Gadolinium Retention in Erythrocytes and Leukocytes From Human and Murine Blood Upon Treatment With Gadolinium-Based Contrast Agents for Magnetic Resonance Imaging Enza Di Gregorio, PhD, Chiara Furlan, MSc, Sandra Atlante, MSc, Rachele Stefania, PhD, Eliana Gianolio, PhD, and Silvio Aime, PhD Objectives: Being administered intravenously, the tissue that gadolinium-based contrast agents (GBCAs) for magnetic resonance imaging mostly encounter is blood. Herein, it has been investigated how much Gd is internalized by cellular blood components upon the in vitro incubation of GBCAs in human blood or upon intravenous administration of GBCAs to healthy mice. We report results that show how the superb sensitivity of inductively coupled plasma–mass spec- trometry (ICP-MS) allows the detection of very tiny amounts of GBCAs entering red blood cells (RBCs) and white blood cells (WBCs). This finding may intro- duce new insights in the complex matter relative to excretion and retention path- way of administered GBCAs. Materials and Methods: The study was tackled by 2 independent approaches. First, human blood was incubated in vitro with 5 mM of GBCAs (gadoteridol, gadobenate dimeglumine, gadodiamide, and gadopentetate dimeglumine) for variable times (30 minutes, 1 hour, 2 hours, and 3 hours) at 37°C. Then, blood cell components were isolated by using the Ficoll Histopaque method, washed 3 times, mineralized, and analyzed by ICP-MS for total Gd quantification. Further- more, blood components derived from human blood incubated with gadodiamide or gadoteridol underwent UPLC-MS (ultra performance liquid chromatography– mass spectrometry) analysis for determination of the amount of intact Gd-DTPA- BMA and Gd-HPDO3A. Second, the distribution of Gd in the blood components of healthy CD-1 mice was administered intravenously with a single dose (1.2 mmol/kg) of gadodiamide or gadoteridol. Blood samples were separated and processed at different time points (24 hours, 48 hours, 96 hours, and 10 days after GBCA administration). As for human blood, ICP-MS quantification of total Gd and UPLC-MS determination of the amount of intact GBCAs were carried out. Results: The amount of Gd taken up by RBCs and WBCs was well detectable by ICP-MS. The GBCAs seem to be able to cross the membrane by diffusion (RBCs) or, possibly, by macropinocytosis (WBCs). Ex vivo studies allowed it to be established that the structure of the different GBCAs were not relevant to determine the amount of Gd internalized in the cells. Although the amount of Gd steadily decreases over time in gadoteridol-labeled cells, in the case of gadodiamide, the amount of Gd in the cells does not decrease (even 10 days after the administration of the GBCA). Moreover, while gadoteridol maintains its structural integrity upon cellular uptake, in the case of gadodiamide, the amount of intact complex markedly decreases over time. Conclusions: The detection of significant amounts of Gd in RBCs and WBCs indicates that GBCAs can cross blood cell membranes. This finding may play a role in our understanding of the processes that are at the basis of Gd retention in the tissues of patients who have received the administration of GBCAs. Key Words: gadolinium, erythrocytes, leukocytes, blood, MRI, GBCAs (Invest Radiol 2020;55: 30–37) G adolinium-based contrast agents (GBCAs) are extensively used in clinical practice as they add physiological information to the ana- tomical images provided by the magnetic resonance imaging tech- nique. 1 Gadolinium-based contrast agents were thought to be among the safest compounds until the discovery of nephrogenic systemic fibro- sis (NSF), a rare disease that affects primarily the skin and joints. Al- though the ethiology of the disease is not completely elucidated, in 2006, a causality link between NSF and GBCAs in patients with end- stage renal disease was established. 2 A number of observations pointed to the association of lower kinetic stability of certain linear GBCAs to their long retention time in the blood of patients with severe renal impairment. 3 Avoiding the administration of low stability linear GBCAs to patients with low glomerular filtration rates, the cases of GBCAs-related NSF have drastically decreased. In 2014, a new source of concern 4 was raised after the report of Kanda et al, 5 who showed an increased signal intensity in the dentate nucleus and in the globus pallidus of patients who have received several GBCA admin- istrations. Furthermore, it was shown that the observed hyperintensity can be associated with tiny amounts of Gd-containing species retained in the involved brain structures. 6 To gain more insight into the occurring mechanisms that lead to Gd retention, a number of studies have been undertaken at preclinical level by administering multiple doses of GBCAs to mice and rats. 7–15 These studies brought important insights about the way through which the GBCAs may accesses the CNS interstitium at the choroid plexus and enter the glymphatic route in its slow excretion process. 8,16–18 During this process, GBCAs endowed with lower kinetic stability may yield chemical transformations that eventually result in the for- mation of insoluble Gd-containing deposits that remain unaltered over long periods. 11,14 Conversely, the higher stability of the macro- cyclic GBCAs allows these systems to remain intact, thus allowing a relatively faster transit pathway. Although no clinical consequence associated with Gd retention has yet been reported, the number of studies developed in the last few years provide new knowledge on how a metal-containing xenobiotic may interact with complex bio- logical matrices. 19 Still, a number of questions have to be tackled. Among them, we 7 and others, 20–23 have been interested to know to what extent Gd may be retained in tissues other than the brain. By analyzing the Gd content in organs and tissues of mice administered with multiple doses of GBCA, it is evident that Gd is present at concen- trations much higher than those found in the brain. In particular, an en- hanced retention was observed in the excretion organs such as the Received for publication June 4, 2019; and accepted for publication, after revision, July 9, 2019. From the Department of Molecular Biotechnologies and Health Sciences, University of Turin, Turin, Italy. Silvio Aime is the senior author of this study. Conflicts of interest and sources of funding: The study was supported by the “Progetto di Ateneo Compagnia di San Paolo” (CSTO160182), EuroBioimaging Italy CNR, and “Regional project Gadoplus (IR2) Industrializzazione dei Risultati della Ricerca (F.E.S.R. 2014/2020).” E.D.G. was supported by FIRC-AIRC (Fondazione Italiana per la Ricerca sul Cancro AIRC) fellowship. The other au- thors declare no conflict of interest. Supplemental digital contents are available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Web site (www.investigativeradiology.com). Correspondence to: Eliana Gianolio, PhD, Department of Molecular Biotechnologies and Health Sciences, University of Turin, Via Nizza 52, Turin, Italy. E-mail: eliana.gianolio@unito.it. Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved. ISSN: 0020-9996/20/5501–0030 DOI: 10.1097/RLI.0000000000000608 ORIGINAL ARTICLE 30 www.investigativeradiology.com Investigative Radiology • Volume 55, Number 1, January 2020 Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.