T1 and T2 of ferritin solutions: Effect of loading factor

Proton magnetic relaxation times T₁ and T₂ were measured at field strengths from 0.05 T to 1.5 T in solutions of ferritin with loading factors from 90 to 3600 iron atoms per molecule. 1/T₂ increased linearly with field strength, as previously observed, and the slope per unit iron was approximately the same in all samples. This latter finding indicates that the field dependence of T₂ may be used as a measure of ferritin-bound iron, regardless of loading factor. A possible explanation is presented, based on the presumed antiferromagnetic structure of the ferritin core and the linear dependence of 1/T₂ on core magnetization. A nonzero contribution to 1/T₂ in the limit of low field and a contribution to 1/T₁ were also found, both of which increase linearly with loading factor for constant protein concentration; these effects represent quantum mechanical dipole-dipole relaxation of water protons either by iron atoms on the surface of the core or by the iron core itself. Finally, the extrapolated intercept at LF = 0 for both 1/T₁ and 1/T₂ indicates a contribution from a small number of iron ions bound to the protein shell. These results may help in the use of MRI to measure brain iron and possibly even ferritin loading factor.     

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.     

转铁蛋白受体1表达在弗朗西斯菌入侵巨噬细胞中的作用

目的 评估土拉弗朗西斯菌LVS感染鼠巨噬细胞期间获取铁的影响因素.方法 用表达绿色荧光蛋白(GFP)的土拉弗朗西斯菌LVS感染鼠巨噬细胞J774A.1.结合单抗的转铁蛋白受体-1用键合了Alexa594的羊抗鼠二抗显色.用实时定量PCR法检测5个铁代谢相关基凶在土拉弗朗西斯菌LVS感染或未感染J774A.1鼠巨噬细胞中的表达.用活的弗朗西斯菌和灭活菌分别感染巨噬细胞,免疫印迹分析比较Tfr1表达水平.用小十扰RNA下调转铁蛋白受体-1的表达,进而用土拉弗朗西斯菌LVS分别感染转铁蛋白受体-1表达下调的细胞和转染无关siRNA的细胞,并进行细菌计数.结果 土拉弗朗西斯菌疫苗株可以诱导转铁蛋白受体-1在宿主巨噬细胞中表达.基因表达分析显示土拉弗朗西斯菌LVS随着时间的增加通过诱导转铁蛋白受体1(Tfr1)~.节蛋白(Irp1和IRP2)主动获取铁.免疫印迹结果表明小干扰RNA对转铁蛋白受体-1的表达下调了大约75%.细菌入侵试验显示,在感染1h时,转铁蛋白受体-1表达下调的细胞内细菌数量等同于对照(F=1.06,P=0.326 5);而在感染24h时,Tfr1下调样本中的细菌数量明显低于对照样本(F=24.12,P=0.000 6).结论 在感染早期Tfr1的上调是由翻译后调节介导的,并随着Irp1和IRP2表达的增加而增加.Tfr1表达的增加通过转铁蛋白介导的铁运输扩充了细胞内动态铁池,使弗朗西斯菌易于获取铁.转铁蛋白受体-1的下调不影响细菌与其他膜蛋白的结合而入侵,但抑制细菌在细胞内的增殖.     

Relaxometry and magnetometry of ferritin

By combining nuclear magnetic relaxometry on 39 ferritin samples with different iron loading with magnetometry, results were obtained that suggest a new interpretation of the core structure and magnetic properties of ferritin. These studies provide evidence that, contrary to most earlier reports, the ferritin core is antiferromagnetic ( AFM ) even at body temperature and possesses a superparamagnetic (SPM) moment due to incomplete cancellation of antiparallel sublattices, as predicted by Néel's theory. This moment also provides a likely explanation for the anomalous T₂ shortening in ferritin solution. However, the number of SPM moments derived from this model is less than the number of ferritin molecules determined chemically, and a similar discrepancy was found by retrospectively fitting previously published magnetometry data. In other words, only a fraction of the ferritin molecules seem to be SPM. The studies also provide evidence for paramagnetic (PM) Curie-Weiss iron ions at the core surface, where the local Néel temperature is lower; these ions are apparently responsible for the weaker T₁ shortening. In fact, the conversion of uncompensated AFM lattice ions to PM i ons could explain the small number of SPM particles. The apparent Curie Law behavior of ferritin thus appears to be a coincidental result of different temperature dependences of the PM and SPM components.     

Toxicity,biodistribution, and ex vivo MRI detection of intravenously injected cationized ferritin

The goal of the work was to establish the toxicity and biodistribution of the superparamagnetic protein cationized ferritin (CF) after intravenous injection. Intravenously injected CF has been used to target the extracellular matrix with high specificity in the kidney glomerulus, allowing measurements of individual glomeruli using T‐weighted MRI. For the routine use of CF as an extracellular matrix‐specific tracer, it is important to determine whether CF is toxic. In this work, we investigated the renal and hepatic toxicity, leukocyte count, and clearance of intravenously injected CF. Furthermore, we studied CF labeling in several organs using MRI and immunohistochemistry. Serum measurements of biomarkers suggest that intravenous injection of CF is neither nephrotoxic nor hepatotoxic and does not increase leukocyte counts in healthy rats at a dose of 5.75 mg/100 g. In addition to known glomerular labeling, confocal and MRI suggest that intravenously injected CF labels the extracellular matrix of the hepatic sinusoid, extracellular glycocalyx of alveolar endothelial cells, and macrophages in the spleen. Liver T values suggest that CF is cleared by 7 days after injection. These results suggest that CF may serve as a useful contrast agent for detection of a number of structures and functions with minimal toxicity. Magn Reson Med, 2013. © 2012 Wiley Periodicals, Inc.     

Magnetic and relaxation properties of multifunctional polymer‐based nanostructured bioferrofluids as MRI contrast agents

A series of maghemite/polymer composite ferrofluids with variable magnetic core size, which show a good efficiency as MRI contrast agents, are presented. These ferrofluids are biocompatible and can be proposed as possible platforms for multifunctional biomedical applications, as they contain anchoring groups for biofunctionalization, can incorporate fluorescent dyes, and have shown low cellular toxicity. The magnetic properties of the ferrofluids have been determined by means of magnetization and ac susceptibility measurements as a function of temperature and frequency. The NMR dispersion profiles show that the low frequency behavior of the longitudinal relaxivity r₁ is well described by the heuristic model of ¹H nuclear relaxation induced by superparamagnetic nanoparticles proposed by Roch and co‐workers. The contrast efficiency parameter, i.e., the nuclear transverse relaxivity r₂, for samples with d > 10 nm assumes values comparable with or better than the ones of commercial samples, the best results obtained in particles with the biggest magnetic core, d = 15 nm. The contrast efficiency results are confirmed by in vitro MRI experiments at ν = 8.5 MHz, thus allowing us to propose a set of optimal microstructural parameters for multifunctional ferrofluids to be used in MRI medical diagnosis. Magn Reson Med, 2011. © 2011 Wiley Periodicals, Inc.     

A multinuclear MR study of Gd-EOB-DTPA: Comprehensive preclinical characterization of an organ specific MRI contrast agent

The characterization of the hepatobiliary contrast agent Gd-EOB-DTPA (gadolinium 3, 6, 9-triaza-3, 6, 9-tris(carboxymethyl)-4-(4-ethoxybenzyl)-undecandicarboxylic acid) in various media (water solution, protein containing solution, phosphorylated metabolites solution, and excised and perfused liver) was performed using different NMR approaches: water ¹H nuclear magnetic relaxation dispersion profiles, ²H NMR longitudinal and transverse relaxation rates of labeled complex, water ¹⁷O transverse relaxation rates and chemical shifts, ³¹P relaxation rates and peak area of phosphorylated metabolites. The higher proton relaxivity of Gd-EOB-DTPA in water compared with Gd-DTPA is related to a shorter distance (r) between the water proton and the gadolinium ion and to a longer rotational correlation time (T_R) of the hydrated complex. Although the thermodynamic stability of Gd-EOB-DTPA is identical to the one of Gd-DTPA, its kinetic stability in solutions containing phosphorylated metabolites (ATP, phosphocreatine, and inorganic phosphate) as measured by ³¹P relaxation rates analysis is higher than for the parent compound. Gd-EOB-DTPA binds noncovalently to serum proteins. Its interaction with human serum albumin is characterized by a dissociation constant of 1-4.1 mM as calculated from proton and deuterium relaxation rates and equilibrium dialysis. This noncovalent interaction involves the subdomain IIA of human serum albumin. ³¹P spectroscopy of the excised and perfused rat livers was used to monitor the uptake of Gd-EOB-DTPA by the hepatocytes where it enhances the nuclear relaxation of the intracellular metabolites without impairing the adenosine triphosphate metabolism of the cells.     

Assessment of iron distribution between liver,spleen, pancreas,bone marrow,and myocardium by means of R2 relaxometry with MRI in patients with β‐thalassemia major

Purpose

To investigate the correlation between the degree of hepatic, splenic, pancreatic, vertebral bone marrow (VBM), and myocardial siderosis, as expressed by relaxation rate (R2 = 1/T2) values, in patients with thalassemia.

Materials and Methods

R2 relaxation rate values of liver, spleen, VBM, pancreas, and myocardium were estimated in 68 consecutive transfusion‐dependent patients with β‐thalassemia major and 10 healthy controls using a respiratory triggered 16‐echo Carr‐Purcell‐Meiboom‐Gill (CPMG) spin echo sequence.

Results

Hepatic R2 values were significantly increased in all 68 patients; VBM, pancreatic, and myocardial R2 values were increased in 67/68, 35/47, and 47/61 patients, whereas five patients showed decreased pancreatic R2 attributed to fatty degeneration. Of the 39 nonsplenectomized patients, splenic R2 values were decreased in 30 and normal in nine patients. Hepatic R2 values correlated with splenic (r = 0.63, P < 0.001), VBM (r = 0.52, P < 0.001), but not with myocardial and pancreatic R2 values.

Conclusion

Despite positive correlations between the degree of hepatic, splenic, and VBM siderosis, as expressed by respective R2 values, there was variability of iron distribution patterns in thalassemic patients. Unpredictable patterns of iron distribution may be seen, such as normal signal of the spleen in the presence of siderotic liver, resembling primary hemochromatosis. Fatty degeneration of the pancreas was not uncommon. J. Magn. Reson. Imaging 2009;29:853–859. © 2009 Wiley‐Liss, Inc.     

Polystyrene microsphere–ferritin conjugates: A robust phantom for correlation of relaxivity and size distribution

In vivo iron load must be monitored to prevent complications from iron overload diseases such as hemochromatosis or transfusion‐dependent anemias. While liver biopsy is the gold standard for determining in vivo iron load, MRI offers a noninvasive approach. MR phantoms have been reported that estimate iron concentration in the liver and mimic relaxation characteristics of in vivo deposits of hemosiderin. None of these phantoms take into account the size distribution of hemosiderin, which varies from patient to patient based on iron load. We synthesized stable and reproducible microsphere‐ferritin conjugates (ferribeads) of different sizes that are easily characterized for several parameters that are necessary for modeling such as iron content and bead fraction. T₁s and T₂s were measured on a 1.41‐T low‐resolution NMR spectrometer and followed a size‐dependent trend. Ferribeads imaged at 4.7 and 14.1 T showed that signal intensities are dependent on the distribution of ferritin around the bead rather than the iron concentration alone. These particles can be used to study the effects of particle size, ferritin distribution, and bead fraction on proton relaxation and may be of use in mimicking hemosiderin in a phantom for estimating iron concentration. Magn Reson Med, 2011. © 2010 Wiley‐Liss, Inc.     

Separate MRI quantification of dispersed (ferritin‐like) and aggregated (hemosiderin‐like) storage iron

A new MRI method is proposed for separately quantifying the two principal forms of tissue storage (nonheme) iron: ferritin iron, a dispersed, soluble fraction that can be rapidly mobilized, and hemosiderin iron, an aggregated, insoluble fraction that serves as a long‐term reserve. The method utilizes multiple spin echo sequences, exploiting the fact that aggregated iron can induce nonmonoexponential signal decay for multiple spin echo sequences. The method is validated in vitro for agarose phantoms, simulating dispersed iron with manganese chloride, and aggregated iron with iron oxide microspheres. To demonstrate feasibility for human studies, preliminary in vivo data from two healthy controls and six patients with transfusional iron overload are presented. For both phantoms and human subjects, conventional R₂ and R₂* relaxation rates are also measured in order to contrast the proposed method with established MRI iron quantification techniques. Quantification of dispersed (ferritin‐like) iron may provide a new means of monitoring the risk of iron‐induced toxicity in patients with iron overload and, together with quantification of aggregated (hemosiderin‐like) iron, improve the accuracy of estimates for total storage iron. Magn Reson Med 63:1201–1209, 2010. © 2010 Wiley‐Liss, Inc.     

Spin-echo MRI underestimates functional changes in microvascular cerebral blood plasma volume using exogenous contrast agent.

While most functional MRI studies using exogenous contrast agent employ gradient-echo (GE) signal, spin echo (SE) imaging would represent an attractive alternative if its detection power were more comparable with GE imaging. This study demonstrates that SE methods systematically underestimate functional changes in microvascular cerebral blood plasma volume (CBV), so that SE detection power in brain tissue cannot match that provided by GE signal. Empirically, the in vivo response of SE-CBV was about 40% smaller than that of GE-CBV in rat brain at low basal values of CBV, a result that is consistent with physics predictions under the simplifying assumption of uniform vessel dilation. However, increasing values of basal CBV were associated with monotonically increasing mean vessel sizes and monotonically decreasing GE to SE ratios of functional changes in CBV (fCBV). This result suggests the presence of large but weakly reactive conduit vessels at high basal values of CBV. Hence, we conclude that GE imaging is the method of choice for functional MRI (fMRI) using exogenous contrast agent in most cases, although SE methods may represent a more spatially linear representation of underlying neural activity that becomes most apparent in regions with high basal CBV, such as the cortical surface.     

Feasibility of in vivo identification of endogenous ferritin with positive contrast MRI in rabbit carotid crush injury using GRASP.

In vivo markers that allow for detection of ferritin within atheromatous plaque may be useful for identifying iron-catalyzed hydroxyl-radical formation and subsequent lipid peroxidation. Recently, a positive contrast MR technique--GRadient echo Acquisition for Superparamagnetic particles/suscePtibility (GRASP)--was used to identify the presence of magnetic entities in phantom models. The aim of the current study was to determine the feasibility of using GRASP in conjunction with conventional T(2) (*)-weighted (T(2) (*)W) gradient-echo (GRE) sequences for identifying ferritin/hemosiderin deposition using in vitro and in vivo models of thrombus. In vitro thrombi were prepared by incubating blood with ferritin. MRI was performed using conventional GRE sequences and GRASP. The results indicate that GRASP was able to verify ferritin deposition in in vitro thrombi. In vivo thrombi were created using a crush injury model in rabbits. The signal enhancement obtained using conventional GRE sequences and GRASP was compared with the location of iron deposition by histology. In all of the animals the GRASP signal correlated with signal loss by conventional GRE, and ferritin/hemosiderin deposition by histology. GRASP sequences in combination with conventional GRE sequences may be used to detect the presence of ferritin deposition in in vitro thrombi and in vivo crush-injured rabbit carotid arteries.     

lacZ as a genetic reporter for real‐time MRI

Molecular imaging based on MRI is currently hampered by the lack of genetic reporters for in vivo imaging. We determined that the commercially available substrate S‐Gal? can be used to detect genetically engineered β‐galactosidase expressing cells by MRI. The effect and specificity of the reaction between β‐galactosidase and S‐Gal? on MRI contrast were determined both in vitro and in vivo. β‐galactosidase activity in the presence of S‐Gal? resulted in enhanced T₂ and T*₂ MR‐contrast, which was amplified with increasing magnetic field strengths (4.7‐17.6 T) in phantom studies. Using both lacZ⁺ transgenic animals and lacZ⁺ tissue transplants, we were able to detect labeled cells in live animals in real time. Similar to phantom studies, detection of the labeled cells/tissues in vivo was enhanced at high magnetic fields. These results demonstrate that the genetic reporter, lacZ, can be used as an in vivo marker gene using high‐field‐strength MRI. Magn Reson Med, 2010. © 2010 Wiley‐Liss, Inc.     

Design and characterization of a new irreversible responsive PARACEST MRI contrast agent that detects nitric oxide.

Irreversible responsive PARAmagnetic Chemical Exchange Saturation Transfer (PARACEST) MRI contrast agents constitute a new type of agent for molecular imaging. To investigate the utility of this approach, a novel PARACEST MRI contrast agent, Yb(III)-(1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid)-orthoaminoanilide (Yb-DO3A-oAA), was developed that detects nitric oxide (NO). The agent exhibited two CEST effects at -11 ppm and +8 ppm, which were assigned to chemical exchange from amide and amine functional groups, respectively. The effects of pH, temperature, and concentration were investigated to characterize the complex and to optimize PARACEST detection. This responsive PARACEST MRI contrast agent incurred an irreversible covalent change in the presence of NO and O(2), which caused an irreversible disappearance of both PARACEST effects from MR images. The NO-dependent response of a relaxivity-based MRI contrast agent, Gd-DO3A-oAA, was investigated for comparison. This report highlights the advantages of irreversible MRI contrast agents, demonstrates that large changes in PARACEST can be used to create a highly responsive agent, and indicates challenges that must be overcome to apply this type of contrast agent to in vivo biomedical applications in molecular imaging.     

珠蛋白生成障碍性贫血妊娠妇女血清转铁蛋白受体的测定

目的通过测定妊娠期珠蛋白生成障碍性贫血(地中海贫血)妇女与正常妊娠妇女、缺铁性贫血妊娠妇女的可溶性血清转铁蛋白受体(sTfR)水平,探讨sTfR值对诊断妊娠期地中海贫血的价值。方法随机选择妊娠20~24周,年龄24~30岁,排除合并其他疾病的妇女,应用溶血百分比和基因检测法诊断α和β地中海贫血,ELISA法检测sTfR值并进行统计学分析。结果妊娠期地中海贫血妇女的sTfR值为18·614±8·464nmol/L,低于临界值(28·1nmol/L,P<0·05),属于正常范围。妊娠期地中海贫血妇女的sTfR值明显低于正常妊娠妇女(P<0·01)。结论通过sTfR值能准确诊断妊娠妇女的缺铁性贫血,但妊娠妇女sTfR浓度降低的原因及其降低对地中海贫血的影响有待于进一步研究。     

Viral capsids as MRI contrast agents.

Viral capsids have the potential for combined cell/tissue targeting, drug delivery, and imaging. Described here is the development of a viral capsid as an efficient and potentially relevant MRI contrast agent. Two approaches are outlined to fuse high affinity Gd(3+) chelating moieties to the surface of the cowpea chlorotic mottle virus (CCMV) capsid. In the first approach, a metal binding peptide has been genetically engineered into the subunit of CCMV. In a second approach gadolinium-tetraazacyclododecane tetraacetic acid (GdDOTA) was attached to CCMV by reactions with endogenous lysine residues on the surface of the viral capsid. T(1) and T(2) ionic relaxivity rates for the genetic fusion particle were R1 = 210 and R2 = 402 mM(-1)s(-1) (R2 at 56 MHz) and for CCMV functionalized with GdDOTA were R1 = 46 and R2 = 142 mM(-1)s(-1) at 61 MHz. The relaxivities per intact capsid for the genetic fusion were R1 = 36,120 and R2 = 69,144 mM(-1)s(-1) (R2 at 56 MHz) and for the GdDOTA CCMV construct were R1 = 2,806 and R2 = 8,662 mM(-1)s(-1) at 61 MHz. The combination of high relaxivity, stable Gd(3+) binding, and large Gd(3+) payloads indicates the potential of viral capsids as high-performance contrast agents.