Chethaka L.
Kahakachchi
* and
Dennis A.
Moore
Covidien, Imaging Solutions, 675 McDonnell Blvd, Hazelwood, MO 63042, USA. E-mail: chethaka.kahakachchi@covidien.com
First published on 4th June 2010
The gadolinium species present in a rat kidney following intravenous administration of a gadolinium-based magnetic resonance contrast agent (Optimark™, Gadoversetamide injection) to a rat was examined in the present study. The major gadolinium species in the supernatant of the rat kidney tissue extracts was determined by reversed-phase liquid chromatography with online inductively coupled plasma optical emission spectrometry (HPLC-ICP-OES). The identity of the compound was established by liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) detection. The principal gadolinium(III) complex in a rat kidney tissue extract was identified as Gd-DTPA-BMEA 24 Hrs and 7 days after a single intravenous injection of Optimark™ (gadoversetamide; Gd-DTPA-BMEA) at a dose of 5 mmol Gd/kg body weight. The study demonstrated for the first time the feasibility of the use of two complementary techniques, HPLC-ICP-OES and HPLC-ESI-MS to study the in vivo behavior of gadolinium-based magnetic resonance contrast media.
Fig. 1 Structures, names and abbreviations of gadolinium(III) complexes.‡ |
Several in vivo studies have shown gadolinium deposition in various organs of animals5–8 and humans9,10 following intravenous administration of these MRCM. Though these studies describe the distribution of the gadolinium, in vivo, they have not determined the form of the gadolinium measured in the various tissues analyzed. Deposited gadolinium could be present in a variety of forms such as a phosphate salt or a complex with endogenous potential ligands such as proteins or carbohydrates. Indeed, the deposited gadolinium may be the sequestered form of the original complex present in the MRCM due to ultrastructural changes associated with vacuolization in the kidney.11,12 Recently several publications have suggested a potential association between gadolinium-based MRCM and nephrogenic systemic fibrosis (NSF), although the exact cause of NSF or mechanisms are yet to be established.13–16 At present NSF is considered as a serious late adverse reaction in patients with severe to terminal renal insufficiency.17 Since the mechanism by which some gadolinium-based MRCM might trigger NSF has not yet been elucidated several researchers have attempted to identify presence of gadolinium in tissue biopsies of patients with NSF. Thus, the biological fate of systemically distributed gadolinium could be of relevance to elucidate a potential link with NSF.
One major limitation with these studies and all others looking at the biodistribution of Gd in tissues is the difficulty of identifying the form of the Gd present. For example, scanning electron microscopy/energy dispersive X-ray spectroscopy (SEM/EDS) has been used for the identification and determination of deposits of Gd and co-precipitated elements in tissue biopsies of patients with NSF.18,19 However, this procedure detected only insoluble elements in the tissues and was reported that any water soluble forms of gadolinium may not be detected due to the sample preparation procedure used by the technique. Bussi et al. have reported the distributed gadolinium content of rat tissues after a single intravenous administration of 1 mmol kg−1 of MRCM (MultiHance, Omniscan and Gadovist), following sacrifice after 24 h post injection. The total gadolinium content after microwave digestion of tissues varied considerably with higher concentrations found in the kidneys, liver, spleen and bone.7 In several of these studies, including this study, researchers have determined the total gadolinium content present in the tissues examined as Gd3+ using inductively coupled plasma optical emission spectrometry (ICP-OES) or inductively coupled plasma mass spectrometry (ICP-MS), these techniques by themselves cannot reveal the form of the gadolinium measured.7–10,20,21 Radiochemical detection and quantification using radioactive 153Gd has also been used to observe the biodistribution of 153Gd-DTPA.6 A dual labeling study of 153Gd-DTPA and Gd-[14C]-DTPA in rats suggested that the gadolinium present in liver, spleen and bone was no longer associated with the original 14C-labeled ligand. Once again, the actual form of the gadolinium was not determined by this approach.22 Behra-Miellet et al. published a procedure for identifying gadolinium(III) complexes in blood samples to study the interaction of contrast agents with neutrophils in blood by HPLC-electro spray ionization mass spectrometry (ESI-MS) based on the gadolinium isotopic distribution.23
Since none of these techniques have been able to determine the form of the gadolinium species present in biological tissues after injection, the present study set out to determine if the form of gadolinium species present in biological tissue is that of the original complex found in the MRCM injected. This technique relies on analyzing tissue extracts allowing determination of the gadolinium(III) complex identity by observation of it's retention time on reversed-phase high-performance liquid chromatography. To enable the sensitive detection of the eluting gadolinium complex, this technique utilizes inductively coupled plasma optical emission spectrometry (HPLC-ICP-OES).24
The importance of the extraction technique employed must be noted. The technique must be one which does not alter the retained gadolinium species in tissues. But rather, merely releases it from the biological sample. There are only a limited number of procedures published for gadolinium(III) complex extraction from biological specimens. Tetramethylammonium hydroxide (TMAH) was used to extract Gd-DTPA2− from a patient's hair following intravenous administration of the corresponding MRCM (Magnevist). However, this resulted in partial decomposition of the complex introducing a potential source of error in the determination of Gd-DTPA2− present in the sample.25
This report presents for the first time the identification of the form of a gadolinium species present in rat kidney extracts following intravenous administration of a MRCM (Optimark™). The water extractable gadolinium species in the tissue extracts were determined by HPLC-ICP-OES and the identity confirmed by HPLC-ESI-MS. Furthermore, in this work the extraction potential of Gd-DTPA2−, Gd-DOTA1−, Gd-HP-DO3A, Gd-DTPA-BMA, Gd-DTPA-BMEA, Gd-BOPTA2− and free Gd3+ from spiked samples of bovine muscle tissue were investigated.
ICP-OES Parameters | |
Forward power | 1.20 kW |
Plasma flow | 15.0 L min−1 |
Auxiliary flow | 1.5 L min−1 |
Nebulizer flow | 0.80 L min−1 |
Analysis wavelength | Gd 342.246 nm |
Replicates | 1 |
Dwell time | 1000 ms |
Acquisition mode | TRS (Transient Real time Signal) |
HPLC Parameters | |
(A) Gd-DTPA-BMEA, Gd-DTPA2−and Gd-HP-DO3A | |
Column | Waters XTerraTM 5 μm RP-C18 (4.6 × 250 mm) |
Mobile phase | 10 mM ammonium acetate in water |
(B) Gd-DTPA-BMA and Gd-DOTA1− | |
Column | Phenomenex SynergiTM 4 μm RP-Hydro C-18 (4.6 × 250 mm) |
Mobile phase | 10 mM ammonium acetate in water |
(C) Gd-BOPTA2− | |
Column | Thermo BDS HypersilTM 3 μm RP-C18 (4.6 × 50 mm) |
Mobile phase | 10 mM ammonium acetate in water with 5% acetonitrile |
Flow rate | 1 mL min−1 |
Injection volume | 50 μL |
Column temperature | Ambient |
An Agilent 6130 single quadrupole HPLC-MS equipped with an API-electrospray interface controlled by ChemStation software (version B.02) was used for data acquisition and processing. The ESI-MS was connected to an Agilent 1200 series High Performance Liquid Chromatographic system (all from Agilent Technologies, CA, USA), consisting of a G1322A degasser, a G1312A quaternary pump, a G1315B diode array detector, a G1316A column compartment, a G1313A autosampler and a variable injector (capable of injecting 1 to 100 μL volumes) for mass spectrometry analysis of the gadolinium(III) complexes. The mass spectrometer was operated in the full scan mode with positive ion polarity, with the following settings: mass range 120 to 1000 m/z, drying temperature 350 °C, nebulizer pressure 35 psi, drying gas flow 10 L min−1, fragmentor 70 and capillary potential 3000 V.
A CEM MARS Xpress closed vessel digestion system with 55 mL PFA vessels (all from CEM Corporation, NC, USA), a VirTis bench top 4.0 K ZL freeze dryer (SP Industries, NY, USA), LABQUAKE® shaker & rotator (Barnstead/Thermolyne, IA, USA), ultrasonic bath (Branson Ultrasonic Corp., CT, USA) and a Centrific® bench top centrifuge (Fisher Scientific, NJ, USA) were used for biological sample preparation.
The standard reference material investigated for the MRCM spike study was NIST SRM 8414, Bovine Muscle Powder (National Institute of Standards and Technology, USA), a lyophilized powder with certified element content for major, minor and trace constituents.
The 1000 mg L−1 gadolinium certified standard was diluted appropriately to prepare calibration working standards for Gd (0.1 to 25 μg mL−1). Yttrium was used as an internal standard for the analysis. Individual working standard stock solutions of gadolinium(III) complexes were prepared by appropriately diluting the 0.5 M gadolinium-based MRCM solutions with de-ionized water in volumetric glassware for HPLC-ICP-OES analysis. For the bovine muscle powder spike recovery study 5 mM of the gadolinium(III) complexes were prepared by diluting 1 mL of the 0.5 M MRCM solution in 100 mL volumetric flasks with de-ionized water.
Rat kidney tissue—the freeze dried rat kidney tissue samples were placed in pyrex glass containers and mechanically crushed by grinding with a glass rod until a fine powder was obtained. The powdered rat kidney tissue samples (0.05 g) were accurately weighed into 15 mL centrifuge tubes for the water extraction, 3 mL of de-ionized water was added and then sonicated for 60 min. The homogenate was then centrifuged at 1500 × g for 5 min at room temperature and the supernatant was recovered and used for total gadolinium determination by ICP-OES. The samples were diluted with water and filtered through a PTFE 0.45 μm filter (Phenomenex, CA, USA) prior to Gd speciation analysis by HPLC-ICP-OES. The sample extracts were stored in the dark at 0–4 °C until analysis.
Rat kidney tissue—for total gadolinium determination 0.05 to 0.1 g of the samples were accurately weighed into the microwave vessels with 2 mL of HNO3 acid and 1 mL of de-ionized water. The samples were subjected to microwave assisted acid digestion by operating the microwave at 1600 W (100% power) ramping to 180 °C over 15 min and then holding at 180 °C for 15 min. The clear solutions were transferred and made up to volume with de-ionized water in 5 mL volumetric flasks with Y3+ as an internal standard.
The total gadolinium concentrations of these samples after microwave assisted acid digestion of samples and after water extractions were measured by ICP-OES are given in Table 2. Microwave assisted acid digestion completely destroys the matrix allowing quantitative determination of total gadolinium content. A mild extraction would preserve the species of interest while allowing the identification of the form of the gadolinium species by HPLC. The spike recoveries for the six gadolinium(III) complexes and free Gd3+ after microwave digestion and total Gd determination were between 96 and 106%. The water extractable gadolinium accounted for about 48–70% of total gadolinium for the different gadolinium(III) complexes. However, only 5% gadolinium was water extractable as free Gd3+. The results for the MRCM and Gd3+ extraction from bovine muscle tissue are shown in Fig. 2. Additional extraction for 24 h on a rotator at room temperature did not improve the extraction efficiency. Filtration through a 0.45 μm filter also did not change the total gadolinium content in the supernatant. Extraction with a solvent like water should release free or loosely bound gadolinium species, however if the gadolinium species is present as a Gd3+ salt or tightly bound to proteins for example its release would be more difficult. The variations in extractability of the different MRCM under similar extraction conditions do not appear to be related to the charge of the complex and is not fully understood presently. Ultrasonic extractions with water and enzymes have been used to extract a variety of elements from biological samples.26,27 Further study of extraction conditions for Gd-DTPA-BMEA from spiked bovine muscle tissue using water and ultrasonic energy is needed to optimize the extraction procedure.
Fig. 2 Gadolinium extraction from MRCM spiked bovine muscle powder using water as an extractant, % of water extractable gadolinium based on total gadolinium content after microwave assisted acid digestion; error bars ± S.D. (n = 3). |
Spiked sample | Quantity of Gd added (μg) | Total Gd (μg g−1)a | % Recovery | ||
---|---|---|---|---|---|
Microwave digestion | Extraction | Microwave digestionc | Extractiond | ||
a The total Gd content mean ± standard error (n = 3), where the μg g−1 results are based on dry tissue weight. b The limit of detection (LOD) was calculated to be 0.5 μg g−1. c % Recovery based on the theoretical total Gd concentration 314.5 μg g−1 spiked for the different MRCM and 400 μg g−1 for the spiked Gd3+. d % Recovery extraction = (total Gd from extraction)/(total Gd from microwave digestion) × 100. | |||||
Blank | 0 | <LODb | <LODb | — | — |
Optimark™ | 157.25 | 333.7 ± 14.5 | 149.5 ± 10.4 | 106 | 48 |
Magnevist™ | 157.25 | 336.0 ± 2.3 | 169.7 ± 13.6 | 107 | 51 |
ProHance™ | 157.25 | 320.3 ± 11.5 | 154.3 ± 1.4 | 102 | 48 |
Omniscan™ | 157.25 | 304.0 ± 3.2 | 188.9 ± 8.3 | 97 | 62 |
Dotarem™ | 157.25 | 302.1 ± 14.9 | 210.5 ± 24.1 | 96 | 70 |
MultiHance™ | 157.25 | 310.3 ± 13.3 | 151.1 ± 18.2 | 99 | 49 |
Gd3+ | 200 | 397.3 ± 8.3 | 19.2 ± 10.3 | 99 | 5 |
Treatment | Time pointa | Sample | Gd content (μg g−1) | Average Gd content (μg g−1)b |
---|---|---|---|---|
a Post injection tissue collection time point. b Mean ± standard error (n = 3), where the μg g−1 results are based on dry tissue weight, the limit of detection (LOD) was calculated to be 0.5 μg g−1. | ||||
10.0 mL/kg saline injection | 24 Hrs | Rat Kidney 1 | <LOD | <LOD |
Rat Kidney 5 | <LOD | |||
Rat Kidney 8 | <LOD | |||
7 days | Rat Kidney 11 | <LOD | <LOD | |
Rat Kidney 13 | <LOD | |||
Rat Kidney 16 | <LOD | |||
5 mmol/kg Optimark injection | 24 Hrs | Rat Kidney 2 | 1884 | 1600 ± 166 |
Rat Kidney 4 | 1605 | |||
Rat Kidney 9 | 1310 | |||
7 days | Rat Kidney 10 | 687 | 673 ± 151 | |
Rat Kidney 15 | 404 | |||
Rat Kidney 18 | 927 |
Rat Kidney 9 | Rat Kidney 18 | |
---|---|---|
a Mean ± standard error for Rat Kidney 9 (n = 2) and Rat Kidney 18 (n = 4) total Gd content, where the μg g−1 results are based on dry tissue weight. b Mean ± standard error (n = 3) for the % amounts in the water extracts, residues and sum. | ||
Post injection tissue collection time point | 24 Hrs | 7 days |
Total Gd content in tissue (μg g−1)a | 1351 ± 41 | 970 ± 31 |
Extracted amount (%)b | 81 ± 5 | 77 ± 2 |
Residual amount (%)b | 21 ± 2 | 30 ± 7 |
Sum of extracted and residual amounts (%)b | 102 ± 5 | 107 ± 8 |
Fig. 3 Typical HPLC-ICP-OES chromatograms for (A) spiked bovine muscle tissue water extracts and a Optimark standard and (B) spiked bovine muscle tissue water extract and a ProHance standard (seeTable 1for separation conditions). |
Fig. 4 (A) HPLC-ICP-OES chromatogram from a rat kidney tissue (Rat Kidney 2) extract after a single intravenous injection of 5 mmol/kg Optimark injection and a Optimark standard (seeTable 1for separation conditions) (B) Mass spectra extracted at the retention time of the (A) peak. |
As shown in Fig. 5, similar gadolinium HPLC-ICP-OES elution profiles were obtained for the rat kidney tissue extracts (water extraction using sonication) 24 Hrs and 7 days post injection of Optimark. Retention times were matched with the Optimark standard HPLC retention time. In the water extracts of rat kidney tissue after Optimark injection, Gd-DTPA-BMEA appeared as the major gadolinium compound (Table 5). The Gd-DTPA-BMEA content in the water extracts were determined by external calibration of 0 to 500 μM of Gd-DTPA-BMEA using the peak area mode and linear regression analysis (correlation coefficients greater than 0.995 and %RSD for standards and samples less than 5%). The Gd-DTPA-BMEA content in the rat kidney tissues 24 Hrs after Optimark injection was higher than the Gd-DTPA-BMEA content present 7 days post injection, consistent with the total Gd results determined above. Again, approximately 80% of total gadolinium from the rat kidney tissues extracted was present as Gd-DTPA-BMEA. These results indicate that gadolinium in the rat kidney tissues is primarily present as the original gadolinium(III) complex and is consistent with vacuolization of the contrast agent in the kidney.29 Extraction and determination of gadolinium(III) complexes quantitatively from a wide range of rat tissue samples after different MRCM dosing regiments may provide valuable information for in vivo bio-distribution studies, therefore future studies will focus on further development of this analytical methodology for other tissues. For example, several recent studies have shown gadolinium retention in the skin of rats8,30 and in the skin of patients31 with NSF. Since these results are based on total gadolinium measurement alone to reveal the form of the gadolinium species it would be necessary to perform speciation studies using chromatographic separation and element specific detection methods such as HPLC-ICP-MS.
Fig. 5 HPLC-ICP-OES chromatograms from rat kidney tissue extracts (Rat Kidney 9 and Rat Kidney 18) after a single intravenous injection of 5 mmol/kg Optimark injection (seeTable 1for separation conditions). |
Sample I.D. | Time point | Total Gda (μg g−1) | Gd-DTPA-BMEAb | ||
---|---|---|---|---|---|
Peak area (%) | Conc. (μg g−1) | % Total Gdc | |||
a Mean ± standard error for Rat Kidney 9 (n = 2) and Rat Kidney 18 (n = 4) total Gd content, where the μg g−1 results are based on dry tissue weight. b Mean ± standard error (n = 3), where the μg g−1 results are based on dry tissue weight. c The % total Gd in the extracts based on total Gd from microwave digestion. | |||||
Rat Kidney 9 | 24 Hrs | 1351 ± 41 | >99 | 1163 ± 75 | 86 ± 6 |
Rat Kidney 18 | 7 days | 970 ± 31 | >99 | 761 ± 10 | 78 ± 2 |
Footnotes |
† Electronic supplementary information (ESI) available: Optimization of ultrasonic water extraction procedure for total gadolinium determination in Optimark™ spiked bovine muscle tissue. See DOI: 10.1039/b915806e. |
‡ Trademarks are the property of their respective owners. |
This journal is © The Royal Society of Chemistry 2010 |