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.     

Evaluation of different models of experimentally induced liver cirrhosis for MRI research with correlation to histopathologic findings

RATIONALE AND OBJECTIVES: Three models of experimentally induced liver cirrhosis were evaluated for MRI research on chronic liver disease. The influence of different histopathologic changes in liver fibrosis and cirrhosis on relaxation times and signal intensities was studied in vitro and in vivo. METHODS: Liver fibrosis and cirrhosis in rats was induced by oral or subcutaneous administration of carbon tetrachloride (CCl4) or by thioacetamide (TAA) in drinking water. On histology, the degree of liver fibrosis and cirrhosis, fatty infiltration, iron accumulation, and inflammatory changes were measured semiquantitatively. The amount of connective tissue was quantitatively determined by morphometry. The results were correlated with T1 and T2 relaxation times and signal intensities of the liver studied in vitro by relaxometry and in vivo by MRI. RESULTS: In both groups with CCl4 administration, histology revealed different degrees of liver fibrosis and cirrhosis. Subcutaneous injection of CCl4 also resulted in increased fatty infiltration. On the contrary, TAA produced complete liver cirrhosis in all animals. Overall, there was a good correlation between the liver T2 relaxation time and the amount of connective tissue in liver fibrosis and cirrhosis. However, the degree of liver fibrosis and cirrhosis was also strongly correlated with the degree of inflammatory changes. In the group with CCl4 administration, there was a good correlation between the fatty infiltration and the T1 relaxation time, as well as with the liver signal intensity on the T1-weighted gradient echo sequence. An increased iron accumulation was also correlated with the degree of liver fibrosis/cirrhosis; however, there was no significant influence of the iron on relaxation times or signal intensities. CONCLUSIONS: The TAA model is easier to perform and more reliable in liver cirrhosis induction than the CCl4 models. Although there is a positive correlation between the T2 relaxation times and the degree of liver fibrosis/cirrhosis, this probably results from the associated inflammatory changes and is not caused by the increased amount of connective tissue.     

Focal manifestations of diffuse liver disease at MR imaging.

Detection and exclusion of focal liver lesions is especially difficult in patients with diffuse liver disease. Magnetic resonance (MR) imaging may be particularly valuable in these patients. By judicious comparison of appropriate pulse sequences, normal and hypertrophic liver may be distinguished from atrophic, neoplastic, or otherwise abnormal hepatic parenchyma. Chemical shift (lipid-sensitive) techniques allow definitive identification of fatty liver, including focal fatty infiltration or focal sparing. T2-weighted and T2*-weighted images allow identification of iron overload, depicting malignancies as focal masses without iron. Analysis of signal intensity and internal morphology allows confident distinction between regenerative nodules and hepatocellular carcinoma in most instances, and allows diagnosis of early carcinoma within regenerative nodules. MR imaging provides capabilities for noninvasive characterization of liver tissue beyond those available with other noninvasive modalities.     

Comparison between signal-to-noise ratio,liver-to-muscle ratio,and 1/T2 for the noninvasive assessment of liver iron content by MRI

PURPOSE: To compare different MRI-derived parameters, i.e., liver signal-to-noise ratio (LSNR), liver-to-muscle ratio (LMR) and liver transversal relaxation rate (R2), in terms of their correlation with the ex vivo determined iron content in an experimental model of liver iron overload. MATERIALS AND METHODS: Multi-echo spin echo (SE) images of the liver were acquired at 4.7 T from a group of 33 male wistar rats subjected to a high iron content diet for feeding periods ranging from 2 to 50 days. Liver transversal relaxation time, liver signal-to-noise ratio, and liver-to-muscle ratio were measured over the same region of interest in order to get a direct comparison between these parameters. After MRI experiments, the rats were sacrificed and the liver iron content was measured ex vivo by atomic absorption spectroscopy. RESULTS: The iron content is better correlated to the LSNR than to the other parameters (LMR, R2). CONCLUSION: The finding that liver signal-to-noise ratio is better correlated to the iron content than the liver T2 relaxation rate is relevant for clinical applications of MRI because a T2 determination is more time-consuming, both for acquisition and postprocessing of images, than a simple SNR determination.     

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.     

Asialoglycoprotein receptor function in benign liver disease: evaluation with MR imaging

An arabinogalactan-coated ultrasmall superparamagnetic iron oxide (AG-USPIO) preparation specific for asialoglycoprotein (ASG) receptors on hepatocytes was used as a magnetic resonance (MR) imaging contrast agent in the evaluation of a spectrum of benign liver diseases in animal models. The activity of hepatocyte ASG receptors, which directly reflects liver function, was directly assessed by measuring liver relaxation times in vitro and MR signal intensity in vivo. The following measurements allowed three-dimensional assessment of liver function: (a) liver relaxation time, (b) native MR signal intensities of liver, (c) response of liver to the AG-USPIO probe (percentage decrease of liver signal intensity after intravenous administration of 10 mumol/kg of AG-USPIO: normal liver 55%, fatty liver 57%, acute hepatitis 36%, chronic hepatitis 29%, and cirrhosis 46%), and (d) redistribution of hepatocyte-specific AG-USPIO to the spleen (present in hepatitis and cirrhosis but not in normal liver and fatty liver). The results of this study indicate that cellular hepatic abnormalities can be detected and quantitated with MR receptor imaging.     

多回波Dixon技术在非酒精性脂肪肝中脂肪定量及铁沉积的应用进展

非酒精性脂肪性肝病(NAFLD)病人的肝脏脂肪含量和铁过载与其病情的进展密切相关。基于MRI的水脂分离技术即Dixon技术不仅可以测量肝脏脂肪分数,还可得出R2*弛豫图来反映铁沉积情况,其具有无创性、可重复性等优点,在NAFLD的早期诊断、病情评估及疗效评价中具有重要的临床价值。就多回波Dixon技术在肝脏脂肪定量及铁沉积中的研究进展予以综述。     

In vivo investigation of hepatic iron overload in rats using T2 maps: quantification at high intensity field (4.7-T)

In vivo quantitation of hepatic iron content is useful in diagnosis and staging of several iron related diseases. We used an experimental model of hepatic iron overload to determine the correlation between iron content and T2 relaxation time in rat liver. Experiments were carried out at 4.7T for high signal-to-noise ratio (SNR) using a spin-echo multiecho sequence with six echoes and minimum echo-time of 5.5 msec. The liver iron content was determined ex vivo by atomic absorption spectrophotometry (AAS). T2 maps were calculated in order to evaluate the space distribution of the iron content. We found good linear correlation between the in vivo liver transversal relaxation rate and the iron content within the range explored (106-4538 microg Fe/g liver wet wt.). T2 maps revealed that the decrease in T2 is not homogeneous through the liver parenchyma. This finding represents a physiological limitation to obtaining better correlation between T2 and iron content.     

Nuclear magnetic resonance of the liver,spleen, and pancreas

This review includes the initial experience with NMR imaging of the liver, spleen, and pancreas at the University of California, San Francisco, using a prototype 0.35 Tesla system. This experience shows great promise for detection of hepatic metastases using T₁-weighted pulse sequences. T₂-weighted pulse sequences appear sensitive for detecting cavernous hemangioma of the liver and may allow tissue specific discrimination of the benign lesion from cancer. NMR is also suitable for evaluating diffuse metabolic alterations and is sensitive and specific for the diagnosis of iron overload. Detection of fatty liver requires use of chemical shift techniques as conventional NMR imaging pulse sequences are relatively insensitive. Motion artifacts and lack of an effective bowel contrast agent limits imaging of the pancreas and retroperitoneum, where CT remains the procedure of choice. The normal spleen has longer T1 and T2 relaxation times than liver or pancreas and NMR has not been successful in diagnosing splenic metastases or lymphoma on a routine basis. We conclude that NMR imaging will be valuable in the diagnosis of focal liver disorders; until fast scan techniques and effective magnetic contrast agents are available for oral and/or intravenous use, other abdominal applications will remain limited.     

Diagnosis of fatty liver with MR imaging.

The diagnosis of fatty liver with magnetic resonance (MR) imaging was evaluated in experimental rat models of simple fatty infiltration and fatty liver with hepatocellular injury. T1 and T2 were measured ex vivo and correlated with the histologic degree of fatty infiltration. Enhancement of fatty liver with four different cells-specific contrast agents was studied with ex vivo relaxometry and in vivo MR imaging. Quantitative analysis of conventional and chemical shift MR images was correlated with biochemically determined fat content of the liver. Diet-induced simple fatty infiltration of the liver caused a decrease in T1 of 15%, whereas the T1 of L-ethionine-induced fatty liver with hepatocellular injury increased by 12%. T2 showed a positive correlation with the degree of fatty infiltration in both models. Cell-specific hepatobiliary contrast agents showed the same liver uptake and relaxation enhancement in fatty livers as in normal livers. Conventional T1-weighted images and chemical shift images showed good correlation (r = .83 and .80, respectively) between signal intensity and the degree of fatty infiltration. However, only chemical shift imaging was reliable in the diagnosis of fatty liver.     

Receptor imaging: application to MR imaging of liver cancer

A new contrast agent for magnetic resonance (MR) imaging, directed to asialoglycoprotein (ASG) receptors on hepatocytes, was used for detection of liver cancer in rats. Ultrasmall superparamagnetic (mean size, 12 nm) particles of iron oxide (USPIOs) were targeted to ASG receptors by coating particles with arabinogalactan (AG). Liver T2 relaxation times decreased more effectively after a single intravenous administration of AG-USPIO than after an equal dose of a conventional superparamagnetic liver MR contrast agent (AMI-25; mean size, 72 nm). Receptor affinity studies demonstrated that receptor-mediated attachment and subsequent cellular endocytosis do not occur in primary malignant (hepatocellular carcinoma) or metastatic (adenocarcinoma) tumors, because the surface ASG receptors are lost during malignant dedifferentiation. In vitro relaxation and in vivo MR imaging experiments of liver tumors show that targeting USPIO to hepatocytes rather than to the mononuclear phagocytic system allows a considerable dose reduction, increases tumor-liver contrast, and potentially allows distinction of ASG-positive (benign hepatocellular) and ASG-negative (malignant hepatocellular) tumors.     

Abdominal lymphadenopathy in beta-thalassemia: MRI features and correlation with liver iron overload and posttransfusion chronic hepatitis C

OBJECTIVE: The objective of our study was to describe the MRI features of abdominal lymphadenopathy in patients with beta-thalassemia major and investigate the relation of abdominal lymphadenopathy with the severity of iron overload and posttransfusion chronic hepatitis C. MATERIALS AND METHODS: Abdominal MRI studies of 60 consecutive patients with beta-thalassemia major, performed for quantification of liver iron overload at a single institution, were retrospectively studied for the presence of lymph nodes and their distribution, size, and number. The signal intensity ratios of liver, spleen, and the largest lymph node to the right paraspinous muscle (L/M, S/M, and LN/M, respectively) were calculated on T1-weighted gradient-echo images. MRI findings for the lymph nodes were compared with the histologically assigned activity level of chronic hepatitis C that was available in 17 patients who had undergone liver biopsy within 1 month of the MRI examination. RESULTS: Hypointense abdominal lymph nodes larger than 7 mm were seen in 19 (32%) of 60 thalassemic patients in perihepatic and paraortic distributions. Lymphadenopathy was related to both the severity of hepatic siderosis, as expressed by the L/M values, and the presence of chronic hepatitis C, given that 18 (95%) of the 19 thalassemic patients with lymphadenopathy had chronic hepatitis C. Moreover, thalassemic patients with a moderate or severe level of hepatic inflammation presented with abdominal lymphadenopathy more frequently than those with mild hepatic inflammation. CONCLUSION: The development of hypointense abdominal lymphadenopathy in patients with beta-thalassemia major who have received multiple transfusions depends both on the severity of liver iron overload and on the presence and the activity level of coexistent chronic hepatitis C.     

Magnetic resonance imaging in chronic liver disease evaluated in relation to hepatic fibrosis--clinical and experimental results

In 21 patients with chronic liver disease, the ratio of liver to muscle signal intensity on T1-weighted images was negatively correlated with the progression of hepatic fibrosis defined according to findings by laparoscopy and liver biopsy, and differentiated six patients with early chronic hepatitis from eight with liver cirrhosis. On T2-weighted images, the number of low intensity nodules comparable in size to regenerating nodules surrounded by connective tissues showed a positive correlation with stage. When hepatic fibrosis with no necrosis or fat infiltration was induced in rats, T2 values were positively correlated with hepatic hydroxyproline content, though there was no such correlation for T1 values. These results suggest that MR imaging may be useful for determining the progression of hepatic fibrosis in chronic liver disease. T2 values may directly reflect hepatic fibrosis.     

Primary liver tumors: diagnosis by MR imaging

MR features of 153 proved primary liver tumors (95 malignant, 58 benign) in 55 patients with hepatocellular carcinoma (21), cholangiocarcinoma (seven), carcinosarcoma (one), hepatoblastoma (one), hemangioma (16), hepatic adenoma (four), focal nodular hyperplasia (three), leiomyoma (one), and hemangioendothelioma (one) were studied retrospectively to determine which techniques are most reliable for lesion detection and which criteria are most useful for differential diagnosis. MR data were correlated with histologic features such as fatty degeneration, fibrosis, and peritumoral edema. Unlike metastatic cancer, hepatocellular carcinoma was best detected (p less than .01) with T2-weighted pulse sequences. The mean tumor-liver T2 difference was 34.4%, while the mean T1 difference was only 21.8%. A tissue-specific diagnosis of hepatocellular carcinoma was possible in 14 of 21 patients by identification of fatty degeneration of the tumor (eight of 17), tumor capsule (five of 21), and/or vascular invasion (six of 21). MR features of peritumoral edema, present in six of 21 patients with hepatocellular carcinoma and in seven of 25 patients with metastases, were exclusively associated with malignant tumors. The large variation in tissue characteristics (relaxation times and proton density) seen in primary liver tumors necessitates the use of multiple pulse sequences to maximize lesion detection. However, the combined use of T1- and T2-weighted spin-echo and T2-weighted phase-contrast images had the advantage of distinguishing benign from malignant primary liver tumors in 48 of 55 patients in this series.     

Chronic carbon tetrachloride and phospholipase D hepatotoxicity in rat: in vivo 1H magnetic resonance, total lipid analysis, and histology

Magnetic resonance (MR) imaging, localized in vivo proton spectroscopy, and T1 relaxation measurements were obtained from the livers of rats treated chronically with carbon tetrachloride and phospholipase D. The MR data correlated well with lipid changes measured biochemically and histologically. MR images appeared generally hyperintense during fatty infiltration, changing to hypointense mottling during cirrhosis. Water T1 relaxation times showed no statistically significant change at any time during the experiments from the control value of 908 ms (SE = 42 ms). Minor changes in lipid T1 values with time were noted. The average lipid T1 curve demonstrated a linear relation with time (r = 0.81), increasing from the control value of 283 ms (+/- 16 ms) to 365 ms (+/- 53 ms) at the end of the third week and decreasing slightly through the end of the experiment. Water-suppressed in vivo spectra showed quantitative changes in liver lipids which correlated well with the biochemical and histologic analysis. From the MR images and spectroscopy results it was possible to distinguish early fatty liver from more advanced cirrhosis.     

The effect of haemosiderosis and blood transfusions on the T2 relaxation time and 1/T2 relaxation rate of liver tissue.

Patients with chronic anaemia need repeated blood transfusions, which eventually lead to iron overload. The excess iron from blood transfusions is deposited in the reticuloendothelial system and in the parenchymal cells of the liver, spleen and other organs. Cellular damage is likely to occur when iron overload in the liver is pronounced. Liver biopsy is still necessary to evaluate the degree of haemosiderosis or haemochromatosis. To avoid this invasive procedure, methods have been sought to determine the concentration of iron in liver tissue and to estimate the effect of the treatment of haemosiderosis or haemochromatosis. In this MRI study, the T2 relaxation time and the 1/T2 relaxation rate of liver were determined in 23 patients who had undergone repeated blood transfusions for chronic anaemia. The first 60 transfusions had the greatest influence on the measured T2 relaxation time, with T2 relaxation time decreasing as haemosiderosis progresses. The 1/T2 relaxation rate increases significantly in a linear fashion when the number of blood transfusions increases up to 60. After 60 transfusions the influence of additional blood transfusions on the T2 value was minimal; the same response, although in reverse, was seen in the 1/T2 relaxation rate curve. One possible explanation for this may be that the MR system could detect the effect of only a limited amount of iron excess and any concentration over this limit gives a very short T2 relaxation time and a very weak signal from the liver, which is overwhelmed by background noise. However, in mild and moderate haemosiderosis caused by blood transfusions, T2 relaxation time and 1/T2 relaxation rate reflect iron accumulation in liver tissue.     

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.     

Quantification of liver iron with MRI: State of the art and remaining challenges

Liver iron overload is the histological hallmark of hereditary hemochromatosis and transfusional hemosiderosis, and can also occur in chronic hepatopathies. Iron overload can result in liver damage, with the eventual development of cirrhosis, liver failure, and hepatocellular carcinoma. Assessment of liver iron levels is necessary for detection and quantitative staging of iron overload and monitoring of iron‐reducing treatments. This article discusses the need for noninvasive assessment of liver iron and reviews qualitative and quantitative methods with a particular emphasis on magnetic resonance imaging (MRI). Specific MRI methods for liver iron quantification include signal intensity ratio as well as R2 and R2* relaxometry techniques. Methods that are in clinical use, as well as their limitations, are described. Remaining challenges, unsolved problems, and emerging techniques to provide improved characterization of liver iron deposition are discussed. J. Magn. Reson. Imaging 2014;40:1003–1021 . © 2014 Wiley Periodicals, Inc .     

Magnetic resonance imaging and spectroscopy of hepatic iron overload

Experimental animals that had been given excess iron in their diet were studied by magnetic resonance (MR) imaging in vivo and by magnetic resonance (MR) spectroscopy in vitro. Hepatic iron overload in patients with transfusional iron excess was studied by MR imaging, and isolated iron protein fractions were studied in vitro by MR spectroscopy. The spin echo image intensity of livers with iron overload was decreased because of the extreme decreases in T2 compared with normal; T1 was decreased only moderately. The relaxation rates 1/T2 and 1/T1 both showed a linear relationship to hepatic iron levels. Ferritin solutions showed moderate decreases in T2 and mild decreases in T1. The T2 relaxivity of ferritin, which is due to the iron core rather than the apoferritin protein shell, does not appear sufficient to account for the extreme decrease in T2 observed in hepatic iron overload. Low molecular weight cytosol iron is present in lower concentrations than ferritin but potentially has much greater relaxivity and may contribute to the MR findings. These techniques may be useful in other studies of iron metabolism.     

1H NMR relaxation measurements of human tissues in situ by spatially resolved spectroscopy

Spatially resolved spectroscopy (SPARS) is a method for obtaining high-resolution NMR spectra of well-defined volumes of human tissues in situ. This method was combined with the Carr-Purcell-Meiboom-Gill multiple-echo sequence for T2, and with inversion recovery for T1 relaxation measurements. Relaxation times obtained by SPARS were compared with standard CPMG and IR relaxation measurements and with imaging methods, using a number of relaxation phantoms. Spectroscopically resolved relaxation data of human bone marrow, muscle, and adipose tissue in situ were obtained. T2 measurements of human adipose tissue gave different results when using single-echo measurements rather than the CPMG method. This difference was interpreted as a J modulation effect, which shows up in fatty acid proton resonances. This J modulation effect influences the intensity of adipose tissue in routine 1H NMR images.