Hyperpolarized esters as metabolic markers in MR
A technology of hyperpolarization and metabolites, applied in the field of 13C-MR research, which can solve problems such as undetectable
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Embodiment 1
[0117] General Carboxylesterase Activity in Example 1 Cancer Cells
[0118] General carboxylesterase activity was determined spectrophotometrically using the carboxylesterase substrate 1-naphthyl acetate (NA) as described above (Rudakova, 2011). Briefly, sonicated cells were incubated with 1 mM substrate in 150 mM phosphate buffer (pH 7.5), 1-naphthol production was monitored at 322 nm and quantified according to its extinction coefficient (2200 M-1 cm-1). The reaction was allowed to proceed at 30 degrees for 10 min. The substrate concentration and temperature are chosen so as to obtain assay conditions under which the assay exhibits linear performance.
[0119] The following is a reaction scheme for a general esterase detection assay:
[0120]
[0121] Cells from different sources (rat hepatoma, Morris; human hepatoma, HepG2; human prostate cancer, PC-3; human breast cancer, MCF-7) were harvested and redissolved in phosphate buffered saline to 10 million cells / ml conce...
Embodiment 2
[0126] Example 2. Using hyperpolarized [1,3- 13 C 2 Carboxylesterase activity determined by ]-ethyl acetoacetate
[0127] 2.a. Hyperpolarized [1,3- 13 C 2 ]-Production of ethyl acetoacetate
[0128] A) The carboxylic acid form of the Finland group (0.85 mg, 0.82 μmol) was dissolved in [1,3- 13 C 2 ]-Ethyl acetoacetate (48 μl, 50.1 mg, 0.378 mmol). To this solution was added the gadolinium complex [[α1,α4,α7-tris[(phenylmethoxy)methyl]-1,4,7,10-tetraazacyclododecane-1,4,7 , 10-tetraacetic acid (4-)] gadolinite (gadolinite) (1-)] hydrogen in DMSO solution (1.1mg100μmol / g solution). The concentrations of radical and gadolinium were 17 mM and 2.3 mM, respectively.
[0129] B) Hyperpolarization of a 30 μmol solution of A). The samples were dissolved in 5 ml of phosphate buffer (40 mM, pH 7.3). The pH after dissolution was 7.3.
[0130] 2.b. Using hyperpolarized [1,3- 13 C 2 ]-Ethyl acetoacetate in rat hepatoma cells Carboxylesterase activity measure...
Embodiment 3
[0136] Example 3. Hyperpolarized [1,3- 13 C 2 ]-Ethyl acetoacetate with hyperpolarized [1- 13 Comparison between C]-pyruvate metabolism
[0137] Copolarized [1,3- 13 C 2 ]-Ethyl acetoacetate and [1- 13 C]-pyruvate experiments were performed which resulted in concentrations of approximately 3.5 mM of each substrate in the experiments. [1,3- 13 C 2 ]- DNP preparation of ethyl acetoacetate was carried out as described in WO2006 / 011809 [1- 13DNP preparation of C]-pyruvate. The two substrates are copolarized without mixing the substrates. The substrate mixture was injected into 10 million cells in suspension after dissolving in 5 ml of phosphate buffer (40 mM pH 7.3) and adding 2.5 μl of NaOH to neutralize pyruvate (thereby converting it to the pyruvate anionic form). Experiments were performed as described in Example 2.b. Data are presented as the area under the metabolite curve divided by the substrate signal in the first spectrum and multiplied by 100 to give units, D...
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