Neutralization of the Acidogenic Western Diet with Potassium Citrate Increases Bone Mass in Postmenopausal Women with Osteopenia J. Am. Soc. Nephrol., November 1, 2006; 17(11): 3213 - 3222. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
