From the Guerbet Research and Innovation Department, Aulnay-sous-Bois.
Institute of Analytical Sciences and Physico-Chemistry for Environment and Materials, UMR 5254, CNRS-UPPA, Pau, France.
Invest Radiol. 2021 Sep 1;56(9):535-544. doi: 10.1097/RLI.0000000000000774.
To date, the analysis of gadolinium (Gd) speciation in the brain of animals administered with macrocyclic and linear Gd-based contrast agents (GBCAs) has been limited to Gd soluble in mild buffers. Under such conditions, less than 30% of the brain tissue was solubilized and the extraction recoveries of GBCAs into the aqueous phase were poor, especially in the case of the linear GBCAs. The aim of this study was to find the conditions to solubilize the brain tissue (quasi-)completely while preserving the Gd species present. The subsequent analysis using size exclusion chromatography-inductively coupled plasma-mass spectrometry (SEC-ICP-MS) was intended to shed the light on the speciation of the additionally recovered Gd.
Four groups of healthy female Sprague Dawley rats (SPF/OFA rats; Charles River, L'Arbresle, France) received randomly 5 intravenous injections (1 injection per week during 5 consecutive weeks) of either gadoterate meglumine, gadobenate dimeglumine, gadodiamide (cumulated dose of 12 mmol/kg), or no injection (control group). The animals were sacrifice 1 week (W1) after the last injection. Brain tissues were solubilized with urea solution, whereas tissues extracted with water served as controls. Total Gd concentrations were determined in the original brain tissue and its soluble and insoluble fractions by inductively coupled plasma-mass spectrometry (ICP-MS) to calculate the Gd accumulation and extraction efficiency. Size exclusion chromatography coupled to ICP-MS was used to monitor the speciation of Gd in the soluble fractions. The stability of GBCAs in the optimum conditions was monitored by spiking the brain samples from the untreated animals. The column recoveries were precisely determined in the purpose of the discrimination of weakly and strongly bound Gd complexes. The identity of the eluted species was explored by the evaluation of the molecular size and retention time matching with Gd chelates and ferritin standard. The speciation analyses were carried out for 2 different brain structures, cortex and cerebellum.
The combination of water and urea extractions (sequential extraction) managed to solubilize efficiently the brain tissue (97% ± 1%) while preserving the stability of the initially injected form of GBCA. For macrocyclic gadoterate, 97% ± 1% and 102% ± 3% of Gd initially present in the cortex and cerebellum were extracted to the soluble fraction. For gadobenate, similar amounts of Gd (49% ± 1% and 46% ± 4%) were recovered from cortex and cerebellum. For gadodiamide, 48% ± 2% of Gd was extracted from cortex and 34% ± 1% from cerebellum. These extraction efficiencies were higher than reported elsewhere. The SEC-ICP-MS and the column recovery determination proved that Gd present at low nmol/g levels in brain tissue was exclusively in the intact GBCA form in all the fractions of brain from the animals treated with gadoterate. For the linear GBCAs (gadobenate and gadodiamide), 3 Gd species of different hydrodynamic volumes were detected in the urea-soluble fraction: (1) larger than 660 kDa, (2) approximately 440 kDa, and (3) intact GBCAs. The species of 440 kDa corresponded, on the basis of the elution volume, to a Gd3+ complex with ferritin. Gd3+ was also demonstrated by SEC-ICP-MS to react with the ferritin standard in 100 mM ammonium acetate (pH 7.4). In contrast to macrocyclic gadoterate, for linear GBCAs, the column recovery was largely incomplete, suggesting the presence of free, hydrolyzed, or weakly bound Gd3+ with endogenous ligands.
The sequential extraction of rat brain tissue with water and urea solution resulted in quasi-complete solubilization of the tissue and a considerable increase in the recoveries of Gd species in comparison with previous reports. The macrocyclic gadoterate was demonstrated to remain intact in the brain 1 week after administration to rats. The linear GBCAs gadobenate and gadodiamide underwent ligand exchange reactions resulting in the presence of a series of Gd3+ complexes of different strength with endogenous ligands. Ferritin was identified as one of the macromolecules reacting with Gd3+. For the linear GBCAs, 3% of the insoluble brain tissue was found to contain more than 50% of Gd in unidentified form(s).
迄今为止，对动物给予大环和线性基于钆的对比剂（GBCA）后的大脑中钆（Gd）形态的分析仅限于在温和缓冲液中可溶解的 Gd。在这种条件下，脑组织的溶解度小于 30%，并且 GBCA 进入水相的提取回收率很差，特别是对于线性 GBCA。本研究的目的是找到一种方法，在保留存在的 Gd 形态的情况下，几乎完全溶解脑组织（准完全溶解）。随后使用尺寸排阻色谱-电感耦合等离子体质谱法（SEC-ICP-MS）进行分析，以阐明另外回收的 Gd 的形态。
四组健康雌性 Sprague Dawley 大鼠（SPF/OFA 大鼠；Charles River，L'Arbresle，法国）随机接受 5 次静脉注射（连续 5 周每周 1 次），分别为钆特酸葡甲胺、钆贝葡胺、钆喷酸葡胺（累积剂量为 12 mmol/kg）或不注射（对照组）。最后一次注射后 1 周（W1）处死动物。用尿素溶液溶解脑组织，而用提取水的组织作为对照。通过电感耦合等离子体质谱法（ICP-MS）测定原始脑组织及其可溶和不可溶部分中的总 Gd 浓度，以计算 Gd 积累和提取效率。使用尺寸排阻色谱与 ICP-MS 监测可溶性部分中 Gd 的形态。通过向未经处理的动物的脑组织样本中添加 GBCA 来监测 GBCA 的稳定性。通过精确测定柱回收率，以区分弱结合和强结合的 Gd 络合物。通过评估洗脱物的分子大小和保留时间与 Gd 螯合物和铁蛋白标准相匹配，探索洗脱物的特征。对 2 种不同的脑结构（皮质和小脑）进行了形态分析。
水和尿素提取（顺序提取）的组合能够有效地溶解脑组织（97%±1%），同时保持初始注射形式的 GBCA 的稳定性。对于大环钆特酸葡甲胺，皮质和小脑中初始存在的 Gd 中有 97%±1%和 102%±3%被提取到可溶部分。对于钆贝葡胺，皮质和小脑中的 Gd 回收量相似（49%±1%和 46%±4%）。对于钆喷酸葡胺，皮质中回收了 48%±2%的 Gd，小脑中回收了 34%±1%的 Gd。这些提取效率高于其他报道。SEC-ICP-MS 和柱回收率测定证明，在给予钆特酸葡甲胺的动物的所有脑区的脑组织中，以低 nmol/g 水平存在的 Gd 仅以完整的 GBCA 形式存在。对于线性 GBCA（钆贝葡胺和钆喷酸葡胺），在尿素可溶部分中检测到 3 种不同水力学体积的 Gd 形态：（1）大于 660 kDa，（2）约 440 kDa，和（3）完整的 GBCA。基于洗脱体积，440 kDa 的物质对应于与铁蛋白形成的 Gd3+络合物。通过 SEC-ICP-MS 也证明，Gd3+在 100 mM 乙酸铵（pH 7.4）中与铁蛋白标准发生反应。与大环钆特酸葡甲胺相反，对于线性 GBCA，柱回收率极不完全，这表明存在游离、水解或与内源性配体结合较弱的 Gd3+。
水和尿素溶液对大鼠脑组织的顺序提取导致组织几乎完全溶解，与以前的报告相比，Gd 形态的回收率大大增加。给予大鼠后 1 周，钆特酸葡甲胺被证明保持完整。线性 GBCA 钆贝葡胺和钆喷酸葡胺经历了配体交换反应，导致存在一系列与内源性配体结合强度不同的 Gd3+络合物。铁蛋白被鉴定为与 Gd3+反应的一种大分子。对于线性 GBCA，3%的不可溶脑组织中含有超过 50%的未识别形式的 Gd。
