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ORIGINAL ARTICLE

Comparison of Gd DTPA-BMA (Omniscan) versus Gd HP-DO3A (ProHance) Retention in Human Bone Tissue by Inductively Coupled Plasma Atomic Emission Spectroscopy
Wendell A. Gibby, MD,* Krissa A. Gibby,† and W. Andrew Gibby‡

Rationale and Objectives: Human bone tissue was collected following administration of a clinical dose of gadolinium chelate (0.1 mmol per kg) to patients undergoing hip joint replacement surgery to determine if measurable differences in Gd deposition occur between 2 widely available magnetic resonance contrast agents. Materials and Methods: Gd HP-DO3A (ProHance), Gd DTPABMA (Omniscan), and an age-matched control population without history of gadolinium chelate administration were compared. Bone samples were collected fresh, placed in refrigeration, and subsequently frozen. Tissue digestion was performed using a microwave tissue digester and concentrated nitric acid. A method of tissue analysis was created for gadolinium using inductivity coupled plasma atomic emission spectroscopy (ICP-AES). Results: Tissue retention was 1.18 Ϯ .787 ␮g Gd/g bone (N ϭ 10) for Omniscan and 0.466 Ϯ .387 ␮g Gd/g bone (N ϭ 8) for ProHance measured by ICP-AES. Conclusion: Omniscan (Gd DTPA-BMA) left 2.5 times more Gd behind in bone than did ProHance (Gd HP-DO3A). Key Words: human bone gadolinium retention, gadolinium ICPAES, Gd HP-DO3A, Gd DTPA-BMA (Invest Radiol 2004;39: 138 –142)

adolinium (Gd) chelates are widely used as contrastenhancing agents for magnetic resonance imaging. The structure of the chelate in part determines the kinetic lability of the metal ligand and, in turn, can affect the release of the metal in vivo. Numerous in vitro studies have indicated

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Received August 29, 2003 and accepted for publication, after revision, November 23, 2003. From the *Riverwoods Advanced Imaging Center, Provo, Utah; the †Department of Tumor Biology, Georgetown University, Washington, DC; and ‡Magnetic Research Inc., Provo, Utah. Reprints: Wendell A. Gibby, MD, Riverwoods Advanced Imaging Center, 280 W. Riverpark Dr., Ste. 100, Provo, UT 84604. E-mail: wgibby@novared.net Copyright © 2004 by Lippincott Williams & Wilkins ISSN: 0020-9996/04/3903-0138 DOI: 10.1097/01.rli.0000112789.57341.01

differences in Gd chelate affinities and in Gd chelate kinetic labilities. These can be affected by a variety of factors, including the pH, the availability of other metal ions that compete with the Gd on the chelate, and the structure of the chelate. Gd DTPA-BMA is a substituted open-chain chelate which, although having somewhat similar thermodynamic stability1 to the ring compound Gd HP-DO3A, has vastly different kinetic stabilities as a result of the relatively rigid ring structure of HP-DO3A.2 Animal studies have indicated repeatedly an increase in Gd retention for the more labile Gd DTPA-BMA material both as a chemical entity and as formulated for clinical use. However, there have been no human studies to date that have attempted to demonstrate a significant difference in Gd retention. The retention of Gd could be important clinically, because Gd is not a naturally occurring biologic constituent and, once within the tissues of animals, persists for long periods of time.3 It has significant toxicities, both in in vitro and in vivo experiments.4 – 6 For example, it is the most potent calcium antagonist known.7,8 Gd has the potential of leeching into membranes, bone, and enzymatic structures, causing as-yet undetermined long-term consequences. Therefore, the release of Gd into the human body is of significant clinical interest.3,9,10 This study was undertaken to compare 2 U.S. Food and Drug Administration-cleared, commonly used Gd-based magnetic resonance imaging chelates, Gd DTPA-BMA and Gd HP-DO3A, in their retentive properties in human bone tissue. Human bone tissue was selected for 2 reasons: 1) it is available in certain orthopedic procedures, whereas other tissues such as the liver, spleen, and so on, are not readily acquired; and 2) bone is one of the target organs in which Gd retention occurs.

METHODS
Patients undergoing a total hip arthroplasty with removal of the femoral head were enrolled after informed consent. Gd DTPA-BMA or Gd HP-DO3A was injected
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intravenously at a dose of .1 mmol/kg not less than 3 days and not more than 8 days before surgery. The study was performed under the auspices of the Institutional Review Board. Table 1 indicates the age distribution and timing of the Gd injection between the 2 groups. An age-matched control population undergoing hip replacement was also obtained. The femoral heads were cut in half. Half of the tissue sample was sent to Magnetic Research Incorporated, Provo, Utah, for analysis. The other half was sent to Bracco Research for independent analysis.

Experimental ICP-AES Equipment and Materials
Argon from Praxair: 5.0 ultra-high purity. Composition: hydrocarbons less than 1 part per million, oxygen less than 2 parts per million, and moisture less than 3 parts per million. ICP AES Instrument: Varian Liberty Series 2, Plasma 96, Fischerbrand-approved pump tubes. Internal diameter: 0.051. Manual sample tube aspiration connected to a short piece of Teflon tubing. Software version 1.12. Operating conditions: Nebulizer set at 180 psi. Gas inlet pressure set at 100 psi. Integration time: 1 second. Peak tracking window: 0.080 nm. Replicates: 3. Grating order: 1. Power: 1 kW. Viewing height: 10 mm. General settings: Scan window first order 0.12 nm. Photo multiplier tube voltage: 640. Plasma flow: 10.5 L/min. Auxiliary flow: 0.75 L/min. Introduction settings: Sample uptake delay: 10 seconds. Pump rate: 15 RPMs. Instrument stabilization delay: 5 seconds. Rinse time: 300 seconds. Elemental analysis parameters for Gd were set at 1-second integration time with a polynomial-plotted background correction and an analog wavelength of 342.247 nm. The parameters for Yttrium were 1-second integration time with automatic background correction and an analog wavelength of 371.030 nm. Wavelength calibration and torch alignment were performed before each run. The 4-Gd standards were also run at the beginning and end of each run. Gd linearity and limit of detection was measured for standard solutions linear and reproducible to .1 ␮g Gd/g solvent (Fig. 1).

laboratory. Yttrium standard for ICP Y2O3 in 2% HNO3; 1000 ␮g/mL, gadolinium standard for ICP both from E. M. Science, Gibbstown, NJ. Yttrium stock solution was prepared by diluting 1:100 with 5 mL of the Yttrium stock at 10 ␮g/g H20 solution added to 495 mL of distilled water, creating a 10.000 ␮g Yt/g H20 solution. Gd standard calibration solutions were prepared at: 10.036, 1.0043, 05031, and .2004 ␮g Gd/g H20 in a washed 500 mL Pyrex volumetric flask at 23°C; along with 36.68 g CaCl2 ϫ 2 H2O; 0.5000 g Yttrium standard solution (1000 ␮g/mL) and sufficient water to constitute 500 mL of stock solution. Standard solutions also contained 100 mL of 70% nitric acid and 36.68 g CaCl2 per 500 mL H2O. Yttrium and Gd standards were weighed with an OHAUS Explorer analytical balance capable of measuring to 0.0001 g weighed in small plastic Dixie cups. Each bone assay was performed in triplicate.

Tissue Digestion
Bone tissue was digested by removing 1 g (Ϯ 0.001 g) from the hip bone samples. Bone samples included a slice of cortex and medullary bone, which contained marrow. We avoided the dome of the femoral bone where cartilage and degeneration were present. This was placed in a Pyrex Star System CEM Digestion Flask. Twenty milliliters of the nitric acid reagent was automatically pipetted by the CEM instrument (software version 86005/1.03). Ramp time was 5 minutes. Target temperature was 95°C and the time at temperature was 15 minutes. The bone samples were watched visually until nearly all the nitric acid boiled away. When approximately 2.0 to 3.0 mL of residual acid and dissolved bone are present, the sample is removed, 1 mL of the diluted Yttrium solution (10 ␮g/g H20) is added, and the volume of the sample is then adjusted to 10 mL with distilled water. (If the sample is not caught before complete loss of the nitric acid, significant charring occurs and the sample must be discarded.)

Reagents
The following reagents were used: micron-filtered distilled water. Nitric acid, 70% (vol/vol): with heavy metals less than 0.2 parts per million. Frozen, unprocessed human bone tissue cut with a standard hacksaw in the pathology

Recovery of Gd From Spiked Bone
The recovery analysis and limit detection of human bone tissue was performed using bone tissue spiked with varying concentrations of Gd from a single control bone between .1 and 1.0 ␮g Gd/gm bone (Fig. 2). The slope error

TABLE 1. Age Distribution and Gadolinium Timing Control (N ‫ ؍‬7) Average patient age* (years) Injection to bone harvest* 55.8 Ϯ 20.2 N/A Omniscan (N ‫ ؍‬10) 67.3 Ϯ 12.8 4.4 days Ϯ 1.4 ProHance (N ‫ ؍‬8) 63.6 Ϯ 9.7 4.5 days Ϯ .9

*There was no significant difference between groups.

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FIGURE 1.

was 16%. The recovery of Gd in the spiked control samples was 54%. The coefficient of determination (r2) was .8845. The ␮g/Gm of Gd in the bone sample is equal to measured ␮g/g of bone divided by the internal Yttrium standard, divided by the bone weight, times 10 mL of solution per 1 g bone, times the density correction factor of water. The

human data was then normalized for Gd recovery using spiked bone samples of Figure 2. The formula used was: Actual Concentration Gd ϭ Measured Gd .54 ϩ .0075

RESULTS

The detection limit of Gd for this system is greater than 0.1 but less than 0.2 ␮g/g bone.

FIGURE 2.

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Comparison of Gd DTPA-BMA vs Gd HP-DO3A Retention

Table 2 represents the normalized, measured Gd content in micrograms of gadolinium per gram of bone. We did not attempt to determine any relationship between the time after Gd injection and residual trace metal present. This was because the numbers of patients was limited, and we tightly controlled the time from injection to bone harvest; 4.4 Ϯ 1.4 days in the case of Gd DTPA-BMA and 4.5 Ϯ .9 days for Gd HPDO3A.

DISCUSSION
Despite claims to the contrary that “there seems to be no dissociation of Gd within the body,”11 all of the currently used magnetic resonance contrast agents have the potential for transmetallation in vivo. Physicians injecting such material into the body should be aware of the potential deleterious effects of free Gd. Gd is one of the most potent inorganic calcium antagonists known.6,7 High concentrations of heavy lanthanide metals in animals are known to be toxic and can cause, among other things, fatty degeneration of the liver, changes in nucleic acid, lipid and carbohydrate metabolism, writhing, ataxia, labored respiration, sedation, hypotension, and death.3 Gd metal injected intramuscularly induces sarcomas4 and “granulomatous neoplasms.”5 These agents are also known to interfere with coagulation.8,11 Unlike metals with a known biologic function, Gd does not have a known pathway for excretion from within the body. Once within the tissues, it can persist for long periods of time,6 primarily within the liver and bone. In small quantities, free Gd initially goes to the liver and is then transferred to bone with negligible elimination over the next 3 weeks.12 In the dosages given clinically, transmetallation is not known or suspected to be an acute problem, although it could be the cause of the wellknown elevated serum Fe levels reported initially with the introduction of Gd DTPA. These were subsequently corrected to a large extent by adding more free ligand to Magnevist’s formulation. However, the potential of transmetallation should be considered for long-term sequelae, and reasonable efforts should be made to select ligands that reduce this effect. One of the primary causes of transmetallation in vitro is the competition by other bioavailable metals

such as zinc and copper that attack the chelate complex. Some chelates such as Gd DTPA-BMA formulate with 5% excess calcium ligand.13 The excess chelate scavenges the bioavailable metals. Studies with human volunteers have shown that a single dose of Gd DTPA-BMA removes approximately 32% of total plasma zinc (albeit a small fraction [.09%] of the total zinc pool in the body).14 However, with repeated high dosages in subacute toxicity studies in animals, monkeys injected with Gd DTPA-BMA demonstrate all of the signs of zinc deficiency; including testicular atrophy, skin lesions with ulceration, and gastritis.15 One of the mistakes made in evaluating chelates for in vivo use is to compare thermodynamic stability constants. Thermodynamic stability is measured by titrating the metal to the chelate at a pH of approximately 11. At this pH, there are no competing hydrogen ions for the chelate and a theoretical maximum stability for the chelate is obtained. However, the more relevant stability constant for in vivo use is the conditional stability constant, which is calculated at a pH of 7.4. In the milieu of the body, there are metals that will attack the chelate and displace the Gd. The concentration of these metals depends on the microenvironment for the chelate. Furthermore, in the body, there is no equilibrium. The thermodynamic equilibrium constant calculated in vitro is simply not germane. Once a Gd ion is released, it is immediately carried away by other biologic material, being bound to membranes, enzymes, bone substrate, or other weak chelates such as citrates and phosphates. It might never “see” the chelate again. The more important aspect of chelate safety is that of kinetic stability. For example, consider 2 chelates of near-equivalent conditional thermodynamic stability: Gd HPDO3A and Gd DTPA. Thermodynamic stability measurements at a pH of 7.4 would indicate that only 1 atom of 1017 (a very small number) of Gd would be loose from the chelate at any given time. Yet a variety of studies, including in vivo transmetallation11,16 –19 and Gd retention in animals20,21 and human case reports,22–24 suggests that there is a significant difference between the propensity of these 2 chelates to give up Gd in vivo. Both chelates have the same number of coordinating sites. However, HP-DO3A is a ring compound that is

TABLE 2. Results of Gadolinium Bone Retention Control Average ␮g/g fresh bone tissue Ϫ0.117 Nϭ7 Standard Deviation .227 t Test Control v ProHance 0.0021 Gd DTPA-BMA Average ␮g/g fresh bone tissue 1.18 N ϭ 10 Standard Deviation .787 t Test Control v Omniscan 0.0004 Gd HP-DO3A Average ␮g/g fresh bone tissue .466 Nϭ8 Standard Deviation .387 t Test Omniscan v ProHance 0.0165

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quite rigid.25 For the Gd to break free, it must simultaneously break 5 to 6 coordination sites. On the other hand, Gd chelated to DTPA can break free sequentially. Thus, it is much less likely for the HP-DO3A to release its Gd. An agent such as Gd DTPA-BMA, which not only has a much lower conditional stability constant (14.9), also has less kinetic stability than HP-DO3A or Gd DTPA. It is therefore more likely to leave more Gd behind as it dissociates in the body. The half-life in the blood for an extracellular contrast agent is approximately 20 minutes. Given that, virtually all of the chelate that could be filtered out of the tissues should be gone by 4 to 5 days. Furthermore, these agents are not taken up by cells. Transmetallation experiments have shown that Gd is readily displaced from weak open chain chelates by competition with other metal ions (especially Zn and Cu) or weak chelates such as phosphate or citrate.18 Furthermore, animal experiments have shown dissociation in vivo.16 Based on this, it is likely that most of the Gd detected 3 to 8 days after administration is released from the original chelate. It is possible, although not likely, that the injected Gd chelate could be responsible for at least part of the residual in the bones. This is the first article in humans to convincingly show a significant difference in the Gd deposition between 2 commonly used magnetic resonance contrast agents. Furthermore, we have provided methodology for the measurement of residual trace metals in human bone. The potential risk from Gd release and long-term retention rises with higher dosage and increased frequency of use. This information is important to consider in certain patient populations in whom excretion of contrast is reduced (eg, renal failure or congestive heart failure) or in patients who will receive repeated exposures to Gd chelates (eg, pediatric brain tumor, patients with multiple sclerosis) or in patients in whom very little risk can be tolerated (eg, pregnant patients).

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