Gundberg C M, Nieman S D, Abrams S, Rosen H
Department of Orthopaedics and Rehabilitation, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
J Clin Endocrinol Metab. 1998 Sep;83(9):3258-66. doi: 10.1210/jcem.83.9.5126.
Recent studies suggest that fracture risk is associated with increased undercarboxylated osteocalcin. Methods use differences in binding of undercarboxylated and fully carboxylated osteocalcin to hydroxyapatite or barium sulfate. We evaluated these methods and found that results varied with the amount and preparation of the salts. Furthermore, patient samples with differing amounts of total osteocalcin could not be directly compared. Errors in the determination of undercarboxylated osteocalcin were minimized by expressing data as the percent of the total osteocalcin in the sample, and correcting for the basal level of osteocalcin using a polynomial equation derived from multiple binding curves. Errors from 5-15% in estimation of undercarboxylated osteocalcin were observed without both of these corrections. When differing types of assays were employed (RIA, intact, N-terminal), results also were affected. In normal adults and children and in patients on long-term warfarin therapy, the percent osteocalcin not bound to hydroxyapatite was lower when measured with an intact assay than by a polyclonal RIA. Differences were related to the amount of N-terminal osteocalcin fragments, which had low affinity for hydroxyapatite and resulted in variable overestimation of undercarboxylated osteocalcin. In a kit specific for uncarboxylated osteocalcin, we found good discrimination between carboxylated and uncarboxylated intact osteocalcin. However, the assay detected large osteocalcin fragments and overestimated their concentration by up to 350%. Values for uncarboxylated osteocalcin were not different in patients on coumadin compared with normal adults with this kit, but when normalized to the total intact osteocalcin, percent uncarboxylated osteocalcin was greater in patients on coumadin than in controls, as would be expected. Kit values for uncarboxylated osteocalcin in normal children were higher than intact values in the same subject, because of the increased reactivity of the kit toward circulating fragments that were elevated in children. Thus, for estimation of undercarboxylated osteocalcin, care must be taken to standardize the hydroxyapatite or barium sulfite used for binding, to correct for the basal level of osteocalcin in the sample, to use immunoassays that do not detect small fragments, and to express the results as the percent of the total osteocalcin in the sample. Without these precautions, the assessment of undercarboxylated osteocalcin is not reliable.
近期研究表明，骨折风险与羧化不足的骨钙素增加有关。方法利用羧化不足和完全羧化的骨钙素与羟基磷灰石或硫酸钡结合的差异。我们评估了这些方法，发现结果因盐的用量和制备方法而异。此外，总骨钙素含量不同的患者样本无法直接进行比较。通过将数据表示为样本中总骨钙素的百分比，并使用从多条结合曲线推导的多项式方程校正骨钙素的基础水平，可将羧化不足骨钙素测定中的误差降至最低。若未进行这两项校正，羧化不足骨钙素估计值会出现5%-15%的误差。当采用不同类型的检测方法（放射免疫分析、完整型、N端型）时，结果也会受到影响。在正常成年人、儿童以及长期接受华法林治疗的患者中，采用完整型检测方法测定时，未与羟基磷灰石结合的骨钙素百分比低于多克隆放射免疫分析测定的结果。差异与N端骨钙素片段的量有关，这些片段对羟基磷灰石亲和力低，导致羧化不足骨钙素的估计值出现不同程度的高估。在一种针对未羧化骨钙素的试剂盒中，我们发现羧化和未羧化的完整骨钙素之间有良好的区分度。然而，该检测方法检测到大量骨钙素片段，并将其浓度高估了高达350%。使用该试剂盒时，服用香豆素的患者与正常成年人相比，未羧化骨钙素的值并无差异，但将其标准化为总完整骨钙素后，服用香豆素的患者中未羧化骨钙素的百分比高于对照组，这与预期相符。正常儿童未羧化骨钙素的试剂盒值高于同一受试者的完整值，这是因为该试剂盒对儿童中升高的循环片段反应性增强。因此，为了估计羧化不足的骨钙素，必须注意标准化用于结合的羟基磷灰石或亚硫酸钡，校正样本中骨钙素的基础水平，使用不检测小片段的免疫分析方法，并将结果表示为样本中总骨钙素的百分比。若不采取这些预防措施，羧化不足骨钙素的评估将不可靠。
