Aluminum's Complex Influence in Provocation Testing: A Multi-Method Validation Characterizing Iatrogenic Artifacts, Endogenous Interactions, and Mitigation Strategies

Provocation testing with chelating agents like Na₂Ca-EDTA serves as a powerful clinical tool that enhances the detection of urinary trace metals and offers valuable insights into the relationships between body burden and environmental exposure. However, the simultaneous mobilization and transport of multiple metals—competing for the same binding sites on provocation chelators and within physiological storage, transport, and excretion mechanisms—can create complex, sometimes counterintuitive, urinary excretion patterns that complicate interpretation. Additionally, trace-metal impurities in the chelating solution can significantly skew results [1], which is a particular concern in high-precision applications such as environmental epidemiology.<br><br>Our investigation was initially prompted by puzzling clinical observations: repeated concurrent urinary spikes of uranium and aluminum, both non-essential and potentially toxic elements [2]. With urinary aluminum typically exceeding uranium by three orders of magnitude and no other apparent confounders, their association most likely indicates shared exposure pathways (either environmental or iatrogenic) or an aluminum-driven enhancement of uranium excretion, rather than the reverse. Besides its association with uranium, aluminum also shows strong positive associations with a range of essential metals (e.g., manganese, iron, chromium) and other non-essential or toxic metals, such as cadmium, lead, and uranium (Figs. 1A–3), raising concerns about the pervasive influence of iatrogenic confounding.<br>The spring 2017 uranium spike [2] presents an intriguing characteristic: it occurred without a concurrent general aluminum surge (Fig. 1B). Despite this, Fig. 1A demonstrates that even minimal aluminum, presumably reflecting an endogenous burden from prior environmental deposition, shows an even stronger association with enhanced uranium excretion. This points to aluminum's influence on uranium excretion being multifaceted, involving at least two distinct components (Fig. 11a): one linked to environmental/endogenous aluminum and another to iatrogenic aluminum from chelators, each contributing with distinct dynamics or efficiencies.Motivated by these compelling clinical observations and the broader challenge of deciphering complex inter-metal correlations in provoked urine, we undertook a systematic, multi-phase investigation. This study aimed to:Rigorously measure trace metals in all infusion solutions in a first QC Phase: DMPS and glutathione showed negligible impurities, but Na₂Ca-EDTA proved to be the dominant source of aluminum, iron, and manganese contamination (Fig. 8a; Excel 'Aluminum_CEMET_QC_Modelling'), corroborating earlier findings [1].Dissect the dual impact of aluminum, both from environmental/endogenous origins and iatrogenic Na₂Ca-EDTA impurities, on post-provocation urinary metal excretion profiles using a multi-method approach. This involved:<br>- Extensive correlation analyses (Table 1, Figs. 4–10, Excel 'Data_Sets_Imputation_Correlation'),<br>- Pharmacokinetic (PK) modeling (Fig. 10),<br>- Gaussian Mixture Models (GMMs; Figs. 11a–c), and<br>- Comparative provocation with cleaner Zn-DTPA to differentiate exposure and biological interactions from<br>iatrogenic impurity artifacts (Figs. 8e, 12).Establish a framework, including potential mitigation strategies, for more robust analysis of provoked urine data in future environmental and clinical research.<br><br><b>Key Findings &amp; Conclusions:</b><br><b>Significant and highly variable Na₂Ca-EDTA impurities:</b> Quality control assays revealed highly variable aluminum (3.2–38.7 mg/L) and iron (1.9–29.0 mg/L) content in commercial Na₂Ca-EDTA solutions (Table 1a, Figs. 5, 8a). This iatrogenic metal load directly drives elevated urinary aluminum and iron excretion in a dose-responsive manner (Table 1b, Figs. 6, 10).<br><br><b>PK modeling quantifies iatrogenic aluminum:</b> PK modeling attributed a substantial portion (up to 70 %) of urinary aluminum to solution-derived contamination (Fig. 10).<br><br><b>Gaussian mixture modeling (GMM) </b><b>reveals differential impact:</b> GMM identified two distinct urinary excretion peaks for both aluminum and iron (optimal N = 2). For aluminum, the two components contributed roughly equally to the overall distribution, whereas for iron, there was a strong predominance of the higher-concentration component (Figs. 11a and b). In contrast, uranium exhibited a single-component distribution (N = 1 optimal), showing no evidence that direct uranium contamination from EDTA solutions significantly altered its population distribution (Fig. 11c).<br><br><b>Al–U interaction</b><b> is primarily biological:</b> Despite negligible direct uranium contamination in Na₂Ca-EDTA solutions, a consistent positive correlation is observed between urinary aluminum and uranium with both Na₂Ca-EDTA provocation (partial Spearman ρ≈0.45; Figs. 4, 8c-e) and even with the less-impurity-prone Zn-DTPA provocation (Spearman ρ≈0.36; Figs. 8e, 12). This persistence suggests an intrinsic Al–U interaction, likely where aluminum (endogenous and, to a lesser extent, iatrogenic) enhances uranium excretion via competitive displacement from transport proteins (e.g., transferrin, albumin), consistent with established aluminum physiology [3–7].<br><br><b>Iatrogenic aluminum modulates urinary metal excretion: </b>both environmental Al and that introduced by Na₂Ca-EDTA jointly govern the Al–U dose–response and alter inter-metal correlations in urine, with the iatrogenic fraction dominating at aluminum levels &gt; 150 µg Al/g creatinine.<br><br><b>Zn-DTPA confirms</b> <b>endogenous aluminum mobilization: </b>Provocation with cleaner Zn-DTPA also reveals persistent and robust Al–metal correlations (e.g., Al–Mn, Al–Fe), highlighting the contribution of endogenous aluminum mobilization and its biological interactions (Figs. 8e, 12, Excel 'Data_Sets_Imputation_Correlation').<br><br><b>Mitigating Iatrogenic Effects by QC:</b> The variability of Na₂Ca-EDTA impurities—primarily in aluminum and, to a lesser extent, iron and manganese—poses significant confounding potential. This multi-modal validation study highlights the critical importance of awareness and management of chelator solution variability in provocation testing. By characterizing these artifacts and proposing mitigation approaches, we aim to provide a framework for more robust and reliable analysis of urinary metal excretion data in both clinical and environmental research settings. For valid downstream environmental epidemiology, mitigation strategies are crucial, such as excluding samples with urinary aluminum concentrations exceeding a threshold indicative of substantial iatrogenic load (e.g., &gt; 140 µg/g creatinine). The datasets and detailed analyses supporting these conclusions are provided herein.<br><br><b>Biological significance of environmental aluminum:</b> The steep initial Al–U dose–response (Fig. 1) and durable Al–metal correlations under Zn-DTPA (Figs. 8e, 12) reveal that low-level, environmentally deposited aluminum can mobilize other metals more powerfully than larger iatrogenic loads. Future provocation testing frameworks should therefore aim to quantify and correct for this baseline endogenous aluminum burden.<br><br><b>References</b><br><br>1) Blaurock-Busch E, Strey R, editors. Chronische Metallbelastungen – Toxikologie, Diagnose und Therapie [Chronic Metal Exposure: Toxicology, Diagnosis and Therapy] [Internet]. Norderstedt (Germany): BoD – Books on Demand GmbH; 2017. p. 155. Available from: https://d-nb.info/1147825653.<br>2) Carmine TC. The Uranium Episode (March–May 2017) in Temporal Context: Associations with CEMET Uranium, Aluminum, and Local PM10 Exposure (2016–2019) [dataset]. figshare; 2024. Available from: https://doi.org/10.6084/m9.figshare.27435639.v5.3) Rahimzadeh MR, Rahimzadeh MR, Kazemi S, Amiri RJ, Pirzadeh M, Moghadamnia AA. Aluminum Poisoning with Emphasis on Its Mechanism and Treatment of Intoxication. Emerg Med Int. 2022 Jan 11;2022:1480553. doi: 10.1155/2022/1480553. PMID: 35070453; PMCID: PMC8767391.<br><br>4) Fatemi SJ, Kadir FH, Moore GR. Aluminium transport in blood serum. Binding of aluminium by human transferrin in the presence of human albumin and citrate. Biochem J. 1991 Dec 1;280 ( Pt 2)(Pt 2):527-32. doi: 10.1042/bj2800527. PMID: 1747128; PMCID: PMC1130580.<br><br>5) Cochran M, Coates J, Neoh S. The competitive equilibrium between aluminium and ferric ions for the binding sites of transferrin. FEBS Lett. 1984 Oct 15;176(1):129-32. doi: 10.1016/0014-5793(84)80926-6. PMID: 6489514.<br>6) El Hage Chahine JM, Hémadi M, Ha-Duong NT. Uptake and release of metal ions by transferrin and interaction with receptor 1. Biochim Biophys Acta. 2012 Mar;1820(3):334-47. doi: 10.1016/j.bbagen.2011.07.008. Epub 2011 Aug 17. PMID: 21872645.7) Golub MS, Han B, Keen CL. Aluminum alters iron and manganese uptake and regulation of surface transferrin receptors in primary rat oligodendrocyte cultures. Brain Res. 1996;719(1-2):72–77. doi:10.1016/0006-8993(96)00087-X.

以乙二胺四乙酸钙二钠（Na₂Ca-EDTA）等螯合剂开展的激发试验，是一项强效临床工具，可提升尿液痕量金属的检测效能，并为机体金属负荷与环境暴露间的关联提供宝贵见解。然而，多种金属同时被动员并转运——在激发性螯合剂以及生理储存、转运与排泄机制中竞争相同结合位点——可形成复杂且有时违背直觉的尿液排泄模式，增加结果解读难度。此外，螯合剂溶液中的痕量金属杂质会显著干扰检测结果[1]，这在环境流行病学等高精密应用场景中尤为值得关注。

本研究最初源于令人困惑的临床观察：铀与铝这两种非必需且具有潜在毒性的元素[2]，在尿液中反复同时出现排泄峰值。通常尿液中铝的浓度比铀高出三个数量级，且无其他明显混杂因素，二者的关联大概率提示存在共同的暴露途径（环境源性或医源性），或是铝驱动铀排泄增强，而非反之。除与铀相关外，铝还与一系列必需金属（如锰、铁、铬）及其他非必需/毒性金属（如镉、铅、铀）呈现强正相关（图1A~3），这引发了人们对医源性混杂因素普遍影响的担忧。

2017年春季出现的铀排泄峰值[2]具有一个有趣的特征：其发生时并未伴随铝的整体升高（图1B）。尽管如此，图1A显示，即使是微量的铝（推测反映了先前环境沉积的内源性负荷），也与铀排泄增强呈现更强的关联。这表明铝对铀排泄的影响是多方面的，至少涉及两种不同的组分（图11a）：一种与环境/内源性铝相关，另一种与螯合剂带来的医源性铝相关，二者各自具有独特的动力学特征或效能。

受这些引人关注的临床观察，以及解析激发尿液中复杂金属间关联这一更广泛的挑战所驱动，我们开展了一项系统性、多阶段的研究。本研究旨在：
1. 在第一阶段质量控制（QC）中严格检测所有输注溶液中的痕量金属：二巯基丙磺酸钠（DMPS）与谷胱甘肽（glutathione）的杂质含量可忽略不计，但乙二胺四乙酸钙二钠（Na₂Ca-EDTA）被证实是铝、铁和锰污染的主要来源（图8a；Excel表格"Aluminum_CEMET_QC_Modelling"），这与此前的研究结果一致[1]。
2. 采用多方法手段，剖析铝（包括环境/内源性来源及医源性Na₂Ca-EDTA杂质）对激发后尿液金属排泄谱的双重影响。具体包括：
- 全面的相关性分析（表1、图4~10、Excel表格"Data_Sets_Imputation_Correlation"），
- 药代动力学（PK）建模（图10），
- 高斯混合模型（Gaussian Mixture Models, GMMs；图11a~c），以及
- 使用更纯净的二乙三胺五乙酸锌（Zn-DTPA）进行对照激发试验，以区分暴露与生物相互作用与医源性杂质伪影（图8e、12）。
3. 建立一套框架，包括潜在的缓解策略，以便在未来的环境与临床研究中更稳健地分析激发尿液数据。

**主要发现与结论：**
**Na₂Ca-EDTA杂质显著且变异度高：** 质量控制检测显示，商业化Na₂Ca-EDTA溶液中的铝（3.2~38.7 mg/L）与铁（1.9~29.0 mg/L）含量存在显著变异（表1a、图5、8a）。这种医源性金属负荷以剂量依赖的方式直接驱动尿液中铝与铁排泄升高（表1b、图6、10）。

**药代动力学建模量化医源性铝：** 药代动力学建模证实，尿液中铝有高达70%的比例源自溶液污染（图10）。

**高斯混合模型（GMM）揭示差异化影响：** GMM识别出铝与铁均存在两种不同的尿液排泄峰（最优组分数量N=2）。对于铝，两种组分对整体分布的贡献大致相当；而对于铁，高浓度组分占据主导地位（图11a和b）。相比之下，铀呈现单组分分布（最优N=1），未发现EDTA溶液中直接的铀污染显著改变其群体分布的证据（图11c）。

**铝-铀相互作用主要为生物学效应：** 尽管Na₂Ca-EDTA溶液中几乎不含直接的铀污染，但在使用Na₂Ca-EDTA进行激发试验时，尿液中铝与铀始终呈现正相关（偏斯皮尔曼ρ≈0.45；图4、8c~e），即使在杂质更少的Zn-DTPA激发试验中亦是如此（斯皮尔曼ρ≈0.36；图8e、12）。这种关联性的持续性提示存在内在的铝-铀相互作用，可能是铝（内源性，以及程度较轻的医源性）通过竞争性置换转运蛋白（如转铁蛋白、白蛋白）结合位点，增强铀的排泄，这与已确立的铝生理机制一致[3~7]。

**医源性铝调节尿液金属排泄：** 环境铝与Na₂Ca-EDTA引入的医源性铝共同调控铝-铀的剂量-反应关系，并改变尿液中的金属间关联，当尿液铝浓度超过150 µg Al/g肌酐时，医源性铝组分占据主导地位。

**Zn-DTPA验证内源性铝动员：** 使用更纯净的Zn-DTPA进行激发试验，同样显示出持久且显著的铝-金属相关性（如铝-锰、铝-铁），凸显了内源性铝动员及其生物相互作用的贡献（图8e、12、Excel表格"Data_Sets_Imputation_Correlation"）。

**通过质量控制减轻医源性影响：** Na₂Ca-EDTA杂质（主要为铝，其次为铁与锰）的变异性具有显著的混杂潜力。这项多模态验证研究强调了在激发试验中关注并管理螯合剂溶液变异性的重要性。通过表征这些伪影并提出缓解方法，我们旨在为临床与环境研究场景中更稳健可靠的尿液金属排泄数据分析提供框架。对于有效的下游环境流行病学研究，缓解策略至关重要，例如排除尿液铝浓度超过指示显著医源性负荷阈值的样本（如>140 µg/g肌酐）。支持本研究结论的数据集与详细分析已随本文一并提供。

**环境铝的生物学意义：** 陡峭的初始铝-铀剂量-反应关系（图1），以及Zn-DTPA试验下持久的铝-金属相关性（图8e、12）表明，低剂量环境沉积的铝，其动员其他金属的能力强于较大剂量的医源性铝负荷。因此，未来的激发试验框架应致力于量化并校正这种基线内源性铝负荷。

**参考文献**

1) Blaurock-Busch E, Strey R, editors. Chronische Metallbelastungen – Toxikologie, Diagnose und Therapie [慢性金属暴露：毒理学、诊断与治疗] [Internet]. Norderstedt (Germany): BoD – Books on Demand GmbH; 2017. p. 155. 可从以下网址获取：https://d-nb.info/1147825653.
2) Carmine TC. The Uranium Episode (March–May 2017) in Temporal Context: Associations with CEMET Uranium, Aluminum, and Local PM10 Exposure (2016–2019) [数据集]. figshare; 2024. 可从以下网址获取：https://doi.org/10.6084/m9.figshare.27435639.v5.
3) Rahimzadeh MR, Rahimzadeh MR, Kazemi S, Amiri RJ, Pirzadeh M, Moghadamnia AA. 以中毒机制与治疗为重点的铝中毒. Emerg Med Int. 2022 Jan 11;2022:1480553. doi: 10.1155/2022/1480553. PMID: 35070453; PMCID: PMC8767391.

4) Fatemi SJ, Kadir FH, Moore GR. 血清中的铝转运：人转铁蛋白在人白蛋白与柠檬酸存在下对铝的结合. Biochem J. 1991 Dec 1;280 ( Pt 2)(Pt 2):527-32. doi: 10.1042/bj2800527. PMID: 1747128; PMCID: PMC1130580.

5) Cochran M, Coates J, Neoh S. 铝与铁离子在转铁蛋白结合位点的竞争平衡. FEBS Lett. 1984 Oct 15;176(1):129-32. doi: 10.1016/0014-5793(84)80926-6. PMID: 6489514.
6) El Hage Chahine JM, Hémadi M, Ha-Duong NT. 转铁蛋白对金属离子的摄取与释放及其与1型受体的相互作用. Biochim Biophys Acta. 2012 Mar;1820(3):334-47. doi: 10.1016/j.bbagen.2011.07.008. Epub 2011 Aug 17. PMID: 21872645.
7) Golub MS, Han B, Keen CL. 铝改变原代大鼠少突胶质细胞培养物中铁与锰的摄取及表面转铁蛋白受体的调控. Brain Res. 1996;719(1-2):72–77. doi:10.1016/0006-8993(96)00087-X.