DOI: 10.6084/m9.figshare.29094914.v3
title: Characterizing and Mitigating Na₂Ca-EDTA Impurity Artifacts in Provocation Testing: A Multi-Method Validation for Environmental Metal Excretion Studies

description: Provocation testing with chelating agents such as Na₂Ca-EDTA is a powerful clinical tool, enhancing urinary trace-metal detection and offering a valuable window into relationships between body burden and environmental exposure. However, this heightened sensitivity comes with a caveat: trace-metal impurities within the chelating solutions themselves can significantly confound results, 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. Given that urinary aluminum concentrations are typically three orders of magnitude higher than uranium, and in the absence of other clear confounders, these co-occurrences suggested that any observed aluminum–uranium association likely arises from shared environmental exposure pathways (e.g., common particulate matter carriers, iatrogenic source) or an aluminum-driven enhancement of uranium excretion, rather than the reverse. Beyond its link with uranium, aluminum also exhibited strong positive associations with a range of essential metals (e.g., manganese, iron, chromium) and other non-essential or toxic metals (e.g., cadmium, lead), intensifying concerns about the pervasive influence of iatrogenic confounding.The narrative grew more complex with an inconsistent concurrence of aluminum and uranium: a pronounced surge in urinary uranium across multiple sites in spring 2017 occurred without a corresponding widespread rise in aluminum [1]. This discrepancy hinted at a more intricate scenario, possibly involving a dual role for aluminum—differentiating between environmental and endogenous sources, as well as iatrogenic contributions—in influencing metal excretion patterns through distinct modes of action.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 quantify multi-element impurities in compounded Na₂Ca-EDTA solutions, identifying the primary sources and magnitudes of iatrogenic metal inputs (confirming Na₂Ca-EDTA as the main contributor of Al, Fe, and Mn, consistent with previous reports [2], while DMPS and glutathione showed minimal impurities).Dissect the dual impact of aluminum—both from environmental/endogenous origins and iatrogenic Na₂Ca-EDTA contamination—on post-provocation urinary metal excretion profiles using a multi-method approach. This involved pharmacokinetic (PK) modeling, Gaussian Mixture Models (GMMs), extensive correlation analyses, and comparative provocation with cleaner Zn-DTPA to differentiate biological interactions from solution artifacts.Establish a framework, including potential mitigation strategies, for more robust analysis of provoked urine data in future environmental and clinical research.To implement this framework, we applied the following analytical approaches:<br>Pharmacokinetic (PK) modeling was employed to estimate the fraction of urinary metals attributable to exogenous contamination from the solution.Gaussian mixture models (GMM) characterized the number and location of urinary excretion peaks for key metals (aluminum, iron, uranium).Extensive correlation analyses compared the behavior of aluminum, iron, and uranium under Na₂Ca-EDTA/DMPS versus a less impurity-prone Zn-DTPA/DMPS provocation protocol, aiming to distinguish iatrogenic artifacts from endogenous biological interactions.<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. This iatrogenic metal load directly drives elevated urinary aluminum and iron excretion in a dose-responsive manner.<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.<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. 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.<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; Fig. 4) and even with the less-impurity-prone Zn-DTPA provocation (Spearman ρ≈0.36; Fig. 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.<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.<br><br><b>Mitigation strategy is essential:</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><b>Reference</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