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Stability of several biochemical markers of bone metabolism.

The determination of bone metabolism markers is useful in monitoring pharmacological therapy for osteoporosis (1-3). 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 N[H.sub.2]-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[degrees]C and 23-25[degrees]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 [+ or -] SD, 36.1 [+ or -] 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[degrees]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 PIMP; Orion Diagnostica) (5,6), and NTx was measured with a competitive-inhibition ELISA (Osteomark; Ostex) (7-9).

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 [+ or -] 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:

* P1NP is stable in stored serum for at least 48 h at 23-25[degrees]C, 7 days at 2-8[degrees]C, and frozen for at least 6 months at -20 to -80[degrees]C.

* NTx is stable in stored urine for at least 48 h at 23-25[degrees]C, 7 days at 2-8[degrees]C, 3 months when frozen at -20[degrees]C, and for at least 1 year at -80[degrees]C.

* PYD and DPD are not stable in urine samples stored at room temperature: even after 24 h, the respective mean concentrations compared with the baseline values were reduced by 30% and 39%,respectively. After 48 h, the PYD and DPD concentrations were reduced by more than 50%.

* PYD and DPD are, however, stable for at least 7 days in urine stored at 2-8[degrees]C, for at least 3 months in urine frozen at -20[degrees]C, and for at least 1 year in urine frozen at -80[degrees]C.

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[degrees]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.

We thank Dr. Romolo Dorizzi from the Laboratorio Analisi Ospedale Civile Maggiore (Verona, Italy) for invaluable assistance.


(1.) Panteghini M, Pagani F. Biological variation in bone-derived biochemical markers in serum. Scand J Clin Lab Investig 1995;55:609-16.

(2.) Giavarina D, Marchi G, Giavarina M, Tonelli M, Schiavon R. Urinary excretion of type I collagen cross-linked N-telopeptide in children: biological variability and reference limits. Eur J Lab Med 1997;5:70-3.

(3.) Price CP, Thompson PW. The role of biochemical tests in the screening and monitoring of osteoporosis. Ann Clin Biochem 1995;32:244-60.

(4.) Gerrits MI, Thijssen JHH, Van Rijn HJM. Determination of pyridinoline and deoxypyridinoline in urine, with special attention to retaining their stability. Clin Chem 1995;41:571-4.

(5.) Risteli L, Risteli J. Biochemical markers of bone metabolism [Review]. Ann Med 1993;25:385-93.

(6.) Eriksen EF, Brixen K, Charles P. New markers of bone metabolism: clinical use in metabolic bone disease [Review]. Eur J Endocrinol 1995;132:25163.

(7.) Gunja-Smith Z, Boucek RJ. Collagen cross-linking compounds in human urine. Biochem J 1981;197:759-62.

(8.) Rosen HN, Dresner-Pollak R, Moses AC, Rosenblatt M, Zeind AJ, Clemens JD, Greenspan SL. Specificity of urinary excretion of cross-linked N-telopeptides of type I collagen as a marker of bone turnover. Calcif Tissue Int 1994;54:26-9.

(9.) Hanson DA, Weis MA, Bollen AM, Maslan SL, Singer F, Eyre DR. A specific immunoassay for monitoring human bone resorption: quantitation of type I collagen cross-linked N-telopeptides in urine. J Bone Miner Res 1992;7: 1251-8.

(10.) Delmas P. Biochemical markers of bone turnover for the clinical assessment of metabolic bone disease. Endocrinol Metab Clin N Am 1990;19:1-18.

(11.) Robins SP, Black D, Paterson CR, Reid DM, Duncan A, Seibel MJ. Evaluation of urinary hydroxypyridinium crosslinks measurements as resorption markers in metabolic bone diseases. Eur J Clin Investig 1991;21:310-5.

(12.) Eyre DR, Koob TJ, Van Ness KP. Quantitation of hydroxypyridinium crosslinks in collagen by high-performance liquid chromatography. Anal Biochem 1984;137:380-8.

Angelo Lomeo and Andrea Bolner *

(Exacta Clinical Trials Service, Vicolo Chiodo 8, 37121 Verona, Italy;

* author for correspondence: fax 39-45-8010868, e-mail
Table 1. Effects of storage length and temperature on the molecular
stability of P1NP, NTx, PYD, and DPD in biological matrices.

 [degrees]C Time % P

 23-25 24 h 101.2 [+ or -] 5.5 0.878
 48 h 103.4 [+ or -] 6.7 0.823
 2-8 48 h 102.9 [+ or -] 4.0 0.878
 7 days 104.4 [+ or -] 8.2 0.786
 -20 1 month 97.9 [+ or -] 4.0 0.871
 3 months 95.2 [+ or -] 6.1 0.716
 6 months 103.7 [+ or -] 2.2 0.841
 -80 1 month 101.3 [+ or -] 5.6 0.883
 3 months 97.7 [+ or -] 9.3 0.777
 6 months 104.3 [+ or -] 3.3 0.819
 12 months

 [degrees]C % P

 23-25 85.8 [+ or -] 12.7 0.793
 76.4 [+ or -] 5.2 0.544
 2-8 80.8 [+ or -] 8.1 0.554
 80.4 [+ or -] 19.2 0.694
 -20 81.6 [+ or -] 19.0 0.541
 100.9 [+ or -] 11.9 0.884

 88.9 [+ or -] 8.9 0.713
 92.2 [+ or -] 38.9 0.749
 85.3 [+ or -] 12.6 0.786

 [degrees]C % P

 23-25 76.7 [+ or -] 16.6 0.311
 49.3 [+ or -] 12.4 0.049
 2-8 111.7 [+ or -] 23.0 0.859
 114.3 [+ or -] 17.4 0.738
 -20 106.2 [+ or -] 36.5 0.889
 87.8 [+ or -] 22.9 0.762

 -80 107.9 [+ or -] 16.4 0.839
 80.2 [+ or -] 21.7 0.661

 84.7 [+ or -] 13.0 0.630

 [degrees]C % P

 23-25 61.4 [+ or -] 21.3 0.196
 31.7 [+ or -] 23.0 0.006
 2-8 116.2 [+ or -] 23.3 0.722
 108.9 [+ or -] 48.8 0.425
 -20 110.9 [+ or -] 47.6 0.424
 114.8 [+ or -] 69.5 0.864

 -80 107.8 [+ or -] 17.6 0.540
 85.5 [+ or -] 35.9 0.644

 79.8 [+ or -] 28.1 0.318
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Title Annotation:Technical Briefs
Author:Lomeo, Angelo; Bolner, Andrea
Publication:Clinical Chemistry
Date:Aug 1, 2000
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