DOI: 10.6084/m9.figshare.29094914.v5
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 such as Na₂Ca-EDTA is a powerful clinical tool that enhances urinary trace-metal detection and provides valuable insights into the relationships between body burden and environmental exposure. However, this heightened sensitivity comes with a caveat: trace-metal impurities within the chelating solutions can significantly confound results, which is a particular concern in precise applications like environmental epidemiology.Our investigation was initially spurred by perplexing clinical observations: repeated concurrent urinary spikes of uranium and aluminum, both non-essential and potentially toxic elements [1]. With urinary aluminum typically exceeding uranium by three orders of magnitude and no other apparent confounders, their association most likely reflects shared exposure pathways (environmental or iatrogenic) or an aluminum-driven enhancement of uranium excretion, rather than the reverse. Beyond its association with uranium, aluminum also exhibits 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 1 and 2), intensifying concerns about the pervasive influence of iatrogenic confounding.The paradoxical spring 2017 uranium spike—occurring without a general aluminum surge [1] yet seemingly potentiated by aluminum, as even minimal Al is associated with enhanced uranium excretion (Fig. 1)—suggests that aluminum’s influence on uranium excretion involves at least two components: one linked to environmental/endogenous aluminum and another to iatrogenic aluminum from chelators (Fig. 11a), 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 measured 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 [2].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, 7, 8b–d, 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>GMMs reveal differential impact:</b> GMM analysis identified two distinct urinary excretion peaks for both aluminum and iron (N = 2 optimal), consistent with a baseline population and an iatrogenically impacted group (Figs. 11a and b). In contrast, uranium displayed 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> is primarily biological: 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 other metal excretion:</b> The high iatrogenic aluminum load appears to further influence these biological interactions, contributing to the complex Al–U dose–response observed with Na₂Ca-EDTA and potentially confounding other inter-metal correlations in urine (Figs. 1 and 2).<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) 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.<br><br>2) 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>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><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.<br>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.