| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Technical Briefs |
B Ligand and Osteoprotegerin in Plasma and Serum
1 Department of Clinical Chemistry, Royal Liverpool University Hospital, The University of Liverpool, and2 Human Bone Cell Research Group, Department of Human Anatomy and Cell Biology, The University of Liverpool, Liverpool, L69 3GA, UK
aaddress correspondence to this author at: Department of Clinical Chemistry, 4th Floor Duncan Bldg., Liverpool L69 3GA, UK; fax 44-151-706-5813, e-mail w.d.fraser{at}liverpool.ac.uk
The discovery of receptor activator for nuclear factor-
B ligand (RANKL) and osteoprotegerin (OPG) as the fundamental factors controlling osteoclast formation and activation has advanced the understanding of the processes involved in osteoclastogenesis and bone remodeling (1)(2)(3). RANKL is important for osteoclast survival, differentiation, maturation, and activation, whereas OPG opposes these actions. Expression of RANKL and OPG is altered in many bone remodeling disorders, suggesting that determination of the role of these proteins in bone diseases is of value in understanding their etiology (4).
Accurate quantification of sRANKL and OPG concentrations in serum samples is paramount in research involving metabolic bone disease. The availability of ELISAs for both sRANKL and OPG has led to investigation of the concentrations of these proteins in human samples collected from patients with several disorders and has allowed monitoring of the effect of treatment in bone-related diseases (4)(5)(6)(7)(8)(9). Previous reports of OPG concentrations in postmenopausal women with osteoporosis have produced discordant results in relation to bone turnover (5)(8). Other reports using the sRANKL:OPG ratio to estimate the extent of Paget disease of bone and the effect of bisphosphonate treatment have also shown inconclusive and inconsistent results (9)(10)(11). These data suggest a possible variability in sRANKL and OPG measurements in human serum or plasma. In this study we aimed to clarify whether this variability is attributable to genuine differences among the groups of patients studied or whether it reflects inaccuracies resulting from the sampling process. We investigated several factors that may influence the concentrations of sRANKL and OPG in human blood samples by (a) collecting blood into different anticoagulants; (b) varying the time between blood collection and plasma/serum separation; (c) altering the length of storage of samples; and (d) altering the temperature at which samples were stored.
The study population consisted of 10 human volunteers with different disease activities, including 4 healthy individuals, 2 newly diagnosed patients with Paget disease of bone, and 4 Pagetic patients receiving treatment with bisphosphonate. Ethical permission for the study was obtained from the local research ethics committee.
Blood was drawn into tubes containing lithium heparin, clot activator, or EDTA, or into protease inhibitor tubes containing aprotinin, leupeptin, pepstatin, and EDTA. All sample collection tubes were purchased from SARSTEDT-MONOVETTE®, except for the protease inhibitor tubes, which were obtained from Nichols Institute Diagnostics.
To investigate the effectiveness of different anticoagulants on stabilizing sRANKL and OPG activity, plasma/serum was separated at 15, 30, and 60 min after venesection by centrifugation at 1814g for 10 min at 4 °C. Separated serum/plasma was stored at 4 and -70 °C for up to 24 h before assay. Samples were then thawed at room temperature before determination of sRANKL and OPG by ELISAs (obtained from Biomedica).
To assess the effect of storage of samples on sRANKL and OPG stability, blood was drawn into lithium-heparin and EDTA collection tubes, allowed to stand at room temperature for 30 min, and centrifuged at 1814g for 10 min at 4 °C before the plasma was collected. A portion of each lithium-heparin- and EDTA-plasma sample was assayed immediately for sRANKL/OPG, and the remainder of each sample was stored at -20 and -70 °C for 6 weeks (lithium heparin and EDTA) and 6 months (EDTA). At the end of these time periods, samples were thawed at room temperature before determination of sRANKL and OPG concentrations.
Statistical analysis was carried out by two-way ANOVA with Tukey’s hypothesis. P values <0.05 and a difference of ± 10% were considered significant. Data are presented as the mean (SE).
Interassay imprecision for sRANKL and OPG was established by measuring quality-control material provided in the assays (n = 16 assays). The sRANKL assay had an interassay CV of 13% across the range 3.3–5.4 pmol/L. The OPG assay had an interassay CV of 15% across the range 3.9–6.1 pmol/L.
The effects on sRANKL concentration of collecting blood into different anticoagulants, varying the time interval between collection and centrifugation, and altering storage conditions of samples are shown in Fig. 1
. Different anticoagulants can significantly affect the concentration of sRANKL. Higher sRANKL concentrations were measured when samples were collected into EDTA compared with both lithium heparin and clot activator (P <0.05), whereas the use of protease inhibitor tubes produced no significant differences in concentration compared with clot activator and lithium heparin. Increasing the time interval between blood collection and centrifugation up to 1 h and altering storage conditions for 24 h did not significantly affect the concentration of sRANKL.
|
Similar results were observed after measurement of OPG. Higher concentrations of OPG were detected when samples were collected into clot activator or EDTA compared with lithium heparin (P <0.05). Use of protease inhibitor tubes produced no significant difference in OPG concentrations compared with samples collected into lithium heparin. The concentration of OPG was not affected by changing the time between collection of sample and centrifugation or by storage at different temperatures for 24 h before assay.
These data suggest that samples for use in sRANKL and OPG assays should be collected into EDTA. sRANKL and OPG concentrations were not, however, affected by a delay in separation of plasma from whole blood up to 60 min after collection, nor were they affected by storage of serum/plasma at either 4 or -70 °C for 24 h before assay.
The effects of storage for 6 weeks or 6 months after collection on sRANKL and OPG concentrations in serum/plasma samples are summarized in Table 1
. Significant decreases in sRANKL concentrations were detected when plasma collected into EDTA or lithium heparin was stored at -20 or -70 °C for 6 weeks. sRANKL in lithium-heparin plasma demonstrated a greater degradation than in EDTA plasma (44.1% decrease in lithium-heparin plasma compared with 23.1% in EDTA plasma when stored at -20 °C; 40.8% decrease in lithium heparin plasma compared with 9.5% in EDTA plasma when stored at -70 °C). Although at the lower temperature degradation of sRANKL was reduced in EDTA plasma, a 56.5% decrease in sRANKL still occurred when EDTA plasma was stored at -70 °C for 6 months compared with a 61.5% decrease at -20 °C.
|
There was no significant difference in OPG concentration before and after 6 weeks of storage at -20 or -70 °C when samples were collected into either lithium heparin or EDTA. Storage of EDTA-plasma samples for up to 6 months at -20 or -70 °C produced a 22.8% decrease in OPG when stored at -20 °C and a 19.7% decrease when stored at -70 °C.
We also investigated the effect of storing separated samples in glass or plastic tubes and observed no significant effect of tube type. When samples were collected into nonsiliconized plastic syringes, significant variability in sRANKL was observed compared with results obtained when siliconized tubes were used (SARSTEDT-MONOVETTE). OPG was not affected by the type of sampling technique used.
The variability in sRANKL concentration under different storage conditions suggests a possible explanation for previous discordant findings (5)(8)(9)(10)(11). The amount of sRANKL measured by the ELISA used in this study can reflect only the free, uncomplexed forms in human plasma; therefore, the assays may not accurately reflect the total concentrations of sRANKL and OPG proteins in the samples. Altered concentrations of the proteins could also result from fluctuations in the sRANKL:OPG ratio in human plasma, which may contribute to the previous discordant results for detection of sRANKL:OPG in human plasma. In some circumstances, determination of the expression of RANKL and OPG mRNA extracted from osteoblastic cells may provide a more accurate and precise estimate of changes in the RANKL:OPG ratio in metabolic bone disease than do measurements of plasma concentrations (11)(12)(13)(14).
We recommend that samples for measurement of sRANKL and OPG should be collected into siliconized syringes or collection tubes and that EDTA is the preferred anticoagulant. Separation within 1 h and measurement of sRANKL and OPG in plasma as soon as possible after collection are also recommended. If required, storage is best done at -70 °C, but even at this temperature significant decreases in OPG and sRANKL were observed at 6 months.
We thank Drs. M.J. Diver and W. Taylor for statistical and technical assistance. We also thank the Arthritis Research Campaign (ARC) for funding of K.A.B.
References
B ligand: modulation of the expression by osteotrophic factors and cytokines. Biochem Biophys Res Commun 2000;275:768-775.[CrossRef][ISI][Medline]
[Order article via Infotrieve]
B ligand and osteoprotegerin: regulators of bone physiology and immune responses/potential therapeutic agents and biochemical markers. Ann Clin Biochem 2002;39:551-556.[CrossRef][ISI][Medline]
[Order article via Infotrieve]
B ligand-osteoprotegerin ratio predicts survival in multiple myeloma. Proposal for a novel prognostic index. Blood 2003;102:1064-1069.
B ligand (RANKL) and osteoprotegerin production by human osteoblastic cells: comparison of the effects of 17-ß oestradiol and raloxifene. J Endocrinol 2003;177:423-433.[Abstract]The following articles in journals at HighWire Press have cited this article:
|
|
 |
|
  M. Nybo and L. M Rasmussen The capability of plasma osteoprotegerin as a predictor of cardiovascular disease: a systematic literature review Eur. J. Endocrinol., November 1, 2008; 159(5): 603 - 608. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Joseph, B. Y. Chan, B. H. Durham, A. M. Ahmad, S. Vinjamuri, J. A. Gallagher, J. P. Vora, and W. D. Fraser The Circadian Rhythm of Osteoprotegerin and Its Association with Parathyroid Hormone Secretion J. Clin. Endocrinol. Metab., August 1, 2007; 92(8): 3230 - 3238. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Stern, G. A Laughlin, J. Bergstrom, and E. Barrett-Connor The sex-specific association of serum osteoprotegerin and receptor activator of nuclear factor {kappa}B legend with bone mineral density in older adults: the Rancho Bernardo Study Eur. J. Endocrinol., May 1, 2007; 156(5): 555 - 562. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Rogers and R. Eastell Circulating Osteoprotegerin and Receptor Activator for Nuclear Factor {kappa}B Ligand: Clinical Utility in Metabolic Bone Disease Assessment J. Clin. Endocrinol. Metab., November 1, 2005; 90(11): 6323 - 6331. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
