Intracellular metabolic flux analysis method using substrate labeled with isotope
AssigneeAjinomoto Co., Inc.
InventorsShintaro Iwatani, Yoshihiro Usuda, Kazuhiko Matsui
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Abstract
A method for intracellular metabolic flux analysis comprising (a) culturing cells in a medium not containing any isotope-labeled substrate to a target phase of the metabolic flux analysis, (b) adding an isotope-labeled substrate to the medium, and continuing culture and collecting samples from the medium in time course, (c) measuring isotope distribution in an intracellular metabolite contained in the samples collected in time course, (d) performing a regression analysis for measured data and calculating an isotope distribution ratio in a steady state, and (e) analyzing a metabolic flux of the cultured cells by using the calculated isotope distribution ratio.
Claims11
A method for intracellular metabolic flux analysis comprising the following steps:
- (a) culturing cells in a medium not containing any isotope-labeled substrate to a target phase of the metabolic flux analysis,
- (b) adding an isotope-labeled substrate to the medium and collecting a plurality of cell samples from the isotope-labeled substrate containing medium over a period of time, wherein each cell sample is collected at a distinct and different time point over the period of time,
- (c) measuring isotope distribution of an intracellular metabolite contained in the cells of the plurality of cell samples collected over a period of time, to produce measured data,
- (d) performing a regression analysis for the measured data comprising calculating to calculate an isotope distribution ratio in a steady state, the regression analysis being performed using a specific function represented by equation (I)
- MDV(Mi)ξ={(a*tλ+b)/(d+c*tλ)}η (I);
- and
- (e) analyzing a metabolic flux of the cultured cells using the calculated isotope distribution ratio to obtain the analyzed metabolic flux, wherein MDV(Mi)ξis a mass distribution vector of a substance ξ, η is an integer of for larger, λ is a positive number, t is time, and a, b, c and d are regression parameters providing a minimum nonlinear regression error.
A method for intracellular metabolic flux analysis comprising:
- (a) adding an isotope-labeled substrate to medium of cells cultured to a target phase of the metabolic flux analysis and collecting cell samples from the isotope-labeled substrate containing medium over a period of time;
- (b) measuring isotope distribution of an intracellular metabolite contained in the cells of the collected cell samples, to produce measured data;
- and
- (c) performing a regression analysis for the measured data comprising calculating to calculate an isotope distribution ratio in a steady state using a function represented by equation (I)
- MDV(Mi)ξ={(a*tλ+b)/(d+c * tλ)}η (I);
- and
- (d) analyzing a metabolic flux of the cultured cells by using the calculated isotope distribution ratio to obtain the analyzed metabolic flux,wherein MDV(Mi)λis a mass distribution vector of a substance ξ, η is an integer of 1 or larger, λ is a positive number, t is time, and a, b, c and d are regression parameters providing a minimum nonlinear regression error.
Dependencies are inferred from the claim text.