Characterizing and Mitigating Na₂Ca-EDTA Impurity Artifacts in Provocation Testing: A Multi-Method Validation for Environmental Metal Excretion Studies

Provocation testing with chelating agents like Na₂Ca-EDTA is a widely used clinical tool that enhances the detection of urinary trace metals, offering promise for studying links between body burdens and environmental exposures. However, this methodology can be significantly confounded by impurities in the chelating solutions themselves, potentially leading to a misinterpretation of results, especially in sensitive applications such as environmental epidemiology.This study offers a comprehensive investigation into the impact of multi-element metal contamination on post-provocation urinary metal profiles within compounded pharmagrade Na₂Ca-EDTA solutions. Our work was initially motivated by unexplained spikes in urinary Uranium and complex inter-metal correlation patterns observed in clinical practice, particularly strong associations involving Aluminum, which raised concerns about iatrogenic influences.Using a retrospective analysis of ~1600 patient samples undergoing a standardized Chelation-Evoked Metal Excretion Test (CEMET; sequential i.v. glutathione, Na₂Ca-EDTA, DMPS), we first quantified elemental impurities in chelation solutions, confirming Na₂Ca-EDTA as the primary source. We then employed a multi-method approach to dissect these effects:<b>Pharmacokinetic (PK) modeling</b> to estimate the fraction of urinary metals attributable to exogenous contamination from the solution.<b>Gaussian Mixture Models (GMM)</b> to characterize the number and location of urinary excretion peaks for key metals (Aluminum, Iron, Uranium).<b>Extensive correlation analyses</b> to compare Aluminum, Iron, and Uranium behavior under Na₂Ca-EDTA versus a "cleaner" Zn-DTPA/DMPS provocation, aiming to differentiate iatrogenic artifacts from endogenous biological interactions.<b>Key Findings &amp; Conclusions:</b><b>Significant and highly variable Na₂Ca-EDTA Impurities:</b> Quality control assays revealed highly variable Aluminum (Al) (e.g., 3.2–38.7 mg/L) and Iron (Fe) (e.g., 1.9–29.0 mg/L) content in commercial Na₂Ca-EDTA solutions. This iatrogenic metal load directly drives elevated urinary Al and Fe excretion in a dose-responsive manner.<b>PK Modeling Quantifies Iatrogenic Al:</b> PK modeling attributed a substantial portion (up to 70%) of urinary Al to this solution-derived contamination.<b>GMMs Reveal Differential Impact:</b> GMM analysis identified two distinct urinary excretion peaks for both Al and Fe (N = 2 optimal), consistent with a baseline population and an iatrogenically impacted group. In contrast, Uranium (U) displayed a single-component distribution (N = 1 optimal), with no evidence of direct U contamination from EDTA solutions significantly altering its population distribution.<b>Al-U Interaction is Primarily Biological:</b> Despite negligible direct U contamination in Na₂Ca-EDTA solutions, a consistent positive correlation between urinary Aluminum and Uranium is observed with both Na₂Ca-EDTA provocation (Partial Spearman ρ≈0.45; Fig. 4) and even with 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.<b>Iatrogenic Al Modulates Other Metal Excretion:</b> The high iatrogenic Al 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.<b>Zn-DTPA Confirms Endogenous Al Mobilization:</b> Provocation with cleaner Zn-DTPA reveals persistent, robust Al-metal correlations (e.g., Al-Mn, Al-Fe), highlighting the contribution of endogenous aluminum mobilization and its biological interactions.<b>Mitigation Strategy is Essential:</b> The variability of Na₂Ca-EDTA makes it a major confounder. 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).This multi-method validation highlights the crucial 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. The datasets and detailed analyses supporting these conclusions are provided herein