DOI: 10.6084/m9.figshare.29094914.v6
title: Aluminum's Complex Influence in Provocation Testing: A Multi-Method Validation Characterizing Iatrogenic Artifacts, Endogenous Interactions, and Mitigation Strategies

description: 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 & 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. 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