ETA: Robust software for determination of cell specific rates from extracellular time courses

Abstract

Accurate quantification of cell specific rates and their uncertainties is of critical importance for assessing metabolic phenotypes of cultured cells. We applied two different methods of regression and error analysis to estimate specific metabolic rates from time-course measurements obtained in exponentially growing cell cultures. Using simulated data sets to compute specific rates of growth, glucose uptake, and lactate excretion, we found that Gaussian error propagation from prime variables to the final calculated rates was the most accurate method for estimating parameter uncertainty. We incorporated this method into a MATLAB-based software package called Extracellular Time-Course Analysis (ETA), which automates the analysis workflow required to (i) compute cell specific metabolic rates and their uncertainties, (ii) test the goodness-of-fit of the experimental data to the regression model, and (iii) rapidly compare the results across multiple experiments. ETA was used to estimate the uptake or excretion rate of glucose, lactate, and 18 different amino acids in a B-cell model of c-Myc-driven cancer. We found that P493-6 cells with High Myc expression increased their specific uptake of glutamine, arginine, serine, lysine, and branched-chain amino acids by 2- to 3-fold in comparison to Low Myc cells, but exhibited only modest increases in glucose uptake and lactate excretion. By making the ETA software package freely available to the scientific community, we expect that it will become an important tool for rigorous estimation of specific rates required for metabolic flux analysis and other quantitative metabolic studies.

Isotopically nonstationary 13C flux analysis of cyanobacterial isobutyraldehyde production

“…The amounts of IBA and IVA in each sample were calculated based on the ratios of the integrated IBA/nBA and IVA/nBA peak areas using linear regression from the corresponding calibration curve. To calculate specific aldehyde productivities, cell density and aldehyde concentrations at t=6hr and t=30hr were used as inputs into the publicly available software package ETA (Murphy and Young, 2013). …”

“…These values were regressed using the ETA software package to determine specific rates (Murphy and Young, 2013). The parental SA590 strain served as control.…”

13C flux analysis of cyanobacterial metabolism

“…For soluble metabolites, this involves measuring the depletion of carbon substrates and the accumulation of carbon products in extracellular medium samples collected over time, followed by regression analysis to assess the uptake/excretion rates of these metabolites (Murphy and Young 2013). Cell-free medium samples should be analyzed in parallel to control for medium evaporation or metabolite degradation.…”

Computer-Guided Metabolic Engineering

“…For instance, measurements of the time course of concentrations of extracellular metabolites can be used to determine cell-specific uptake and production rates [29]. The resulting rates can then be used as constraints for the corresponding exchange reactions used to reduce the solution space of the metabolic model describing the metabolism of the cell (see Sects.…”

“…(16)]. Uptake and formation rates r for a substance S not only correspond to the respective time point of the measurement but also to the already generated biomass c Bt [29]. The temporal alteration of biomass [Eq.…”