Medical Research Laboratory, Department of Clinical Medicine, Health, Aarhus University, Aarhus DK-8200, Denmark.
Department of Pulmonary Medicine, Aarhus University Hospital, Aarhus DK-8000, Denmark.
Growth Horm IGF Res. 2020 Apr;51:38-45. doi: 10.1016/j.ghir.2020.01.003. Epub 2020 Jan 21.
Insulin-like growth factor binding-protein 2 (IGFBP-2) was originally identified as an IGF-carrier, governing IGF half-life, tissue accessibility and biological effects. Later, IGFBP-2 was discovered to possess IGF-independent effects. IGFBP-2 circulates in several forms, as free protein, complexed with IGF-I or IGF-II, or as IGFBP-2 fragments. The various IGFBP-2 forms are all included when measuring serum IGFBP-2 concentrations by immunoassay (i.e., immunoreactive (ir-)IGFBP-2). In this study, we describe a novel method to measure the amount of IGF that circulates bound to IGFBP-2.
IGFBP-2 was immunoprecipitated from human serum using magnetic beads, which were subsequently eluted by acidification. After neutralization, eluates were assayed for ir-IGFBP-2, IGF-I and IGF-II and compared to serum concentrations. This allowed measurement of IGFBP-2-compexed IGF-I and IGF-II, respectively. To test the method clinically, serum from 146 patients with lung cancer, 151 patients with non-cancer pulmonary diseases and 28 healthy controls were analyzed.
We immuno-precipitated 97 ± 3.3% of serum IGFBP-2 and recovered > 75% of IGFBP-2-complexed IGFs, with intra- and inter-assay coefficient of variations (CVs) averaging < 5% and < 13%, respectively. No co-precipitation with IGFBP-1, -3 or - 4 was detected. Serum levels of ir-IGFBP-2 (median [25;75%]) differed between groups (cancer patients vs. non-cancer patients vs. healthy controls): 342 [260;480] vs. 262 [189;388] vs. 190 [141;269] μg/l (p < .0001). In parallel with this, concentrations of IGF-II carried by IGFBP-2 averaged: 45.0 [33.3;52.5] vs. 34.2 [25.4;46.1] vs. 19.8 [14.1;26.0] μg/l (p < .0001), and concentrations of IGF-I 8.0 [5.2;11.8] vs. 5.4 [3.6;7.3] vs. 7.0 [3.8;13.0] μg/l (p < .0001). Thus, IGFBP-2 carried more IGF-II than IGF-I in all groups (p < .0001). When expressed relative to IGF-concentrations, IGFBP-2 carried 9.0 [5.3;15.5] % of the IGF-I and 4.8 [2.9;5.8] % of the IGF-II in serum from healthy subjects. Notably, in patients, IGFBP-2 carried relatively less IGF-I, but more IGF-II (p < .0001).
Using our novel assay, we demonstrate: that IGFBP-2 carries ≈10% of circulating IGF-I and ≈5% of circulating IGF-II in healthy subjects; that IGF-II is the primary ligand for IGFBP-2; and that IGFBP-2 carries even more IGF-II in patients than in healthy subjects. Thus, our assay may provide information on IGFBP-2 beyond what is achievable by simply measuring ir-IGFBP-2.
胰岛素样生长因子结合蛋白 2（IGFBP-2）最初被鉴定为 IGF 载体，控制 IGF 半衰期、组织可及性和生物学效应。后来，IGFBP-2 被发现具有 IGF 非依赖性效应。IGFBP-2 以几种形式循环，包括游离蛋白、与 IGF-I 或 IGF-II 结合的形式，或 IGFBP-2 片段。通过免疫测定（即免疫反应性（ir-）IGFBP-2）测量血清 IGFBP-2 浓度时，包括所有这些 IGFBP-2 形式。在这项研究中，我们描述了一种测量与 IGFBP-2 结合的循环 IGF 量的新方法。
使用磁性珠从人血清中免疫沉淀 IGFBP-2，随后通过酸化洗脱。中和后，测定洗脱液中的 ir-IGFBP-2、IGF-I 和 IGF-II，并与血清浓度进行比较。这允许分别测量 IGFBP-2 结合的 IGF-I 和 IGF-II。为了临床测试该方法，分析了 146 例肺癌患者、151 例非癌症肺部疾病患者和 28 例健康对照者的血清。
我们免疫沉淀了 97 ± 3.3%的血清 IGFBP-2，并回收了 > 75%的 IGFBP-2 结合的 IGFs，内和间测定的变异系数（CV）平均 < 5%和 < 13%。未检测到与 IGFBP-1、-3 或 -4 的共沉淀。血清 ir-IGFBP-2 （中位数 [25；75%]）在各组之间存在差异（癌症患者与非癌症患者与健康对照组）：342 [260；480] 比 262 [189；388] 比 190 [141；269] μg/l（p < 0.0001）。与此平行的是，IGFBP-2 携带的 IGF-II 浓度平均为：45.0 [33.3；52.5] 比 34.2 [25.4；46.1] 比 19.8 [14.1；26.0] μg/l（p < 0.0001），IGF-I 浓度为 8.0 [5.2；11.8] 比 5.4 [3.6；7.3] 比 7.0 [3.8；13.0] μg/l（p < 0.0001）。因此，IGFBP-2 在所有组中携带的 IGF-II 多于 IGF-I（p < 0.0001）。当相对于 IGF 浓度表示时，IGFBP-2 在健康受试者的血清中携带 9.0 [5.3；15.5] %的 IGF-I 和 4.8 [2.9；5.8] %的 IGF-II。值得注意的是，在患者中，IGFBP-2 携带的 IGF-I 较少，但携带的 IGF-II 较多（p < 0.0001）。
使用我们的新测定法，我们证明：IGFBP-2 在健康受试者中携带约 10%的循环 IGF-I 和约 5%的循环 IGF-II；IGF-II 是 IGFBP-2 的主要配体；并且 IGFBP-2 在患者中携带的 IGF-II 甚至比健康受试者中更多。因此，我们的测定法可能提供比简单测量 ir-IGFBP-2 更能了解 IGFBP-2 的信息。
