Mimicking liver iron overload using liposomal ferritin preparations.

Close monitoring of liver iron content is necessary to prevent iron overload in transfusion-dependent anemias. Liver biopsy remains the gold standard; however, MRI potentially offers a noninvasive alternative. Iron metabolism and storage is complicated and tissue/disease-specific. This report demonstrates that iron distribution may be more important than iron speciation with respect to MRI signal changes. Simple synthetic analogs of hepatic lysosomes were constructed from noncovalent attachment of horse-spleen ferritin to 0.4 microm diameter phospholipid liposomes suspended in agarose. Graded iron loading was achieved by varying ferritin burden per liposome as well as liposomal volume fraction. T1 and T2 relaxation times were measured on a 60 MHz NMR spectrometer and compared to simple ferritin-gel combinations. Liposomal-ferritin had 6-fold stronger T2 relaxivity than unaggregated ferritin but identical T1 relaxivity. Liposomal-ferritin T2 relaxivity also more closely matched published results from hemosiderotic marmoset liver, suggesting a potential role as an iron-calibration phantom.     

MR evaluation of liver iron overload

Children and young adults with hemolytic anemias requiring frequent transfusions develop increased liver iron content. We evaluated 15 chronically transfused children with sickle cell disease to determine whether spin-echo magnetic resonance (MR) imaging was useful in assessing the degree of iron overload. Quantitative MR parameters were correlated with liver biopsy iron determinations and serum ferritin levels. The best predictor of liver iron was the ratio of the intensities between the liver and paraspinal musculature on somewhat T1 weighted sequence (repetition time 0.5 s, echo time 28 ms), R2 = 0.58. Magnetic resonance was able to separate those patients with liver iron levels greater than 100 micrograms/mg (intensity ratios approximately 0.4), from those with levels less than 100 micrograms/mg (intensity ratios near 1). However, MR was unable to quantitate liver iron in patients with values ranging from 100 to 400 micrograms/mg since similar intensity ratios were present in this range. Thus, MR provides a qualitative rather than quantitative assessment of liver iron overload.     

Evaluation of iron overload by single voxel MRS measurement of liver T2

PURPOSE: To overcome the difficulty of poor signal-to-noise ratio of magnetic resonance imaging (MRI) in evaluating heavy iron overload by using a single voxel magnetic resonance spectroscopy (MRS) technique. MATERIALS AND METHODS: A single voxel STEAM pulse sequence with a minimum TE of 1.5 msec and a sampling volume of 36.6 cm(3) was developed and applied to 1/T2 measurement of the liver in 14 patients with thalassemia whose liver iron concentration was determined through biopsy. RESULTS: The iron level ranged from 0.23 to 37.15 mg Fe/g dry tissue with a median value of 18.06. In all cases, strong MR signals were obtained. 1/T2 was strongly correlated with the liver iron concentration (r = 0.95, P < 0.00005). CONCLUSION: The single voxel MRS measurement of T2 in liver iron overload overcomes the difficulty of lack of detectable signals in conventional MRI when the iron level is high. There is an excellent correlation between the iron level and 1/T2.     

Nuclear magnetic resonance imaging of experimentally induced liver disease

Experimental animal models of hepatitis, fatty liver, and hepatic iron overload were evaluated using a 3.5-kGauss nuclear magnetic resonance (NMR) imaging system. Increases in image intensity measurements and in T2 relaxation times equalled the sensitivity of histologic findings for the detection of early stages of hepatitis. A significant shift in T1 relaxation times characterized the early stages of hepatic necrosis. Liver triglyceride content correlated significantly with increases in NMR intensity measurements (p less than 0.01); however, changes in liver water content had a much greater influence on intensity, T1, and T2. Thus, it may be possible to distinguish hepatitis from benign fatty liver. Liver iron content correlated with decreases in NMR intensity measurements (p less than 0.001), and iron levels as low as 1.2 mg/g were detected. NMR may more specifically identify hepatocellular iron overload than do other techniques that do not distinguish hepatocellular from reticuloendothelial iron.     

Role of iron and ferritin in MR imaging of the brain: a study in primates at different field strengths

The authors measured in vivo signal intensity on magnetic resonance (MR) images and postmortem iron concentrations in the brains of three young and two old rhesus monkeys. T2-weighted MR imaging was done at 0.5, 1.5, 2.0, and 4.7 T. Relative assessment of iron concentration was made from the optical density of brain sections stained with the Perls' method intensified with diaminobenzidine. MR imaging and optical density measurements were made in the centrum semiovale (white matter) and in four gray matter areas: the insular cortex, caudate nucleus, putamen, and globus pallidus, the latter three of which accumulate significant iron deposits with age. High optical density and decreased signal intensity were found in these areas, and the inverse correlation between gray matter/white matter signal ratio and optical density was in good agreement with the theory of T2 shortening caused by diffusion of water through magnetic inhomogeneities. However, the dependence of T2 shortening on field strength was not quadratic, as expected for paramagnetic iron, but instead showed a marked leveling off at higher field strengths. This magnetic "saturation" is explainable by antiferromagnetism and superparamagnetism of the ferritin core and has been observed in ferritin solutions at low temperatures. Similar observations at body temperature are needed before the iron-ferritin explanation for T2 shortening can be considered proved.     

Paramagnetic macrocyclic complexes as contrast agents for MR imaging: proton nuclear relaxation rate enhancement in aqueous solution and in rat tissues

Paramagnetic macrocyclic chelates show promise as magnetic resonance (MR) imaging contrast agents due to stability and relaxivity comparable to those of DTPA-type chelates. For the three copper and manganese macrocyclic complexes studied in aqueous solution, T1 and T2 relaxivities ranged from 0.14 to 5.88 mM-1sec-1 at 6.25 MHz. In rats, the intravenous administration of 16 mumol/kg of Mn(cyclam) caused the liver T1 relaxation rate to double at 15 minutes after injection. T1 measurements by pulsed MR imaging and manganese analyses on excised tissue showed that both relaxation rate (1/T1) and manganese content of liver and kidney increase linearly with the dosage of Mn(cyclam). The linear relationship between 1/T1 and manganese content can be considered an "in tissue" relaxivity plot for the agent. The resulting relaxivity is 54 mM-1sec-1 in liver, compared with 3.1 mM-1sec-1 in aqueous solution. Although this work is preliminary, the implication for medical MR imaging applications is that macrocyclic contrast agents can be effective at approximately one-tenth the current typical dose used for gadolinium DTPA.     

Value of magnetic resonance imaging in diffuse liver diseases

CLINICAL PROBLEM: Diffuse liver diseases show an increasing prevalence. The diagnostic gold standard of liver biopsy has several disadvantages. There is a clinical demand for non-invasive imaging-based techniques to qualitatively and quantitatively evaluate the entire liver. STANDARD RADIOLOGICAL METHODS: Ultrasound, computed tomography (CT) and magnetic resonance imaging (MRI) are routinely used. METHODICAL INNOVATIONS: Steatosis: chemical shift and frequency selective imaging, MR spectroscopy (MRS). Hemochromatosis: MR-based iron quantification. Fibrosis: MR elastography, diffusion, intravoxel incoherent motion (IVIM) and MR perfusion. PERFORMANCE/ACHIEVEMENTS/PRACTICAL RECOMMENDATIONS: T1-weighted in and opposed phase imaging is the clinically most frequently used MR technique to noninvasively detect and quantify steatosis. New methods for quantification that are not influenced by confounders like iron overload are under investigation. The most sensitive method to measure the fat content of the liver is MRS. As data acquisition and analysis remain complex and there is no whole organ coverage, MRS of the liver is not a routine method. With an optimized protocol incorporating T2* sequences, MRI is the modality of choice to quantify iron overload in hemochromatosis. Standard MR sequences cannot depict early stages of liver fibrosis. Advanced MR techniques (e.g. elastography, diffusion, IVIM and perfusion) for noninvasive assessment of liver fibrosis appear promising but their role has to be further investigated.     

Assessment of liver iron overload in thalassemic patients by MR imaging.

The use of MR imaging has been proposed for the assessment of the hepatic iron overload in transfusion-dependent thalassemic patients treated with desferrioxamine. The aim of the study was to correlate serum ferritin levels and MR signal intensity of the liver parenchyma. Results on 12 patients showed that the ratios between the signal intensity of liver parenchyma and muscle and fat are promising parameters for predicting iron overload.     

T2 values in the human brain: comparison with quantitative assays of iron and ferritin

Magnetic resonance (MR) imaging with a whole-body imager was performed in 10 fresh, unfixed whole human brains selected randomly from cadavers. All subjects were neurologically intact before death. T2 time constants were measured within the caudate nucleus, putamen, globus pallidus, cortical gray matter, subcortical white matter, and optic radiation. These regions were then excised, and T2 values were measured again with a 1.5-T MR spectrometer. Quantitative assays of iron, ferritin, and protein from these areas were then performed. Iron concentration varied significantly among brain regions, whereas ferritin and protein concentrations were constant among brain regions and among individuals. Neither iron nor ferritin concentration showed any consistent correlation with T2 values. Histologic examination of brain micro-sections with iron- and ferritin-specific stains of demonstrated poor correlation with biochemical assays of ferritin and iron concentrations. Results indicate that T2 values correlate poorly with iron and ferritin concentrations found in neurologically intact brains.     

Frequency dependence of MR relaxation times II. Iron oxides

The frequency dependence of T1 and T2 was measured for homogeneous suspensions of magnetite and iron oxyhydroxide particles in water with various concentrations of gelatin. The transverse relaxivity showed two types of behavior: (a) For magnetite particles, there was a rapid increase in T2 relaxivity with frequency, followed by a saturation plateau, which accorded with the Langevin magnetization function. From these curves, the magnetic moment of the particle domains was estimated to range from 0.8 to 6.3 104 Bohr magnetons, (b) For iron oxyhydroxide (fer-ritin, ferrihydrite, and akaganeite) particles, T2 relaxivity increased linearly with frequency, the slope of the increase characteristic for each particle. T2 relaxivity generally increased with increasing gelatin concentration, corresponding to the measured decrease in the water diffusion coefficient. For iron oxides, homogeneously distributed either as iatrogenic agents or endogenous biominerals, these findings may aid in the interpretation of in vivo relaxivity and the effect on MR imaging.     

Hepatic iron overload: quantitative MR imaging

Iron deposits demonstrate characteristically shortened T2 relaxation times. Several previously published studies reported poor correlation between the in vivo hepatic 1/T2 measurements made by means of midfield magnetic resonance (MR) units and the hepatic iron content of iron-overloaded patients. In this study, the authors assessed the use of in vivo 1/T2 measurements obtained by means of MR imaging at 0.5 T using short echo times (13.4 and 30 msec) and single-echo-sequences as well as computed tomographic (CT) attenuation as a measure of liver iron concentration in 10 severely iron-overloaded patients with beta-thalassemia major. The iron concentrations in surgical wedge biopsy samples of the liver, which varied between 3 and 9 mg/g of wet weight (normal, less than or equal to 0.5 mg/g), correlated well (r = .93, P less than or equal to .0001) with the preoperative in vivo hepatic 1/T2 measurements. The CT attenuation did not correlate with liver iron concentration. Quantitative MR imaging is a readily available noninvasive method for the assessment of hepatic iron concentration in iron-overloaded patients, reducing the need for needle biopsies of the liver.     

Simplified synthesis and relaxometry of magnetoferritin for magnetic resonance imaging

Magnetoferritin nanoparticles have been developed as high‐relaxivity, functional contrast agents for MRI. Several previous techniques have relied on unloading native ferritin and re‐incorporation of iron into the core, often resulting in a polydisperse sample. Here, a simplified technique is developed using commercially available horse spleen apoferritin to create monodisperse magnetoferritin. Iron oxide atoms were incorporated into the protein core via a step‐wise Fe(II)Chloride addition to the protein solution under low O₂ conditions; subsequent filtration steps allow for separation of completely filled and superparamagnetic magnetoferritin from the partially filled ferritin. This method yields a monodisperse and homogenous solution of spherical particles with magnetic properties that can be used for molecular magnetic resonance imaging. With a transverse per‐iron and per‐particle relaxivity of 78 mM^?1 sec^?1 and 404,045 mM^?1 sec^?1, respectively, it is possible to detect ～10 nM nanoparticle concentrations in vivo. Magn Reson Med, 2010. © 2010 Wiley‐Liss, Inc.     

MR quantification of hepatic iron concentration

PURPOSE: To evaluate the accuracy of magnetic resonance (MR) imaging in the quantification of hepatic iron concentration. MATERIALS AND METHODS: Between April 1999 and June 2001, 112 patients were recruited prospectively. All had undergone liver biopsy and hepatic iron concentration quantification with spectrophotometry, followed by MR imaging. MR imaging involved use of four gradient-echo sequences and one spin-echo sequence. Signal intensity (SI) was measured on images obtained with each sequence by means of regions of interest placed in the liver and paraspinal muscle to obtain the liver-to-muscle SI ratio. The relationship between hepatic iron concentration and SI ratio for each sequence was analyzed with multiple linear regression. Receiver operating characteristic analysis was performed to find the diagnostic thresholds. RESULTS: Sixty-eight patients had normal hepatic iron levels (<36 micromol/g), 23 had hemosiderosis (36-80 micromol/g), and 21 had hemochromatosis (>80 micromol/g). With all sequences, an inverse linear relationship between iron concentration and SI ratio was apparent. The authors generated a mathematic model to estimate the iron concentrations from MR imaging data (r = 0.937). For estimated concentrations of more than 85 micromol/g, the positive predictive value for hemochromatosis was 100%; for those less than 40 micromol/g, the negative predictive value for hemochromatosis was 100%. For estimated concentrations of more than 58 micromol/g, the positive predictive value for iron overload was 100%; for those less than 20 micromol/g, the negative predictive value for iron overload was 100%. CONCLUSION: MR imaging is a useful and noninvasive diagnostic tool for quantification of hepatic iron concentration.     

Ferrite particles for bowel contrast in MR imaging: design issues and feasibility studies

Diverse materials with varying physical and magnetic properties have been evaluated as gastrointestinal contrast agents for magnetic resonance (MR) imaging. Uniform marking of the small bowel remains the greatest challenge. Ferrites are magnetically active iron oxide particles that are miscible with water and cause loss of signal on MR images. The decrease in MR signal intensity produced by ferrites occurs with a wide range of iron concentrations (0.1-10 mM) and with both T1- and T2-weighted pulse sequences. These effects of ferrites are explained by predominant T2 shortening with negligible T1 effects. The ferrite preparation used in this study was stable in vitro, with little iron solubilized by acid. Intragastric administration of ferrite (5 mg of iron per kg in 6 ml) routinely marked the small bowel of rats. The authors conclude that ferrites represent a promising new class of contrast agents for gastrointestinal MR imaging.     

Iron oxide nanoparticle-labeled rat smooth muscle cells: cardiac MR imaging for cell graft monitoring and quantitation

PURPOSE: To perform a quantitative analysis of anionic maghemite nanoparticle-labeled cells in vitro and determine the effect of labeling on signal intensity at magnetic resonance (MR) imaging. MATERIALS AND METHODS: The study was approved by the institutional animal care and use committee at H?pital Bichat. In vitro cell proliferation, iron content per cell, and MR signal intensity of cells were measured in agarose phantoms for 0-14 days of culture after labeling of rat smooth muscle cells with anionic maghemite nanoparticles. Next, iron oxide-labeled smooth muscle cells were injected into healthy hearts and hearts with ischemic injury in seven live Fisher rats. Ex vivo MR imaging experiments in excised hearts 2 and 48 hours after injection were performed with a 1.5-T medical imaging system by using T2-weighted gradient-echo and spin-echo sequences. Histologic sections were obtained after MR imaging. Correlation analyses between division factor of iron load and cell amplification factor and between 1/T2 and number of labeled cells or number of days in culture were performed by using linear regression. RESULTS: Viability of smooth muscle cells was not affected by magnetic labeling. Transmission electron micrographs of cells revealed the presence of iron oxide nanoparticles in vesicles up to day 14 of culture. Intracellular iron concentration decreased in parallel with cell division (r2 = 0.99) and was correlated with MR signal intensity (r2 = 0.95). T2*-weighted MR images of excised rat hearts showed hypointense signal in myocardium at 2 and 48 hours after local injection of labeled cells. Subsequent histologic staining evidenced iron oxide nanoparticles within cells and confirmed the presence of the original cells at 2 and 48 hours after implantation. CONCLUSION: Magnetic labeling of smooth muscle cells with anionic maghemite nanoparticles allows detection of cells with MR imaging after local transplantation in the heart.     

Adrenal glands in beta-thalassemia major: magnetic resonance (MR) imaging features and correlation with iron stores

This study aimed at describing the magnetic resonance (MR) imaging features of the adrenal glands in beta-thalassemic patients and at investigating the relation between adrenal and hepatic siderosis. Adrenal signal intensity (SI) was retrospectively assessed on abdominal MR studies of 35 patients with beta-thalassemia major undergoing quantification of hepatic siderosis and 12 healthy controls, using T1- (120/4/90), intermediate - (120/4/20), and T2*- (120/15/20) weighted GRE sequences. Adrenal SI was graded as grade 0 (normal SI on all sequences), grade 1 (hypointensity on T2* alone), or grade 2 (hypointensity on at least T2*). Adrenal size was measured in the thalassemic patients and compared with normative data. Liver-to-muscle (L/M) SI ratios, expressing hepatic siderosis, were estimated on each sequence. Serum ferritin levels were recorded. Adrenal hypointensity (grades 1 and 2) was noted in 24/35 (68.6%) patients. L/M ratios correlated significantly with adrenal SI in all sequences. Patients with grade 1 and grade 2 adrenal SI had significantly decreased L/M ratios compared with grade 0. Serum ferritin correlated significantly with L/M values but not with adrenal SI. Adrenal size was within normal limits. Diffuse hypointensity in normal-sized adrenals is a common MR finding in beta-thalassemic patients and correlates with the degree of hepatic siderosis.     

1/T2 and magnetic susceptibility measurements in a gerbil cardiac iron overload model

PURPOSE: To measure the transverse relaxation rate (1/T2) and magnetic susceptibility of the heart in conditions of iron overload by using magnetic resonance (MR) imaging and to correlate these with the tissue iron concentration in a gerbil model. MATERIALS AND METHODS: With prior approval by the institutional animal care and use committee, iron overload was induced with one to 15 weekly subcutaneous injections of iron dextran. Nine gerbils had one to five injections, 10 had six to 10, and eight had 13-15. T2 of the whole heart was measured ex vivo (n=27), and the magnetic susceptibility of the tissue was estimated through measurement of the tissue lysate (n=25). The iron level was measured (in milligrams of iron per gram of wet tissue) with chemical analysis after MR imaging. While 1/T2 and magnetic susceptibility are not equivalent measures of the chemically determined tissue iron level, correlations were expected and were identified by using linear regression models. RESULTS: Iron concentration range was 0.28-1.95 mg/g wet tissue. Iron concentration was strongly correlated with 1/T2 (r=0.92, P <.001, and the root of the mean squares error of the linear prediction, epsilonRMS, was 0.17 mg Fe/g wet tissue with a repetition time of 700 msec). Iron concentration also was strongly correlated with magnetic susceptibility (r=0.90, P <.001, epsilonRMS=0.19 mg Fe/g wet tissue). Multiple regression analysis with combined 1/T2 (with repetition time of 700 msec) and magnetic susceptibility data led to a slight increase in r and decrease in epsilon(RMS) (r=0.93, P <.001, epsilonRMS=0.16 mg Fe/g wet tissue). CONCLUSION: The results of this animal model study demonstrate that 1/T2 and magnetic susceptibility values can be used for estimation of the iron level in the heart.     

Comparison of magnetic properties of MRI contrast media solutions at different magnetic field strengths

RATIONALE AND OBJECTIVES: To characterize and compare commercially available contrast media (CM) for magnetic resonance imaging (MRI) in terms of their relaxivity at magnetic field strengths ranging from 0.47 T to 4.7 T at physiological temperatures in water and in plasma. Relaxivities also were quantified in whole blood at 1.5 T. METHODS: Relaxivities of MRI-CM were determined by nuclear magnetic resonance (NMR) spectroscopy at 0.47 T and MRI phantom measurements at 1.5 T, 3 T, and 4.7 T, respectively. Both longitudinal (T1) and transverse relaxation times (T2) were measured by appropriate spin-echo sequences. Nuclear magnetic resonance dispersion (NMRD) profiles were also determined for all agents in water and in plasma. RESULTS: Significant dependencies of relaxivities on the field strength and solvents were quantified. Protein binding leads to both increased field strength and solvent dependencies and hence to significantly altered T1 relaxivity values at higher magnetic field strengths. CONCLUSIONS: Awareness of the field strength and solvent associated with relaxivity data is crucial for the comparison and evaluation of relaxivity values. Data observed at 0.47 T can thus be misleading and should be replaced by relaxivities measured at 1.5 T and at 3 T in plasma at physiological temperature.     

Multicentre dose-ranging study on the efficacy of USPIO ferumoxtran-10 for liver MR imaging

AIM: A dose ranging multicentre phase-II clinical trial was conducted to evaluate the efficacy of ultrasmall superparamagnetic iron oxide (USPIO) ferumoxtran-10 for magnetic resonance (MR) imaging of focal hepatic lesions. MATERIAL AND METHODS: Ninety-nine patients with focal liver lesions received USPIO at a dose of 0.8 (n = 35), 1.1 (n = 32), or 1.7 (n = 32) mg Fe/kg. Liver MR imaging was performed before and after USPIO with T1-weighted and T2-weighted pulse sequences. Images were analysed by two independent readers for additional information (lesion detection, exclusion, characterization and patient management). Signal intensity (SI) based quantitative measurements were also taken. RESULTS: Post-contrast medium MR imaging showed additional information in 71/97 patients (73%) for reader one and 83/96 patients (86%) for reader two. The results with all three doses were statistically significant (P < 0.05). Signal intensity analysis revealed that all three doses increased liver SI on T1-weighted images and decreased liver SI on T2-weighted images. On T2-weighted images metastases increased in contrast relative to normal hepatic parenchyma whereas haemangiomas decreased in contrast. On T2-weighted images there was statistically improved efficacy at the intermediate dose, which did not improve at the highest dose. CONCLUSION: Ultrasmall superparamagnetic iron oxide was an effective contrast agent for liver MR imaging at all doses and a dose of 1.1 mg Fe/kg was recommended for future clinical trials.     

MRI measurement of hepatic magnetic susceptibility-phantom validation and normal subject studies.

A magnetic resonance (MR) imaging method with the potential for assessing hepatic iron overload from measurements of hepatic magnetic susceptibility in vivo is described. Using the blood in the portal and hepatic veins as an internal reference, this technique uses the orientation dependence of signal phase to measure the susceptibility of the liver parenchyma. Computer simulations were done to investigate the requirements on spatial resolution and contrast ratio between the vessels and the background liver tissue for data acquisition. Validation studies were conducted using tube-embedded gel phantoms doped with iron-dextran from 0 to 10 mg Fe/mL to mimic healthy and iron-overloaded livers. The phantom measurements were conducted without motion and flow, under respiration-like oscillatory motion, and with flow. Studies on six normal human subjects demonstrated excellent reproducibility and precision. All images were collected at 1.5 T using a 3D T(1)-weighted turbo field echo sequence for inflow MR angiographies with full flow compensation and capable of cardiac synchronization, navigator gating, and motion correction.