9 1987 by The Hurnana Press Inc. All rights of any nature, whatsoever, reserved. 0163-4984/87/1300-0229502.20
Contrast Agents for Nuclear Magnetic Resonance Imaging M. H. MENDONCA DIAS* AND PAUL C. LAUTERBUR Department of Chemistry, State University of New York at Stony Brook, Stony Brook, NY 11794 ABSTRACT Nuclear magnetic resonance imaging relies upon differences in relaxation times for much of its ability to resolve anatomical structures and to detect changes in tissue. The natural differences can be changed by the administration of paramagnetic substances, such as metal complexes and stable organic free radicals, and ferromagnetic materials, such as small particles of magnetite. Detailed studies of the chemistry and biophysics of such substances in the body are required if they are to become safe and effective contrast agents for use in medical NMR imaging. Index Entries: Nuclear magnetic resonance imaging, contrast agents for; paramagnetic metal ions and complexes; stable organic free radicals; ferromagnetic particles; medical NMR imaging; longitudinal relaxation time (T1); transverse relaxation time (T2); contrast agents; nitroxide free radicals; manganese compounds; gadolinium compounds; iron compounds.
INTRODUCTION Nuclear magnetic resonance imaging is n o w widely used in clinical diagnosis and biomedical research (1-3). Most images are made using the h y d r o g e n (proton) NMR signals from water and from the lipids in adipose tissue. Regions containing air, such as lung tissue, and hard structures, such as some bone, give relatively weak signals, and there are differences in observable water and fat content even a m o n g ordinary soft tissues. The most useful differences, however, are in the NMR relaxation times of different organs and between normal and abnormal tissues. The *Author to whom all correspondence and reprint requests should be addressed. Biological Trace Element Research
229
Vol. 13, 1987
230
Dias and Lauterbur
relaxation times are usually symbolized and described as T1, the spin-lattice relaxation time, and T2, the spin-spin relaxation time. They depend on the molecular environments of the nuclei and on their motions. It has been known since the first studies of NMR in liquids, almost 40 yr ago, that the addition of low concentrations of paramagnetic solutes could drastically shorten the relaxation times of water protons, and this phenomenon has been widely used in chemical and biochemical investigations (4). The first proposal that paramagnetic substances could be used to obtain the same results in vivo, and hence could be used as contrast agents in medical NMR imaging, was made in 1978 and accompanied by experiments on the application of the idea to the detection of myocardial infarction, using injected manganous salts (5). One of the reasons for choosing manganese was the availability of a convenient and accurate analytical method, using acid extraction and atomic absorption spectroscopy, for that element in tissue samples. Because the relaxation efficacy of an element depends on its chemical form and its chemical and physical environment, analytical data on the distribution of contrast agents in tissues is necessary if the experimental results are to be fully interpreted. Two general classes of magnetic contrast agent are known: (1) Paramagnetic ions, complexes and molecules create rapidly fluctuating, tocally inhomogeneous magnetic fields, with frequency components in a range that can cause increases in both relaxation rates RI (l/T1) and R2 (1/T2); and (2) magnetic particles, such as ferromagnetic powders, generate local nearly static magnetic field inhomogeneities that cause increases in the apparent relaxation rate R2, but not in R1. In this paper we will briefly review work done with paramagnetic compounds, as well as present some new results of work with ferromagnetic particles. The need for better analytical methods to guide these investigations will be noted.
REVIEW ON PARAMAGNETIC IONS AND MOLECULES AS CONTRAST AGENTS FOR NMR IMAGING The paramagnetic contrast agents can be either paramagnetic molecules or metal ions and their complexes (6). They can be classified, by route of administration, as intravascular agents, oral agents, and inhalation agents. We will consider these three classes separately below. Most intravascular agents have been based on manganese, as aqueous manganous ion (Mn § 2), or as its complexes with chelating agents, such as EDTA (6,7) or on gadolinium as its complex with diethylenetriamine pentacetic acid, (DTPA) (8,9). The use of other elements, such as chromium, iron, and copper, as compounds of Cr +3, Fe +3, and Cu § has also been proposed (10,11). Although there has been considerable concern about the high toxicity of hydrated, uncomplexed metal ions and their slow clearance from the body, stable chelated forms of several metBiological Trace Element Research
VoL 13, 1987
Contrast Agents for Nuclear Magnetic Resonance Imaging
231
als may be relatively nontoxic, physiologically stable, and rapidly eliminated. One such complex already being tested in humans is Gd DTPA (9). Another goal is to increase the specificity of these agents so that they will affect primarily particular organs or normal or abnormal tissues. One possible method, already investigated for enhancing the visibility of myocardial infarctions (12), is to bind the paramagnetic complexes to specific monoclonal antibodies. Unfortunately, it seems that many hundreds of metal ions must be bound to each antibody molecule to obtain a large enough effect if the amounts of bound antibody are limited by the number of antigenic sites on cell surfaces. The design and evaluation of contrast agents for medical NMR imaging is a complicated problem. Effects on relaxation rates are immediately apparent, as is acute toxicity, but the interpretation of these effects is not straightforward. The time course of uptake and elimination of the agents must be known and mechanisms understood. Possible changes in chemical form and binding are important because the relaxation effects of metals depend on their molecular environments and on molecular motion, so that total concentrations may not always be inferred from the observed relaxation effects (13-15). Furthermore, sites of deposition, toxicity, and routes and rates of elimination depend upon the chemical form of the metal, including its oxidation state. Microscopic inhomogeneities in distribution, such as differences among concentrations in intravascular, extracellular, intracellular, and subcellular spaces, may be significant, requiring sensitive microscopic analytical techniques. Some specific examples of the uses of various metal compounds are given below. Manganese, as manganous ion and as complexes, has usually been injected in doses of 0.01-0.1 mM/kg body weight for studies of myocardial ischemia and infarction (5,7,12) and of uptake and clearance by other organs. A summary of some experiments, more fully discussed elsewhere (6), is given in Table 1. Other forms of manganese used have been a Mn(III) porphyrin (16), in a study that also included the corresponding compounds of Fe(III) and Cu(II), and Mn DTPA entrapped in liposomes
(17). The DTPA complex of gadolinium (III), in dosages similar to those mentioned above for manganese, has been used in animal (18) and human (9) investigations. It appears to be less toxic than manganese complexes, perhaps because it remains undissociated in the body. As yet, studies of gadolinium distribution and relaxation efficacy in tissues similar to those done for manganese have not been reported. An attempt to achieve similar results with Cr EDTA has been published (19), but it was found that much higher dosages were required. Stable nitroxide free radicals have been proposed as paramagnetic contrast agents. They are less effective on a molar basis than are compounds for Mn +2 and Gd § 3, being rather closer to Cu § and Cr +3 in that respect (10). They seem to have very low toxicities, however, and may Biological Trace Element Research
VoL 13, I987
Dias and Lauterbur
232
TABLE 1 Manganese Concentrations and Water Proton Spin-lattice Relaxation Rates"
Organ Pancreas
Liver
Spleen
Kidney
Heart
Solution injected, dose 0.1 mM/kg MnC1, Mn EDTA Mn citrate Saline control MnC12 Mn EDTA Mn citrate Saline control MnC12 Mn EDTA Mn citrate Saline control MnC12 Mn EDTA Mn citrate Saline control MnC12 Mn EDTA Mn citrate Saline control
Range of tissue concentrations of Mn, mM/kg
R ~, s-1
0.23-0.68 0.20-0.51 0.33-1.9 (0.06y' 0.24-0.78 0.23-0.49 0.42-0.64 (0.06)" 0.08-0.54 0.03-0.09 0.02-0.07 (0.045y' 0.20-0.81 0.21-0.42 0.20-0.36 (0.05)" 0.09-0.60 0.06-0.52 0.10-0.37 (0.03y
12-28 11-21 17-26 (4y' 16-40 15-31 24-42 (5)" 4-30 4-5 3.8-4 (4)" 16-32 7.5-14 11-16 (3.6)" 7.6-25 5-19 7-17 (3)"
"Found in organs of rats injected with various forms of manganous ion. Manganese concentrations were determined by atomic absorption spectrosocpy, and relaxation rates were measured on tissue specimens at 4 MHz at room temperature. Some of the spread in the values reported is a result of the different lengths of time that elapsed between injection and sacrifice. The following numbers of animals were used in each series: MnC12 (50), Mn EDTA (14), Mn citrate (3), saline control
(5). ~'Average value
have other advantages, as in renal clearance studies, even at the required dosages of about 1 g/kg body weight. Oral paramagnetic contrast agents have ben used for the study of the gastrointestinal tract. A commercial dietary preparation containing ferric iron has been used successfully (20). Insoluble complexes, such as gadolinium oxalate, have also been proposed for such use (10) on the grounds that they would remain localized and exhibit little toxicity. Molecular oxygen has been proposed as an inhalation contrast agent (21), but a convincing demonstration of its practical usefulness has yet to be published. Biological Trace Element Research
Vol. 13, 1987
Contrast Agents for Nudear /Vlagnetic Resonance Imaging
233
Ferromagnetic Particles as Contrast Agents for NMR Imaging Ferromagnetic particles are a rather different class of NMR imaging contrast agents. Large magnetic fields are produced by ferromagnetic objects. If small ferromagnetic particles are distributed throughout an NMR sample, there will be a distribution of local static magnetic field values, similar to that discussed by Drain (22) for metallic particles. The shape and width of the resulting NMR peak will depend upon diffusion, if the particle sizes and separations are small compared to the average distance of diffusion during a time comparable to the reciprocal of the NMR line width. Particulate ferromagnetic agents, such as magnetite (F304), are expected to increase in line width and, hence, to decrease its reciprocal, the apparent T2, but not to change TI. When the usual spin-echo technique is used for magnetic resonance imaging, it is expected that the decrease in the apparent T2 caused by such particles, in tissues or in other materials, will decrease the image intensity. After intravenous injection, it is expected that the particles will be cleared by the reticuloendothelial system and will then appear mostly in the liver, spleen, and lungs. Their ultimate fate is not known. They could be metabolized, and the dissolved iron then stored largely in the liver, or the particles could be permanently trapped in cells of the reticuloendothelial system or elsewhere. If the particles have a long biological half-life it may be a disadvantage in medical imaging because the effect of the contrast agent would linger and affect subsequent NMR images and spectra. Detailed investigations of the deposition and effects of these particles will require analytical methods capable of distinguishing between particulate and dissolved iron and among different forms of iron-containing particles. Microscopic NMR imaging (23,24) may permit the sites of entrapment of the particles to be found, but must be accompanied by microanalytical studies. Studies of the compartmentation of various substances in tissue may also be possible (25). Methods for the Studies with Ferromagnetic Particle Studies with Phantoms Samples of magnetite of three average sizes (0.05, 0.5, and 3.5 t~m) were uniformly dispersed by careful stirring and ultrasonication in 1.6% agar gels, and the relaxation behavior analyzed at 4 MHz (0.094 T), using a conventional SEIMCO pulsed NMR spectrometer and at 25 MHz (0.6 T), using a Technicare whole-body imaging system. The spin-spin or transverse relaxation times, T2, were measured using either the CarrPurcell (CP) or the Carr-Purcell-Meiboom-Gill (CPMG) sequences. Animal Studies
Three rats were used for nonimaging tissue studies after canulation of the femoral vein and injections of suspensions of magnetite (0.05 i~m) Biological Trace Element Research
VoL 13, 1987
234
Dias and Lauterbur
at concentrations of 4.6, 7.8, and 0.0 mg/kg of body weight. The three animals were sacrificed I h after injection, and the organs and tissues excised for determinations of the transverse relaxation time T2 (at 4.0 MHz), using a CPMG sequence. One rat and one dog were imaged before and 1 h after iv injection of suspensions containing 10 mg/kg of body weight of 0.05 p,m magnetite particles; 3-D spin-echo images of the rat were obtained at 4 MHz and 2-D multislice spin echo images of the dog were obtained at 25 MHz. The repetition time used in both cases was 2.5 s, and the interpulse interval was 15 ms.
Results with Ferromagnetic Particles Results with Phantoms
Because a nonlinear d e p e n d e n c e of the relaxation rate on the concentration of the particles was found, the relative effects cannot be described simply. The larger particles were effective at very low concentrations. For example, 9 x 10 6 of such particles in a liter can change the relaxation rate by about 4.5 s ] (about 40% by comparison with the pure gel). On a molar basis, however, all the particles seem about equally effective in the concentration range studied, as shown in Fig. 1.
2.4 + 0.05 MICRON
"7
tj
9 0.5
MICRON
9 3.5
MICRON
"1.6
ol A
9
I
./T.,r
,
0.8
~I
§
I
t.6
I
I
2.4
I
I
5.2
Log ( CONC. Fe304 ), #M /liler Fig. 1. A comparison of changes in apparent transverse relaxation rates caused by three different particle sizes of magnetite. The relaxation times were calculated using only the first echo of a multiecho image obtained at 25 MHz (0.6 T). The repetition time was 5 s, the echo time was 30 ms.
Biological Trace Element Research
Vol. 13, 1987
235
Contrast Agents for Nudear /Vlagnetic Resonance Imaging
Results with Animals The results obtained on tissues at 4 M H z are p r e s e n t e d in Table 2. A s s u m i n g that the behavior of the magnetic particles is the same in gels a n d tissues, we calculated, for the injected dose of 4.6 mg/kg, the a m o u n t of m a g n e t i t e p r e s e n t on the liver from the observed value of R2 to be about 200 Dmol/kg of liver. Also, as s h o w n in Fig. 2, this contrast agent seems to be selective, p r o d u c i n g the greatest effect in the liver, less in the spleen, a n d relatively little in the kidneys. M e a s u r e m e n t s on lungs a n d other tissues were not m a d e . Coronal (left) a n d sagittal (right) sections from 3D spin-echo 4 MHz (0.094 T) v o l u m e images of a rat injected 1 h previously with a s u s p e n sion containing 10 m g / k g b o d y w e i g h t of 0.05 ~ m m a g n e t i t e particles are s h o w n in Fig. 3. From these images it appears that s o m e deposition of the particles has taken place in the lungs as well as in the liver a n d spleen.
Discussion of the Results with Ferromagnetic Particles Particles of m a g n e t i t e are effective at rather low concentrations in reducing the a p p a r e n t transverse relaxation times of water p r o t o n s in TABLE 2 Effects on the Apparent Transverse Relaxation Rates of Rat Organs" Tissue
Conc of Fe304, mg/kg weight
2T, ms
R2
AR2
0.0 4.6 7.8 0.0 4.6 7.8 0.0 4.6 7.8 0.0 4.6 7.8
10 10 10 10 10 10 10 10 10 10 10 10
20 41 44 13 19 23 16 24 16 15 16 18
0 21 24 0 6 10 0 8 0 0 1 2
0.0 4.6 7.8
10 10 10
5 5 4
0 0 -1
Liver
Spleen
Heart
Kidney
Blood
"Injections of two different concentrations of suspensions of 0.05~m magnetite particles. The relaxation times were measured at 4 MHz (0.094 T), using a conventional SEIMCO pulsed NMR spectrometer with the CPMG sequence.
Biological TraceElementResearch
VoL 13, 1987
Dias and Lauterbur
236
2.4 9 LIVER 9 SPLEEN 9 KIDNEY I
r Q) r
i.6
N
n
Contrast Agents for Nuclear Magnetic Resonance Imaging M. H. MENDONCA DIAS* AND PAUL C. LAUTERBUR Department of Chemistry, State University of New York at Stony Brook, Stony Brook, NY 11794 ABSTRACT Nuclear magnetic resonance imaging relies upon differences in relaxation times for much of its ability to resolve anatomical structures and to detect changes in tissue. The natural differences can be changed by the administration of paramagnetic substances, such as metal complexes and stable organic free radicals, and ferromagnetic materials, such as small particles of magnetite. Detailed studies of the chemistry and biophysics of such substances in the body are required if they are to become safe and effective contrast agents for use in medical NMR imaging. Index Entries: Nuclear magnetic resonance imaging, contrast agents for; paramagnetic metal ions and complexes; stable organic free radicals; ferromagnetic particles; medical NMR imaging; longitudinal relaxation time (T1); transverse relaxation time (T2); contrast agents; nitroxide free radicals; manganese compounds; gadolinium compounds; iron compounds.
INTRODUCTION Nuclear magnetic resonance imaging is n o w widely used in clinical diagnosis and biomedical research (1-3). Most images are made using the h y d r o g e n (proton) NMR signals from water and from the lipids in adipose tissue. Regions containing air, such as lung tissue, and hard structures, such as some bone, give relatively weak signals, and there are differences in observable water and fat content even a m o n g ordinary soft tissues. The most useful differences, however, are in the NMR relaxation times of different organs and between normal and abnormal tissues. The *Author to whom all correspondence and reprint requests should be addressed. Biological Trace Element Research
229
Vol. 13, 1987
230
Dias and Lauterbur
relaxation times are usually symbolized and described as T1, the spin-lattice relaxation time, and T2, the spin-spin relaxation time. They depend on the molecular environments of the nuclei and on their motions. It has been known since the first studies of NMR in liquids, almost 40 yr ago, that the addition of low concentrations of paramagnetic solutes could drastically shorten the relaxation times of water protons, and this phenomenon has been widely used in chemical and biochemical investigations (4). The first proposal that paramagnetic substances could be used to obtain the same results in vivo, and hence could be used as contrast agents in medical NMR imaging, was made in 1978 and accompanied by experiments on the application of the idea to the detection of myocardial infarction, using injected manganous salts (5). One of the reasons for choosing manganese was the availability of a convenient and accurate analytical method, using acid extraction and atomic absorption spectroscopy, for that element in tissue samples. Because the relaxation efficacy of an element depends on its chemical form and its chemical and physical environment, analytical data on the distribution of contrast agents in tissues is necessary if the experimental results are to be fully interpreted. Two general classes of magnetic contrast agent are known: (1) Paramagnetic ions, complexes and molecules create rapidly fluctuating, tocally inhomogeneous magnetic fields, with frequency components in a range that can cause increases in both relaxation rates RI (l/T1) and R2 (1/T2); and (2) magnetic particles, such as ferromagnetic powders, generate local nearly static magnetic field inhomogeneities that cause increases in the apparent relaxation rate R2, but not in R1. In this paper we will briefly review work done with paramagnetic compounds, as well as present some new results of work with ferromagnetic particles. The need for better analytical methods to guide these investigations will be noted.
REVIEW ON PARAMAGNETIC IONS AND MOLECULES AS CONTRAST AGENTS FOR NMR IMAGING The paramagnetic contrast agents can be either paramagnetic molecules or metal ions and their complexes (6). They can be classified, by route of administration, as intravascular agents, oral agents, and inhalation agents. We will consider these three classes separately below. Most intravascular agents have been based on manganese, as aqueous manganous ion (Mn § 2), or as its complexes with chelating agents, such as EDTA (6,7) or on gadolinium as its complex with diethylenetriamine pentacetic acid, (DTPA) (8,9). The use of other elements, such as chromium, iron, and copper, as compounds of Cr +3, Fe +3, and Cu § has also been proposed (10,11). Although there has been considerable concern about the high toxicity of hydrated, uncomplexed metal ions and their slow clearance from the body, stable chelated forms of several metBiological Trace Element Research
VoL 13, 1987
Contrast Agents for Nuclear Magnetic Resonance Imaging
231
als may be relatively nontoxic, physiologically stable, and rapidly eliminated. One such complex already being tested in humans is Gd DTPA (9). Another goal is to increase the specificity of these agents so that they will affect primarily particular organs or normal or abnormal tissues. One possible method, already investigated for enhancing the visibility of myocardial infarctions (12), is to bind the paramagnetic complexes to specific monoclonal antibodies. Unfortunately, it seems that many hundreds of metal ions must be bound to each antibody molecule to obtain a large enough effect if the amounts of bound antibody are limited by the number of antigenic sites on cell surfaces. The design and evaluation of contrast agents for medical NMR imaging is a complicated problem. Effects on relaxation rates are immediately apparent, as is acute toxicity, but the interpretation of these effects is not straightforward. The time course of uptake and elimination of the agents must be known and mechanisms understood. Possible changes in chemical form and binding are important because the relaxation effects of metals depend on their molecular environments and on molecular motion, so that total concentrations may not always be inferred from the observed relaxation effects (13-15). Furthermore, sites of deposition, toxicity, and routes and rates of elimination depend upon the chemical form of the metal, including its oxidation state. Microscopic inhomogeneities in distribution, such as differences among concentrations in intravascular, extracellular, intracellular, and subcellular spaces, may be significant, requiring sensitive microscopic analytical techniques. Some specific examples of the uses of various metal compounds are given below. Manganese, as manganous ion and as complexes, has usually been injected in doses of 0.01-0.1 mM/kg body weight for studies of myocardial ischemia and infarction (5,7,12) and of uptake and clearance by other organs. A summary of some experiments, more fully discussed elsewhere (6), is given in Table 1. Other forms of manganese used have been a Mn(III) porphyrin (16), in a study that also included the corresponding compounds of Fe(III) and Cu(II), and Mn DTPA entrapped in liposomes
(17). The DTPA complex of gadolinium (III), in dosages similar to those mentioned above for manganese, has been used in animal (18) and human (9) investigations. It appears to be less toxic than manganese complexes, perhaps because it remains undissociated in the body. As yet, studies of gadolinium distribution and relaxation efficacy in tissues similar to those done for manganese have not been reported. An attempt to achieve similar results with Cr EDTA has been published (19), but it was found that much higher dosages were required. Stable nitroxide free radicals have been proposed as paramagnetic contrast agents. They are less effective on a molar basis than are compounds for Mn +2 and Gd § 3, being rather closer to Cu § and Cr +3 in that respect (10). They seem to have very low toxicities, however, and may Biological Trace Element Research
VoL 13, I987
Dias and Lauterbur
232
TABLE 1 Manganese Concentrations and Water Proton Spin-lattice Relaxation Rates"
Organ Pancreas
Liver
Spleen
Kidney
Heart
Solution injected, dose 0.1 mM/kg MnC1, Mn EDTA Mn citrate Saline control MnC12 Mn EDTA Mn citrate Saline control MnC12 Mn EDTA Mn citrate Saline control MnC12 Mn EDTA Mn citrate Saline control MnC12 Mn EDTA Mn citrate Saline control
Range of tissue concentrations of Mn, mM/kg
R ~, s-1
0.23-0.68 0.20-0.51 0.33-1.9 (0.06y' 0.24-0.78 0.23-0.49 0.42-0.64 (0.06)" 0.08-0.54 0.03-0.09 0.02-0.07 (0.045y' 0.20-0.81 0.21-0.42 0.20-0.36 (0.05)" 0.09-0.60 0.06-0.52 0.10-0.37 (0.03y
12-28 11-21 17-26 (4y' 16-40 15-31 24-42 (5)" 4-30 4-5 3.8-4 (4)" 16-32 7.5-14 11-16 (3.6)" 7.6-25 5-19 7-17 (3)"
"Found in organs of rats injected with various forms of manganous ion. Manganese concentrations were determined by atomic absorption spectrosocpy, and relaxation rates were measured on tissue specimens at 4 MHz at room temperature. Some of the spread in the values reported is a result of the different lengths of time that elapsed between injection and sacrifice. The following numbers of animals were used in each series: MnC12 (50), Mn EDTA (14), Mn citrate (3), saline control
(5). ~'Average value
have other advantages, as in renal clearance studies, even at the required dosages of about 1 g/kg body weight. Oral paramagnetic contrast agents have ben used for the study of the gastrointestinal tract. A commercial dietary preparation containing ferric iron has been used successfully (20). Insoluble complexes, such as gadolinium oxalate, have also been proposed for such use (10) on the grounds that they would remain localized and exhibit little toxicity. Molecular oxygen has been proposed as an inhalation contrast agent (21), but a convincing demonstration of its practical usefulness has yet to be published. Biological Trace Element Research
Vol. 13, 1987
Contrast Agents for Nudear /Vlagnetic Resonance Imaging
233
Ferromagnetic Particles as Contrast Agents for NMR Imaging Ferromagnetic particles are a rather different class of NMR imaging contrast agents. Large magnetic fields are produced by ferromagnetic objects. If small ferromagnetic particles are distributed throughout an NMR sample, there will be a distribution of local static magnetic field values, similar to that discussed by Drain (22) for metallic particles. The shape and width of the resulting NMR peak will depend upon diffusion, if the particle sizes and separations are small compared to the average distance of diffusion during a time comparable to the reciprocal of the NMR line width. Particulate ferromagnetic agents, such as magnetite (F304), are expected to increase in line width and, hence, to decrease its reciprocal, the apparent T2, but not to change TI. When the usual spin-echo technique is used for magnetic resonance imaging, it is expected that the decrease in the apparent T2 caused by such particles, in tissues or in other materials, will decrease the image intensity. After intravenous injection, it is expected that the particles will be cleared by the reticuloendothelial system and will then appear mostly in the liver, spleen, and lungs. Their ultimate fate is not known. They could be metabolized, and the dissolved iron then stored largely in the liver, or the particles could be permanently trapped in cells of the reticuloendothelial system or elsewhere. If the particles have a long biological half-life it may be a disadvantage in medical imaging because the effect of the contrast agent would linger and affect subsequent NMR images and spectra. Detailed investigations of the deposition and effects of these particles will require analytical methods capable of distinguishing between particulate and dissolved iron and among different forms of iron-containing particles. Microscopic NMR imaging (23,24) may permit the sites of entrapment of the particles to be found, but must be accompanied by microanalytical studies. Studies of the compartmentation of various substances in tissue may also be possible (25). Methods for the Studies with Ferromagnetic Particle Studies with Phantoms Samples of magnetite of three average sizes (0.05, 0.5, and 3.5 t~m) were uniformly dispersed by careful stirring and ultrasonication in 1.6% agar gels, and the relaxation behavior analyzed at 4 MHz (0.094 T), using a conventional SEIMCO pulsed NMR spectrometer and at 25 MHz (0.6 T), using a Technicare whole-body imaging system. The spin-spin or transverse relaxation times, T2, were measured using either the CarrPurcell (CP) or the Carr-Purcell-Meiboom-Gill (CPMG) sequences. Animal Studies
Three rats were used for nonimaging tissue studies after canulation of the femoral vein and injections of suspensions of magnetite (0.05 i~m) Biological Trace Element Research
VoL 13, 1987
234
Dias and Lauterbur
at concentrations of 4.6, 7.8, and 0.0 mg/kg of body weight. The three animals were sacrificed I h after injection, and the organs and tissues excised for determinations of the transverse relaxation time T2 (at 4.0 MHz), using a CPMG sequence. One rat and one dog were imaged before and 1 h after iv injection of suspensions containing 10 mg/kg of body weight of 0.05 p,m magnetite particles; 3-D spin-echo images of the rat were obtained at 4 MHz and 2-D multislice spin echo images of the dog were obtained at 25 MHz. The repetition time used in both cases was 2.5 s, and the interpulse interval was 15 ms.
Results with Ferromagnetic Particles Results with Phantoms
Because a nonlinear d e p e n d e n c e of the relaxation rate on the concentration of the particles was found, the relative effects cannot be described simply. The larger particles were effective at very low concentrations. For example, 9 x 10 6 of such particles in a liter can change the relaxation rate by about 4.5 s ] (about 40% by comparison with the pure gel). On a molar basis, however, all the particles seem about equally effective in the concentration range studied, as shown in Fig. 1.
2.4 + 0.05 MICRON
"7
tj
9 0.5
MICRON
9 3.5
MICRON
"1.6
ol A
9
I
./T.,r
,
0.8
~I
§
I
t.6
I
I
2.4
I
I
5.2
Log ( CONC. Fe304 ), #M /liler Fig. 1. A comparison of changes in apparent transverse relaxation rates caused by three different particle sizes of magnetite. The relaxation times were calculated using only the first echo of a multiecho image obtained at 25 MHz (0.6 T). The repetition time was 5 s, the echo time was 30 ms.
Biological Trace Element Research
Vol. 13, 1987
235
Contrast Agents for Nudear /Vlagnetic Resonance Imaging
Results with Animals The results obtained on tissues at 4 M H z are p r e s e n t e d in Table 2. A s s u m i n g that the behavior of the magnetic particles is the same in gels a n d tissues, we calculated, for the injected dose of 4.6 mg/kg, the a m o u n t of m a g n e t i t e p r e s e n t on the liver from the observed value of R2 to be about 200 Dmol/kg of liver. Also, as s h o w n in Fig. 2, this contrast agent seems to be selective, p r o d u c i n g the greatest effect in the liver, less in the spleen, a n d relatively little in the kidneys. M e a s u r e m e n t s on lungs a n d other tissues were not m a d e . Coronal (left) a n d sagittal (right) sections from 3D spin-echo 4 MHz (0.094 T) v o l u m e images of a rat injected 1 h previously with a s u s p e n sion containing 10 m g / k g b o d y w e i g h t of 0.05 ~ m m a g n e t i t e particles are s h o w n in Fig. 3. From these images it appears that s o m e deposition of the particles has taken place in the lungs as well as in the liver a n d spleen.
Discussion of the Results with Ferromagnetic Particles Particles of m a g n e t i t e are effective at rather low concentrations in reducing the a p p a r e n t transverse relaxation times of water p r o t o n s in TABLE 2 Effects on the Apparent Transverse Relaxation Rates of Rat Organs" Tissue
Conc of Fe304, mg/kg weight
2T, ms
R2
AR2
0.0 4.6 7.8 0.0 4.6 7.8 0.0 4.6 7.8 0.0 4.6 7.8
10 10 10 10 10 10 10 10 10 10 10 10
20 41 44 13 19 23 16 24 16 15 16 18
0 21 24 0 6 10 0 8 0 0 1 2
0.0 4.6 7.8
10 10 10
5 5 4
0 0 -1
Liver
Spleen
Heart
Kidney
Blood
"Injections of two different concentrations of suspensions of 0.05~m magnetite particles. The relaxation times were measured at 4 MHz (0.094 T), using a conventional SEIMCO pulsed NMR spectrometer with the CPMG sequence.
Biological TraceElementResearch
VoL 13, 1987
Dias and Lauterbur
236
2.4 9 LIVER 9 SPLEEN 9 KIDNEY I
r Q) r
i.6
N
n
