Evaluation of two new gadolinium chelates as contrast agents for MRI

Two new gadolinium chelates were investigated for potential use as tissue-specific contrast agents for magnetic resonance imaging. In vitro measurements of stability constants, octanol/water partition coefficients and relaxation times in solutions of water and human serum albumin (HSA) were performed with each new chelate and compared with gadolinium-diethylenetriamine pentaacetic acid, Gd(DTPA). Biodistribution studies and magnetic resonance imaging in rats were used to evaluate the new chelates in vivo. The stability constants (log K) of gadolinium-N,N″-bis(3-hydroxy-6-methyl-2-pyridylmethyl)diethylenetriamine-N,N′,N″-triacetic acid, Gd(DTTA-HP), and gadolinium-1,7-13-triaza-4,10-16-trioxacyclooctadecane-N,N′,N″-triacetic acid, Gd(TTCT), were determined to be 23.65 and 18.07, respectively. These can be compared to a literature value of 22.46 for Gd(DTPA). Octanol/water partition coefficients for both complexes showed they were more lipophilic than Gd(DTPA). Gd(DTTA-HP) exhibited a smaller relaxivity in water but a larger relaxivity in 4% HSA than Gd(DTPA). Gd(TTCT) exhibited a lower relaxivity than Gd(DTPA) in both water and 4% HSA. Both complexes showed similar biodistributions to Gd(DTPA) no carrier-added concentrations. Gd(DTTA-HP) had a greater percent change in signal intensity than Gd(DTPA) on T₁-weighted spin-echo images in the heart, liver, and kidney. Percent change in signal intensity for Gd(TTCT) was lower than Gd(DTPA) in heart, liver, and kidney.     

Preparation,physico-chemical characterization,and relaxometry studies of various gadolinium(III)-DTPA-bis(amide) derivatives as potential magnetic resonance contrast agents

Macroscopic protonation constants were measured for a series of DTPA mono- and bis-amide ligands using potentiometric titrations. Proton NMR pH titrations yielded protonation populations of the various nitrogen and oxygen basic sites of the ligands for the different protonation stages. Amide formation decreased the basicity of the backbone nitrogens of the ligands and the thermodynamic stability of the corresponding Gd³⁺ chelates. Nuclear magnetic relaxation dispersion (NMRD) profiles and ESR linewidths were measured for the Gd³⁺ chelates. Some of these exhibited an elevated high field relaxivity relative to Gd(DTPA)²⁻, in response to their high molecular weight. As opposed to Gd(DTPA)²⁻, at 5°C the chemical exchange process of the single inner-sphere water molecule of the bis-amide complexes becomes so slow that it governs the paramagnetic relaxation process, causing the observed NMRD profiles to be close to those expected for the outer-sphere contribution. The chelates containing long alkyl side chains, such as Gd(DTPA-HPA₂), showed increased relaxivity values in the presence of human serum albumin (HSA), indicative of noncovalent interaction with the protein. These chelates could be useful as nonionic hepatobiliary contrast agents.     

Enhancing the relaxivity of paramagnetic coordination complexes through the optimization of the molecular electrostatic potential

The low relaxivity of paramagnetic coordination complexes limits their use as contrast agents in magnetic resonance imaging (MRI). To address this problem, we study the relationship between the molecular structure of these complexes and their relaxivity. While others have investigated the vibrational modes as molecular determinants of the electronic spin relaxation time, we focus on the analysis of the molecular electrostatic potential (MEP) of the paramagnetic coordination complex. Electrostatic forces dominate the interaction between the coordination complex and water. Hence, in addition to steric forces, the molecular electrostatic potential should be a determinant of the lifetime of the water-metal link (t_m), the internuclear distance between the water hydrogens and the metal (R), and the number of water molecules attached to the metal in the inner and outer spheres of coordination. We compute the molecular electrostatic potential for a series of model metalloporphyrins because their physical and biologic properties are well known, and they are putative magnetic resonance imaging contrast agents with affinity to neoplastic tissue. Replacing the sulfonato groups in MnTPPS4 with carboxylate groups in the ortho position of the phenyl rings attached to the meso carbons results in an electrostatic focusing field that should reduce R and increase t_m. Similar substitutions involving polar groups, including one modeled after a well-known picket-fence porphyrin, are not strong enough to generate a focusing field. Instead, these polar groups should modulate the water-metal interactions through steric interactions. Molecular dynamic simulations show a large outer sphere of coordination around the paramagnet that extends almost three times the distance of the inner sphere of coordination.     

Paramagnetic water proton relaxation enhancement: from contrast agents in MRI to reagents for quantitative "in vitro" assays.

The use of stable complexes of paramagnetic metal ions as probes in analytical applications of the NMR technique is discussed. Two examples are given that involve the determination of the total Fe3+ content of human serum transferrin (Tf) and the extent of glycation of human serum albumin (HSA). The method is based on the measurement of the longitudinal water proton relaxation times, conveniently altered following a selective chemical interaction between the substrate and the paramagnetic probe.     

Proton relaxation enhancement by means of serum albumin and poly-L-lysine labeled with DTPA-Gd3+: relaxivities as a function of molecular weight and conjugation efficiency.

A series of poly-L-lysine chains, with molecular weight ranging from 3300 up to 102,000 Da, were labeled with DTPA-Gd3+. No significant differences in longitudinal and transversal relaxivity, could be demonstrated as a function of the chain length. The R1 and R2 relaxivities were respectively 2.5 and 5 times superior to those of plain DTPA-Gd3+ (at 2.4 T). Bovine serum albumin was also labeled in a way that a wide (DTPA-Gd3+)/BSA range (3-39) was obtained. The longitudinal relaxivity, of these paramagnetically labeled albumins, increased with increasing (DTPA-Gd3+)/BSA ratios. This effect was most pronounced at very low (DTPA-Gd3+)/BSA ratios.     

An albumin-based gold nanocomposites as potential dual mode CT/MRI contrast agent

In pursuit of the biological detection applications, recent years have witnessed the prosperity of novel multi-modal nanoprobes. In this study, biocompatible bovine serum albumin (BSA)-coated gold nanoparticles (Au NPs) containing Gd (III) as the contrast agent for both X-ray CT and T₁-weighted MR imaging is reported. Firstly, the Au NPs with BSA coating (Au@BSA) was prepared through a moderate one-pot reduction route in the presence of hydrazine hydrate as reducer. Sequentially, the BSA coating enables modification of diethylenetriaminepentaacetic acid (DTPA) as well as targeting reagent hyaluronic acid (HA), and further chelation of Gd (III) ions led to the formation of biomimetic nanoagent HA-targeted Gd-Au NPs (HA-targeted Au@BSA-Gd-DTPA). Several techniques were used to thoroughly characterize the formed HA-targeted Gd-Au NPs. As expected, the as-prepared nanoagent with mean diameter of 13.82 nm exhibits not only good colloid stablility and water dispersibility, but also satisfying low cytotoxicity and hemocompatibility in the tested concentration range. Additionally, for the CT phantoms, the obtained nanocomplex shows an improved contrast in CT scanning than that of Au@BSA as well as small molecule iodine-based CT contrast agents such as iopromide. Meanwhile, for the T₁-weighted MRI images, there is a linear increase of contrast with concentration of Gd for the two cases of HA-targeted Gd-Au NPs and Magnevist. Strikingly, the nanoagent we explored displays a relatively higher r₁ relaxivity than that of commercial MR contrast agents. Therefore, this newly constructed nanoagent could be used as contrast agents for synergistically enhanced X-ray CT and MR phantoms, holding promising potential for future biomedical applications.     

Chromium(III) complexes as intermolecular probes

Metal ion complexes provide flexible paramagnetic centers that may be used to define intermolecular contacts in a variety of solution phase environments because both the charge and electronic relaxation properties of the complex may be varied. For most complex ions, there are several proton equilibria that may change the effective charge on the complex as a function of pH which in turn affects the efficacy of application for defining the electrostatic surfaces of co-solute molecules. We report here spectrophotometric and nuclear spin relaxation studies on aqueous solutions of chromium(III) complexes of EDTA, DTPA, and bis-amides of both. The effective charges available from these paramagnetic centers range from -3 to +1 and we report the pH ranges over which the effective charge is defined with confidence for application in magnetic relaxation experiments.     

Fluorescence Study of Sinapic Acid Interaction with Bovine Serum Albumin and Egg Albumin

The mechanism of interaction of protein with compounds used for preparation of matrices for matrix-assisted laser desorption ionization–mass spectrometry (MALDI-MS) methods is unknown. This paper reports the investigation of this mechanism for sinapic acid and bovine serum albumin and egg albumin. To examine these interactions in water a fluorescence method was applied. Sinapic acid can exist in three different forms, depending on pH: undissociated and with one or two deprotonated groups. pK_as of these states are: 4.47 for the COOH group and 9.21 for the OH group [1]. Therefore the interactions were examined at pH: 2.0, 6.4, and 10.5. The results show that sinapic acid at pH 10.5, being a bivalent anion, does not form any complex with these two proteins. At pH 2.0, sinapic acid, being undissociated, interacts weakly with egg albumin. Sinapic acid does not interact with bovine serum albumin at this pH. At pH 6.4, sinapic acid interacts only with bovine serum albumin. Parameters of the sinapic acid and bovine serum albumin complex were calculated based on the theory of multiple equlibria: the total number of binding sites, N = 15; the binding constant, K = 600 M ^–1; and the Hill's coefficient, j = 0.97. These parameters indicate (but not definitively because a large saturation was not obtained) that this is a simple binding of sinapic acid to bovine serum albumin with the binding sites of the same type.     

含硫氨基葡萄糖金属配合物的合成及与DNA和蛋白作用研究

合成了四种含硫氨基葡萄糖金属配合物(M-GLUS,M=Co, Cu, Ni和Zn),利用元素分析,摩尔电导,核磁共振氢谱进行了结构表征。结果表明含硫配体与这四种二价金属离子均形成了2∶1型非电解质配合物。在pH 7.08 Tris缓冲液中,采用紫外吸收光谱和荧光光谱研究了金属配合物与小牛胸腺DNA的作用机制,发现随着金属配合物量的逐渐增加,DNA电子吸收光谱的最大吸收峰呈增色效应,对配合物DNA-EB体系也能产生荧光猝灭作用,说明四种金属配合物均可与DNA发生相互作用,结合方式为部分插入;在近似生理酸度条件下,利用荧光光谱对金属配合物与HSA/BSA的结合特性进行了初步分析,发现配合物均能猝灭HSA/BSA的荧光强度,利用Scatchard方程计算了四种金属配合物与HSA/BSA的结合常数和结合位点数,表明金属配合物与血清白蛋白有较强的结合且只有1类键合位,其中Co-GLUS与蛋白的结合力最强。     

Estimation of free copper ion concentrations in blood serum using T(1) relaxation rates

The water proton relaxation rate constant R(1)=1/T(1) (at 60 MHz) of blood serum is substantially increased by the presence of free Cu2+ ions at concentrations above normal physiological levels. Addition of chelating agents to serum containing paramagnetic Cu2+ nulls this effect. This was demonstrated by looking at the effect of adding a chelating agent-D-penicillamine (D-PEN) to CuSO4 and CuCl2 aqueous solutions as well as to rabbit blood serum. We propose that the measurement of water proton spin-lattice relaxation rate constants before and after chelation may be used as an alternative approach for monitoring the presence of free copper ions in blood serum. This method may be used in the diagnosis of some diseases (leukaemia, liver diseases and particularly Wilson's disease) because, in contrast to conventional methods like spectrophotometry which records the total number of both bound and free ions, the proton relaxation technique is sensitive solely to free paramagnetic ions dissolved in blood serum. The change in R(1) upon chelation was found to be less than 0.06 s(-1) for serum from healthy subjects but greater than 0.06 s(-1) for serum from untreated Wilson's patients.     

Comparison of agarose and cross-linked protein gels as magnetic resonance imaging phantoms

Measurements of the magnetic field dependence of spin-lattice relaxation rates and the response of the water-proton signal intensity to off-resonance radio frequency fields show that the commonly used agarose phantom provides a less faithful representation for the magnetic response of tissue than does a cross-linked protein system. The origin of these differences lies in the structure and intramolecular dynamics of the macromolecular system used to make the gel. These distinctions will also cause differences in the magnetic response of the water spin system when paramagnetic relaxation agents or contrast agents are incorporated. Use of a thermally cross-linked bovine serum albumin phantom is suggested.     

High resolution NMR imaging: Gd-DTPA labeled enzyme as a probe for permeability studies in polyacrylamide gels.

The penetration of horse liver alcohol dehydrogenase (HLAD) molecules into polyacrylamide gel beads, which are used to immobilize the enzyme, was studied. HLAD was labeled with gadolinium diethylene-triamine-pentaacetic acid (Gd-DTPA), using the N-hydroxy-succinimide active ester of DTPA as a chelating agent. The HLAD-(Gd-DTPA)27 has a 3.7-fold larger longitudinal (R1) and a 14-fold larger transversal relaxivity (R2) (at 2.4 T) than the plain Gd-DTPA. A series of dry polyacrylamide gel beads, with total monomer concentration ranging from 5% to 30% were synthesized and swollen in a buffered solution of HLAD-(Gd-DTPA)27. The gel beads were examined with high resolution NMR imaging. The T1- and T2-weighted images revealed that the permeability for the labeled HLAD decreased with increasing total monomer concentration of the gel beads. These imaging results correlate fairly well with the enzymatic reactivities measured for the same range of gel beads but swollen in a solution of non labeled HLAD and NAD+ (nicotinamide adenine dinucleotide). It is concluded that Gd-labeling can be used to monitor the distribution of weakly concentrated, water soluble products in a solid matrix.     

Proton magnetic resonance imaging of diffusion of high- and low-molecular-weight contrast agents in opaque porous media saturated with water

Besides their use in contrast-enhanced proton magnetic resonance imaging (MRI), contrast agents were found to be useful as tracer molecules. Since paramagnetic ions in water have the ability to reduce the T1 of protons around them, MRI can determine the locations of Mn2+ and Gd3+ of ppm concentration in water. In opaque porous media saturated with water, MRI revealed diffusional motions of three contrast agents: MnCl2 (molecular-weight [M.W.], 126), gadolinium-diethylene-triaminepenta acetic acid (Gd-DTPA) (M.W., 743) and albumin (Gd-DTPA) (M.W., 94,000) at a diffusional displacement ratio of 9:5:2. With the aid of these contrast agents, the transport of low- to high-molecular-weight molecules in opaque water media such as living bodies can be observed using MRI.     

DTPA双酯Gd (Ⅲ)配合物的合成及其弛豫率

二乙三胺五乙酸双酐与甘醇单醚反应,得到含多氨基多羧基双酯配体,配体与GdCl₃·6H₂O反应生成非离子配合物.配体及配合物用IR、¹H NMR和元素分析进行了表征,并测试了配合物的驰豫率,其中3种配合物的弛豫率比已用于临床诊断的Gd (DTPA)强,     

An alternate mode of binding of the polyphenol quercetin with serum albumins when complexed with Cu(II)

Polyphenols find wide use as antioxidants, cancer chemopreventive agents and metal chelators. The latter activity has proved interesting in many aspects. We have probed the binding characteristics of the polyphenol quercetin–Cu(II) complex with human serum albumin (HSA) and bovine serum albumin (BSA). Fluorescence studies reveal that the quercetin–Cu(II) complex can quench the fluorescence of the serum albumins. The binding constant (K_b) values are of the order of 10⁵ M^?1 which increased with rise in temperature in case of HSA and BSA interacting with the quercetin–Cu(II) complex. Displacement studies reveal that both the ligands bind to site 1 (subdomain IIA) of the serum albumins. However, thermodynamic parameters calculated from temperature dependent studies indicated that the mode of interaction of the complexes with the proteins differs. Both ΔH° and ΔS° were positive for the interaction of the quercetin–Cu(II) complex with both proteins but the value of ΔH° was negative in case of the interaction of quercetin with the proteins. This implies that after chelation with metal ions, the polyphenol alters its mode of interaction which could have varying implications on its other physicochemical activities.     

有/无金属离子Cu2+或Zn2+参与时中药有效成分伞形花内酯与生物大分子BSA的相互作用

运用荧光光谱(FS)及紫外光谱(UV)研究了有/无金属离子Cu2 或Zn2 参与时中药有效成分伞形花内酯与生物大分子牛血清白蛋白(BSA)的相互作用。实验结果表明:有/无金属离子参与时,伞形花内酯均与BSA形成基态复合物从而猝灭BSA的内源性荧光,猝灭原因主要为静态猝灭和非辐射能量转移;金属离子参与使得KA增大,n仍维持在2左右;伞形花内酯分子能够扦插入BSA分子内部,温度以及金属离子参与对伞形花内酯与BSA分子中荧光性氨基酸残基间的空间距离r影响不大。有/无金属离子参与时伞形花内酯与BSA的作用过程均是一个熵增加和Gibbs自由能降低的自发超分子作用过程。伞形花内酯与BSA之间以静电相互作用为主,金属离子参与使伞形花内酯与BSA分子间静电相互作用增强,故ΔH对ΔG的贡献增大。     

Shear-wave detection of structural effects in aqueous solutions of bovine serum albumin and hemoglobin.

Using cylindrical quartz crystal torsional resonators operating at 39 and 75 kHz to generate shear waves in aqueous solutions of the proteins bovine serum albumin and hemoglobin and the polypeptide poly l-glutamic acid, it has been possible to determine the complex dynamic shear viscosities of the solutions. The effects of concentration, pH, and denaturation using various agents have been studied. It is possible to relate the viscosity and configurational elasticity of the solutions, to the intramolecular and intermolecular forces associated with the of the proteins at frequencies between 60 and 400 kHz and attributed to conformational changes of bovine serum albumin and the quaternary doublet interactions of hemoglobin have been confirmed and emphasized by the use of shear waves.     

Efficiency of paramagnetic relaxation enhancement in off-resonance rotating frame

Transferring from laboratory frame to off-resonance rotating frame for the (1)H spin can compensate the relaxivity loss for paramagnetic agents at the magnetic field strength higher than 3 Tesla and enhance water relaxation rate constant significantly. A comprehensive theory for calculating the relaxation rate constants in the off-resonance rotating frame is described. This theory considers the contributions from both inner shell and outer shell water. The derived relaxation rate constants and relaxation enhancement efficiency as a function of the magnetic field strength and the effective field parameters are directly correlated to the structures, dynamics and environments of paramagnetic agents. To validate the theoretical predictions, we have measured the relaxation enhancement efficiency for a series of macromolecule conjugated gadolinium chelates at 9.4 Tesla. The experimental results confirmed the theoretical predictions. The theory also predicts the relaxation enhancement for T(2)-type paramagnetic agents at high magnetic fields. Promising fields of applications include situations where T(1)- or T(2)-type paramagnetic agents are used for labeling molecular/cellular events.     

Reaction of gadolinium chelates with endogenously available ions

The extent of reaction of 153Gd-radiolabeled Gd(L) chelates with 25 mM CO23- (25 mF), PO34-, Zn2+ and Cu2+ at pH 7 was determined for L = EDTA, DTPA, DOTA, HP-DO3A, and DO3A. Gd(EDTA)- and Gd(DTPA)2- reacted (greater than 20% in 10 min) with Cu2+ and Zn2+ in the presence of PO34-. These double replacement reactions yielded precipitated GdPO4 and chelated Cu(L). Gd(HP-DO3A), Gd(DO3A) and Gd(DOTA)- were inert to reaction with all four ions at room temperature (less than or equal to 1% reaction detected). The thermodynamic binding constants of the ligands for Gd3+ and Cu2+ were found to be equal (10(20) M-1) for DO3A, while DOTA and HP-DO3A favored Gd3+ over Cu2+ by greater than or equal to 10(2) M-1. The low order of reactivity of Gd(DOTA)- and Gd(HP-DO3A) was anticipated by the binding constants, but the lack of reactivity of Gd(DO3A) is attributed to kinetic inertia. This latter property, desirable in MRI contrast agents, is promoted by the conformational stability of the tetraazacyclododecane macrocycle, which forms the backbone of the ligand. It is concluded that this class of chelates is exceptionally inert in solutions of endogenously available ions, and that thermodynamics alone is an insufficient predictor of the reactivity of the highly inert Gd complexes based on the tetraazamacrocycle.