Iron-mobilizing properties of the gadolinium–DTPA complex: clinical and experimental observations

Marina Vorobiov1,, Anna Basok1, David Tovbin1, Alla Shnaider1, Leonid Katchko2 and Boris Rogachev1

1 Department of Nephrology and 2 Department of Pathology, Soroka Medical Center, Ben Gurion University of the Negev, Faculty of Health Sciences, Beer Sheva, Israel



   Abstract
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Gadolinium (Gd) magnetic resonance imaging (MRI) contrast agents are considered to be safe in patients with impaired renal function. Our study investigates a mechanism of severe iron intoxication with life-threatening serum iron levels in a haemodialysis patient following MRI with Gd–diethylenetriaminepentaacetic acid (Gd–DTPA) administration. His previous history was remarkable for multiple blood transfusions and biochemical evidence of iron overload. We hypothesized that Gd–DTPA may have an iron-mobilizing effect in specific conditions of iron overload combined with prolonged exposure to the agent.

Methods. For the in vitro study, Gd–DTPA was added to mice liver homogenate and iron metabolism parameters were measured after incubation in comparison with the same samples incubated with saline only. For the in vivo study, an experimental model of acute renal failure in iron-overloaded rats was designed. Previously iron-overloaded and normally fed rats underwent bilateral nephrectomy by renal pedicle ligation, followed by Gd–DTPA or saline injection. Iron and iron saturation levels were checked before and 24 h after Gd–DTPA or vehicle administration.

Results. Significant mobilization of iron from mice liver tissue homogenate in mixtures with Gd in vitro was seen in the control (saline) and in the experimental (Gd) groups (513±99.1 vs 1117.8±360.8 µg/dl, respectively; P<0.05). Administration of Gd–DTPA to iron-overloaded rats after renal pedicle ligation caused marked elevation of serum iron from baseline 143±3.4 to 570±8 µg/dl (P<0.0001). There were no changes of the named parameter, either in iron-overloaded anuric rats after saline injection or in normal diet uraemic animals, following Gd–DTPA administration.

Conclusions. The combination of iron overload and lack of adequate clearance of Gd chelates may cause massive liberation of iron with dangerous elevation of free serum iron. It is highly recommended that after Gd contrast study, end-stage renal disease patients with probable iron overload should undergo prompt and intensive haemodialysis for prevention of this serious complication.

Keywords: chelate; dialysis; gadolinium; iron overload; renal failure; Gd–DTPA



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Gadolinium (Gd)-containing agents are widely used as contrast media for magnetic resonance imaging (MRI) studies and are thought to be safe [1] even in patients with impaired renal function. Although they are rapidly cleared with a half-life of <2 h in patients with normal renal function, in chronic renal failure half-life is prolonged and may exceed 30 h [2]. While haemodialysis enables more effective clearance of Gd-containing contrast material than peritoneal dialysis, lack of adequate dialysis may significantly prolong Gd clearance [3]. Possible side effects may occur due to in vivo dissociation of the Gd–ligand complex into metal ion and ligand [4]. This phenomenon is thought to launch a ligand competition reaction and metal ion exchange (transmetallation) [5,6]. Very little has appeared in the literature relating to the clinical significance of these transmetallation processes in conditions of prolonged exposure, such as occurs in end-stage renal disease (ESRD) patients. We present here our clinical observations and the results of our laboratory investigation in relation to this phenomenon.



   Subjects and methods
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Patient
A 19-year-old male presented for his routine haemodialysis treatment with headache, arthralgia and fever 1 day after he had undergone an MRI with Gd–diethylenetriaminepentaacetic acid (Gd–DTPA) because of recurrent episodes of abdominal pain. Haemodialysis was discontinued soon after it was begun because of high fever.

The patient has a history of reflux nephropathy and kidney transplant from a living related donor in 1998. Six months after kidney transplantation, non-Hodgkin's lymphoma and Kaposi's sarcoma with lymph node involvement were diagnosed. Immunosuppressive therapy was stopped and interferon (IFN) started. IFN therapy was followed by severe autoimmune haemolytic anaemia, requiring multiple blood transfusions and eventual graft rejection. Iron overload was diagnosed with values of blood ferritin >5000 ng/ml. After treatment with erythropoietin and chelation therapy by deferoxamine, ferritin levels descended to ~1000 ng/ml. Haemodialysis treatment was reinstated after rejection of the graft.

On admission his clinical examination was unremarkable. Laboratory values were consistent with those expected in haemodialysis patients. Chest X-ray was normal. Blood cultures were negative. Expanded chemistry analysis showed an extremely elevated serum iron level of 741 µg/dl, compared with 146 µg/dl several days prior to admission, which continued to rise up to 1377 µg/dl. It should be noted that transferrin values were normal. Elevated levels of liver enzymes of serum glutamic oxaloacetic transaminase (SGOT) and lactate dehydrogenase (LDH) were also found. LDH level increased to 1752 U/l 1 day after his admission, dropping to normal values (482 U/l) after 4 days. Similarly, SGOT elevated to a maximal point of 73 U/l, returning to normal levels (39 U/l) on the third day of hospitalization.

Two days after admission there was a marked rise in ferritin levels, reaching >30 000 ng/ml. After three consecutive haemodialysis sessions, iron levels dropped and returned to baseline within 1 week. The patient also improved clinically with complete remission of symptoms and fever and was discharged from the hospital.

The course of this patient's clinical status led us to hypothesize that Gd may have an iron-mobilizing effect in patients with kidney failure and severe iron overload. This may be due to the prolonged exposure to Gd as a result of impaired renal clearance in uraemic patients. In order to prove this supposition we designed two experimental models: an in vitro model of Gd–DTPA exposure with iron-rich tissue and an in vivo model of acute renal failure in iron-overloaded animals combined with Gd–DTPA administration.

Study design
In vitro study. Mouse liver was homogenized in tissue culture medium with the addition of 20% autologous serum. The samples were divided into equal aliquots in the control and the experimental tissue culture tubes. The basal level of iron-metabolism parameters was taken and 0.05 ml of 469 mg/ml solution Gd–DTPA to 10 ml of homogenate (experimental) or equal amount of saline (control) tubes were added. The tubes were then incubated in a humidified atmosphere of 5% CO2 in air at 37°C. Consecutive samples of medium after short and gentle centrifugation were taken at the beginning of the experiment and after 2, 4, 24 and 72 h of incubation. Iron and transferrin levels were measured. Serum iron levels were measured by in vitro colorimetric assay for the quantitative determination of iron in human serum on an automated clinical chemistry analyser (Roche Diagnostics, GmbH, Mannheim, Germany). Transferrin determination was performed by in vitro immunoturbidimetric assay for the quantitative determination of transferrin in human serum on automated clinical chemistry analyser (Roche Diagnostics).

All the tests were carried out according to the manufacturer's protocol.

In vivo study. Three groups were included in the experiment. In group 1 we examined Gd–DTPA action on serum iron parameters in iron-overloaded anuric rats. Group 2 animals were similarly prepared (iron overload + anuria) and treated by vehicle instead of Gd–DTPA. In group 3, Gd–DTPA was administered to normal feeding anephric rats in order to investigate the influence of Gd–DTPA on iron metabolism in renal failure without previous manipulation of iron intake.

Iron-overload rat model. Male Sprague–Dawley rats weighing 200–220 g were used. They were housed in cages at room temperature of 21–23°C, kept alight from 08.00 to 20.00 h, and were given commercial rat pellet chow with free access to water during the observation period. All animals were divided into three groups: groups 1 and 2 received iron supplementation in drinking water (as a carbonyl iron (II), 50 mg/ml solution), the total dose being 300–350 mg/rat over a period of 3 months [7]. Group 3 was given only regular diet and water.

Acute-renal-failure rat model. After intraperitoneal anaesthesia by ketamine/xylasine mixture, both kidneys of all animals were exposed from midline laparotomy and renal pedicles were ligated. Absence of urine and macroscopic appearance of ischaemic kidney evidenced adequacy of ligation. Immediately after closure of the abdominal cavity, blood samples were taken from the internal jugular vein and 0.15 mmol/kg of Gd–DTPA (groups 1 and 3) or 0.5 ml 0.9% NaCl (group 2) was injected. Twenty-four hours later the rats were again anaesthetized and blood samples were drawn by heart puncture. The animals were then sacrificed by anaesthetic overdose. Liver samples were taken for Prussian blue staining. Serum biochemistry parameters were measured: urea, creatinine, iron and transferrin levels as described previously.

Statistics
Statistical analysis of iron and transferrin levels in supernatant and in blood plasma was performed using ANOVA test followed by Tuckey's multiple comparison test. Results are expressed as means±SEM. A P-value of <0.05 was considered significant.



   Results
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 Abstract
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 Subjects and methods
 Results
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 References
 
As shown in Figure 1Go, after 72 h of exposure of liver homogenate to Gd–DTPA, iron concentrations in the supernatant compared with control tubes were 1117.8±360.8 and 513±99 µg/dl, respectively (P<0.05).



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Fig. 1.  Iron level in mice liver tissue homogenate after 24 h incubation with Gd or saline. *P<0.05.

 
In the in vivo studies, following ligation of the renal pedicle, all animals became anuric and after 24 h, serum creatinine rose to a concentration of 3.442±0.126 mg/dl from a baseline of 0.699±0.02, thus guaranteeing the absence of renal clearance of Gd. The histology of the liver from the iron-fed animals showed extensive haemosiderosis.

Plasma iron levels rose from 143±3.44 to 570±8 µg/dl before and 24 h after administration of Gd to the oral iron-loaded renal failure animals (group 1), respectively (P<0.0001). No changes were observed in the other two groups (Figure 2Go). Liver enzyme levels (data not shown) were identical in all three groups of animals.



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Fig. 2.  Serum iron level in anuric rats after Gd or saline administration: group 1, iron overload + Gd; group 2, iron overload + saline; group 3, normal fed + Gd. *P<0.0001.

 
There were no changes in transferrin levels in any of the groups.



   Discussion
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
We describe a haemodialysis patient who developed extremely high iron concentrations in the plasma. The patient had undergone an MRI investigation a few days prior to admission in which Gd–DTPA was administered as a contrast agent. We hypothesized that the iron intoxication was a result of chelation of stored iron accumulated from previous numerous blood transfusions. This hypothesis was confirmed by in vitro experiments in which a Gd-containing agent was incubated with liver homogenates and in an in vivo model of chronic iron-overloaded renal failure rats to whom Gd–DTPA had been administered.

Free Gd ion [Gd(III)] solubility is poor and can form in vivo precipitates of salts with anions phosphate, carbonate or hydroxyl, which are deposited in liver, bone and muscle. Incorporation of Gd ion within organic ligand forms an ionically stable compound (chelate) with improved solubility, tissue distribution and renal clearance, making it safe for clinical use. Many agents presently in use as contrast material have a linear polyaminocarboxylate molecule derived from ethylenediaminetetraacetic acid (EDTA). DTPA is the first derivative that contains five negatively charged carboxylate moieties. This agent is known as ‘gadopentetate dimeglumine’ (MagnevistTM) [1]. In vivo dissociation of Gd complexes into metal ion and ligand are possibly responsible for certain adverse effects of the agent. This process can be facilitated both by endogenous metals (zinc [Zn(II)], copper [Cu(II)], calcium [Ca(II)] and iron [Fe(III)]) and endogenous acids, destabilizing the complex and leading to its dissociation. Displacement of Gd from its ligand by other metals through competitive ionic binding is known as transmetallation [4].

Several chelating agents are used to remove iron from its body stores, namely deferoxamine, Ca–DTPA, Ca3Na–DTPA and EDTA. These agents differ in their sites of activity (intra- or extracellular) and potency, which is in inverse correlation with complex stability [8]. Gd–DTPA, in spite of its generic relationship with DTPA, is thought to be a very weak chelator without any known effects on iron metabolism [1,4].

Iron cations tend to be tightly bound to haemosiderin and ferritin in vivo and are consequently thought to be poor candidates for transmetallation because of their low free plasma concentration. Acidaemia, resulting from inflammation or tissue hypoxia, may promote conversion of haemosiderin to iron donor, especially in iron-overload conditions [8,9]. In renal failure, the combination of metabolic acidosis and the absence of adequate clearance of Gd-containing agent may favour clinically significant transmetallation with formation of iron–ligand complexes. Abundance of iron ions may also promote concurrent cation exchange in ligand site [4].

Simple addition of Gd–DTPA to the whole blood in our preliminary experiments did not change the iron or iron-saturation levels either immediately or after 24 h of incubation. Furthermore, we did not find changes of iron metabolism after the MRI procedure in other dialysis patients even 48 h after Gd administration (data not shown).

In vitro experiments demonstrated a significant increase of iron concentrations in the supernatant of liver homogenates incubated with Gd–DTPA as compared with experiments in which the liver homogenate was incubated with saline. This increase may indicate a process of transmetallation.

Our in vivo study showed that a combination of iron overload and prolonged Gd–DTPA exposure causes significant elevation of serum iron and iron-saturation levels in uraemic rats. This rise in plasma iron concentrations was not detected in the iron-loaded uraemic animals treated with saline only or in the uraemic non-iron-loaded animals after Gd–DTPA administration.

This observation supports our hypothesis that Gd–DTPA displays its chelating properties only in iron-overload conditions. Rapid Gd–DTPA clearance in normal renal function probably hinders the transmetallation process which, in our experiments, became more pronounced after prolonged exposure to Gd–DTPA.

Haemodialysis enables more effective clearance of Gd-containing agents in ESRD patients than peritoneal dialysis [3,10]. Lack of effective dialysis in our patient (due to fever and general discomfort) probably was the first participating factor of the subsequent clinical situation.

Abnormal liver function tests can be explained by endogenous iron liberation and its toxic effect on the liver [11]. The transient character of this abnormality renders other aetiologies for the liver impairment most unlikely.

Since elevated ferritin levels in our patient appeared only after multiple blood transfusions, a diagnosis of primary haemochromatosis is very unlikely [12,13]. Reaching the initial ferritin level after three haemodialysis sessions could indicate chelated iron (by Gd–ligand) complex elimination, since iron can be removed by haemodialysis only in chelated form [1416]. The sharp elevation of iron levels in our patient 1 day after the MRI study with Gd–DTPA and the rapid drop in iron levels after haemodialysis sessions support our theory that chelation of iron indeed occurred and that the complex iron–chelate was removed by dialysis.

We conclude from our study that prolonged exposure to Gd chelates in conditions of iron overload may cause a transmetallation phenomenon with release of iron from stores. Patients with advanced renal failure on haemodialysis who are suffering from iron overload combined with metabolic acidosis may develop significant elevation of serum iron levels with clinical signs of iron toxicity. Prompt and intensive haemodialysis is essential for the prevention of this complication.



   Acknowledgments
 
We are truly grateful to Prof. Cidio Chaimovitz for his encouragement and invaluable input, to Mrs Batia Vesler for her important contribution in the clinical pathology laboratory, and Mrs Rita Soullam for her excellent technical assistance.



   Notes
 
Correspondence and offprint requests to: Dr Marina Vorobiov, Department of Nephrology, Soroka Medical Center, Ben Gurion University of the Negev, Center of Health Sciences, PO Box 151, Beer Sheva 84101, Israel. Email: marinavorobiov{at}netscape.net Back



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 26. 2.02
Accepted in revised form: 20.12.02