Synthesis and evaluation of novel polysaccharide-Gd-DTPA compounds as contrast agent for MRI

Macromolecular conjugates of two kinds of natural polysaccharides, that from Panax quinquefolium linn (PQPS) and Ganoderma applanatum pat (GAPS), with gadolinium-diethylenetriaminepenta-acetic acid (Gd-DTPA) have been synthesized and characterized by means of FTIR, elementary analysis and ICP-AES. Their stability was investigated by competition study with Ca2+, EDTA (ethylenediaminetetraacetic acid) and DTPA. Polysaccharide-bound complexes exhibit T-1 relaxivities of 1.5-1.7 times that of Gd-DTPA in D2O at 25degreesC and 9.4T. MR imaging of Sprague-Dawley (SD) rats showed remarkable enhancement in rat liver and kidney after i.v. injection of these two complexes: liver parenchyma 60.9+/-5.6%, 57.8+/-7.4% at 65-85 min; kidney 144.9+/-14.5%, 199.9+/-25.4% at 10-30 min for PQPS-GdDTPA, GAPS-Gd-DTPA at gadolinium dose of 0.083 and 0.082 mmol/kg, respectively. Our preliminary in vivo and in vitro study indicates that the two kinds of polysaccharide-bound complexes are potential tissue-specific contrast agents for MRI.

Comparison between Gd-DTPA and several bisamide derivatives as potential MRI contrast agents

Four neutral gadolinium complexes of diethylenetriaminepentaacetic acid (DTPA)-bisamide derivatives have been synthesized and characterized. Their potential application as tissue-specific and low-osmolarity MRI contrast agents has been evaluated by in vitro and in vivo experiments. Their measured relaxivities in D2O, bovine serum albumin and human serum transferrin solutions showed favorable relaxation ability. In vivo studies have proven that Gd(DTPA-BDMA), Gd(DTPA-BIN), and Gd(cyclic-DTPA-1,2-pn) could be promising liver-specific MRI contrast agents and Gd(DTPA-BDMA), and Gd(cyclic-DTPA-1,2-pn) have favorable renal excretion capability. Among them, Gd(cyclic-DTPA-1,2-pn) is a more powerful hepatic contrast agent and Gd(DTPA-BIN) provides the stable imaging contrast for several hours. They also show a lower toxicity.

Evaluation of bioeffects of a long-term administering ChangLe on rat by proton NMR method and biochemical examination

High resolution H-1 nuclear magnetic resonance ( NMR) spectroscopy has been employed to assess long-term toxicological effects of ChangLe (a kind of rare earth complex applied in agriculture). Male Wistar rats were administrated orally with ChangLe at doses of 0, 0.1, 0.2, 2.0, 10 and 20 mg/kg body weight daily, respectively, for 6 months. Urine was collected at-day 30, 60, go and serum samples were taken after 6 months. Many low-molecular weight metabolites were identified by H-1 NMR spectra of rat urine. A decrease in citrate and an increase in ketone bodies, creatinine, DMA, DMG, TMAO, and taurine in the urine of the rats. receiving high doses were found by H-1 NMR spectra. These may mean that high-dosage of ChangLe impairs the specific region of liver and kidney, such as renal tubule and mitochondria. The decrease in citrate and the increase in succinate and alpha-ketoglutarate were attributed to a combination of the inhibition of certain citric acid enzymes, renal tubular acidosis and the abnormal fatty acid catabolism. The information of the renal capillary necrosis could be derived from the increase in DMIA, DMG and TMAO. The increase in taurine was due to hepatic mitochondria dysfunction. The conclusions were supported by the results of biochemical measure. merits and enzymatic assay.

Layer-by-layer assembly of multilayer films composed of avidin and biotin-labeled antibody for immunosensing

Protein multilayers composed of avidin and biotin-labeled antibody (bio-Ab) were prepared on gold surface by layer-by-layer assembly technology using the high specific binding constant (K-a: approximate to 10(15) M-1) between avidin and biotin. The assembly process of the multilayer films was monitored by using real-time BIA technique based on surface plasmon resonance (SPR). The multilayer films were also characterized by electrochemical impedance spectroscopy (EIS) and reflection absorption Fourier transform infrared spectroscopy (FTIR). The results indicate that the growth of the multilayer is uniform. From response of SPR for each layer, the stoichiometry S for the interaction between avidin and bio-Ab is calculated to be 0.37 in the multilayer whereas 0.82 in the first layer. The protein mass concentration for each layer was also obtained. The schematic figure for the multilayer assembly was proposed according to the layer mass, concentration and S value. The utility of the mutilayer films for immunosensing has been investigated via their subsequent interaction with hIgG. The binding ability of the multilayer increased for one to three layers of antibody, and then reach saturation after the fourth layer. These layer-by-layer constructed antibody multilayers enhance the binding ability than covalently immobilized monolayer antibody. This technology can be also used for construction of other thin films for immunosensing and biosensor.

Comparison between GdDTPA and two gadolinium polyoxometalates as potential MRI contrast agents

Two gadolinium polyoxometalates, Gd2P2W18O62 and K-15[(GdO)(3)(PW9O34)(2)], have been evaluated by in vivo as well as in vitro experiments as the candidates of tissue-specific magnetic resonance imaging (MRI) contrast agents. T-1-relaxivities of 28.4 mM(-1)-s(-1) for Gd2P2W18O62 and 11.2 mM(-1)-s(-1) for K-15[(GdO)(3)(PW9O34)(2)] (400 MHz, 25 degreesC) were higher than that of the commercial MRI contrast agent (GdDTPA). Their relaxivities in bovine serum albumin and human serum transferrin were also reported. The favorable liver-specific contrast enhancement and renal excretion capability in in vivo MRI with Sprague-Dawley rats after i.v. administration of K-15[(GdO)(3)(PW9O34)(2)] was demonstrated. In vivo and in vitro assay showed that K-15[(GdO)(3)(PW9O34)(2)] is a promising liver-specific MRI contrast agent. However, Gd2P2W18O62 did not show the favorable quality in vivo as expected from its high relaxivity in vitro, which was attributed to low bioavailability, indicating that it is of limited value as tissue-specific MRI contrast agent.

Arabinogalactan as a carrier for contrast agent in magnetic resonance imaging

Arabinogalactan-Gd-DTPA was synthesized by the reaction of diethylenetriaminepenta-acetic acid (DTPA) bisanhydride with polysaccharide in dry DMSO and characterized by FTIR, elemental analysis and ICP-AES. Its stability was investigated by competition with Ca2+, EDTA, DTPA. The t(1)-relaxivity is 8.06 mmol(-1) . L . s(-1) in D2O, 8.48 mmol(-1) . L . s(-1) in 0.725 mmol . L-1 BSA, respectively. t(1)-weighted MR imaging of rat kidney and liver showed a remarkable enhancement post injection of Arabinogalactan-Gd-DTPA. The results indicate that the arabinogalactan-Gd-DTPA is a potential contrast agent for MRI.

An evaluation of gadolinium polyoxornetalates as possible MRI contrast agent

Two gadolinium polyoxometalates, K9GdW10O36 and K-11 [Gd(PW11O39)(2)], have been evaluated both in vivo and in vitro as candidates for tissue-specific MRI contrast agents. T-1-relaxivities of 6.89 mM(-1) . s(-1) for K9GdW10O36 and 5.27 mM(-1) . s(-1) for K-11[Gd(PW11O39)(2)] are slightly higher than that of the commercial MRI contrast agent (Gd-DTPA). Both compounds bind with bovine serum albumin and human serum transferrin and favorable liver-specific contrast enhancement in in vivo MRI with Sprague-Dawley rats after i.v. administration has been demonstrated. Imaging studies demonstrate that the two agents have a long residence time, showing MR signal enhancement in the liver for more than 40 min, longer than commercially available contrast agents. In vivo and in vitro assays showed that GdW10 and Gd(PW11)(2) are promising liver-specific MRI contrast agents and GdW10 may be used in the diagnosis of the pathological state. However, with the higher acute toxicity, the two gadolinium polyoxometalates need to be modified and studied further before clinical use.

Amplified immunoassay of human IgG using real-time biomolecular interaction analysis (BIA) technology

An automated biomolecular interaction analysis instrument (BI-Acore) based on surface plasmon resonance (SPR) has been used to determine human immunoglobulin G (IgG) in real time. Polyclonal anti-human IgG antibody was covalently immobilized to a carboxymethyldextran modified gold film surface. The samples of human IgG prepared in HBS buffer were poured over the immobilized surface. The signal amplification antibody was applied to amplify the response signal. After each measurement, the surface was regenerated with 0.1 mol/L H3PO4. The assay was rapid, requiring only 30 min for antibody immobilization and 20 min for each subsequent process of immune binding, antibody amplification and regeneration. The antibody immobilized surface had good response to human IgG in the range of 0.12-60 nmol/L with a detection limit of 60 pmol/L. The same antibody immobilized surface could be used for more than 110 cycles of binding, amplification and regeneration. The results demonstrate that the sensitivity, specificity and reproducibility of amplified immunoassay using real-time BIA technology are satisfactory.

Layer-by-layer assembly of multilayer films composed of streptavidin and biotinylated antibody by real-time biomolecular interaction analysis

Layer-by-layer assembly of multilayer films of streptavidin and biotinylated antibody was completed on the streptavidin coated surface. Real-time biomolecular interaction analysis (BIA) based on surface plasmon resonance technique was used to monitor the multilayer assembly in solution continuously. The results indicate that the uniform multilayer film can be fabricated successfully based on the strong interaction between streptavidin and biotin. The mean surface mass concentration of each adsorption layer is 1. 32 ng/mm(2) for biotinylated antibody, 2. 93 ng/mm(2) for streptavidin, according to the correlation of SPR response with surface concentration.

Photoluminescent multilayer films based on polyoxometalates

Ultrathin multilayer films consisting of the polyoxotungstoeuropate cluster K-12[EuP5W30O110] (EuP5W30) and poly( allylamine hydrochloride) (PAH) have been prepared by the layer-by-layer self-assembly method. The (EuP5W30 /PAH) multilayer films have been characterized by small-angle X-ray reflectivity measurements, X-ray photoelectron spectra, and atomic force microscopy (AFM). From the AFM images, the thickness of the {PEI/PSS/PAH(EuW30/PAH)} multilayer film was estimated to be 6.5 nm, corresponding to an average thickness of ca. 1.1 nm for a EuW30/PAH layer pair. The photoluminescent behavior of the film at room temperature was investigated to show the characteristic Eu3+ emission pattern of D-5(0)-->F-7(J). The fluorescence behavior of the multilayer film is essentially identical to that of H-n[EuP5W30O110]((12-n)-) in a concentrated aqueous solution, except for the relative intensities and peak bandwidths. The occurence of photoluminescent activity confirms the potential for creating luminescent multilayers with polyoxometalates (see ref. 23).

Enhanced surface plasmon resonance immunoassay for human complement factor 4

Using an enhanced surface plasmon resonance (SPR) immunosensor, we have determined the concentration of human complement factor 4 (C4). Antibody protein was concentrated into a carboxymethyldextran-modified gold surface by electrostatic attraction force and a simultaneous covalent immobilization of antibody based on amine coupling reaction took place. The sandwich method was applied to enhance the response signal and the specificity of antigen binding assay. The antibody immobilized surface had good response to C4 in the range of 0.02-20 mug/ml by this enhanced immunoassay. The regeneration effect by pH 2 glycine-HC1 buffer was also investigated. The same antibody immobilized surface could be used more than 80 cycles of C4 binding and regeneration. In addition, the ability to determinate C4 directly from serum sample without any purification was investigated. The sensitivity, specificity and reproducibility of the enhanced immunoassay are satisfactory. The results clearly demonstrate the advantages of the enhanced SPR technique for C4 immunoassay.

H-1 NMR analysis for metabolites in serum and urine from rats administrated chronically with La(NO3)(3)

H-1 NMR spectroscopy has been used to assess long-term toxicological effects of a rare earth. Male Wistar rats were administrated orally with La(NO3)(3) at doses of 0.1, 0.2, 2.0, 10, and 20 mg/kg body wt, resp., for 3-6 months. Urine was collected at 1, 2, and 3 months and serum samples were taken after 6 months. Numerous low-M-r metabolites in rats serum and rats urine, including creatinine, citrate, glucose, ketone bodies, trimethylamine N-oxide (TMAO), and various amino acids, were identified on 400- and 500-MHz H-1 NMR spectra. La3+-induced renal and liver damage is characterized by an increase in the amounts of the excreted ketone bodies, amino acids, lactate, ethanol, succinate, TMAO, dimethylamine, and taurine and a decrease in citrate, glucose, urea, and allantoin. Information on the molecular basis of the long-term toxicity of La(NO3)(3) was derived from the abnormal patterns of metabolite excretions. An assay of some biochemical indexes and analysis of some enzymes in plasma supported NMR results.

Effect of rare earth element on the metabolism in rats by high resolution proton nuclear magnetic resonance spectroscopy

The high-field nuclear magnetic resonance (NMR) spectra can be used for the rapid multicomponent analysis in small amounts of biological fluids. In this paper, the effect of La (NO3)(3) on the rats' metabolism in urine was investigated by H-1 NMR analysis. The experimental groups of wistar rats were injected intraperitoneally with La(NO3)(3) at doses of 0.2, 2.0, 10 and 20mg/kg body weight. The remarkable variation of low molecular weight metabolites in urine has been identified by H-1 NMR spectra, in which dimethylamine, N, N-dimethylglycine, urea, alpha -ketoglutarate, trimethylamine N-oxide, succinate, citrate and amino acids have been suggested as NMR markers for renal damage and ethanol, lactate, taurine as the markers for liver damage. This work may assess its possible use in the early detection of biochemical changes associated with Rare Earth induced kidney and liver dysfunction.

Real-time immunoassay of antibody activity in serum by surface plasmon resonance biosensor

A surface plasmon resonance biosensor has been used to determine antibody activity in serum. As a model system, the interaction of mouse IgG and sheep anti-mouse IgG polyclonal antibody was investigated in real time. The factors, including pH value, ionic strength, protein concentration, influencing electrostatic adsorption of mouse IgG protein onto carboxylated dextran-coated sensor chip surface, were studied. The procedures of mouse IgG protein immobilization and immune reaction were monitored in real time. The regeneration effect using the different elution reagents was also investigated. The same mouse IgG immobilized surface can be used for 100 cycles of binding and elution with only 0.38% loss per regeneration in reactivity. The results show that the surface plasmon resonance biosensor is a rapid, simple, sensitive, accurate and reliable detection technique for real-time immunoassay of antibody activity. The assay allows antibodies to be detected and studied in their native form without any purification. (C) 2000 Elsevier Science B.V. All rights reserved.

Detection of anti-human serum albumin antibody using surface plasmon resonance biosensor

The biosensor based on surface plasmon resonance(SPR) technology is a very useful tool to study the interaction between biomolecles. The main advantages of this technique is to "visualize" macromolecular interactions directly in real time, and in a label-free mode rather than indirect methods like enzyme-linked immunosorbent assays (ELISAs). We immobilize human serum albumin (HSA) to the carboxymethyldextran-modified sensor chip surface covalently to detect the activity of anti-HSA in serum, and regenerate the surface with .1 mol/L phosphoric acid. The results show that SPR biosensor can detect the activity of anti-HSA in real-time quickly and the sensor chip can be used over 100 cycles.