Author ORCID Identifier

0000-0001-7113-5111

Document Type

Other

Disciplines

Biophysics

Publication Details

Uploaded on ResearchGate

Abstract

Glycolysis pathway kinetics in cells are known to be modulated for several disorders (cancers, neurodegenerative disorders, diabetes, etc.) and are currently been targeted as a biomarker for disease diagnostics and therapy. Although there are several approaches for monitoring the pathway kinetics, ranging from simple kinetic biochemical assays to sophisticated omics techniques, there remains a significant gap in the field, as there is a noticeable compromise between the extent of insights achievable from approaches which are destructive to the sample, and the real-time, kinetic analysis of a system.

Vibrational spectroscopy is a powerful, label-free, non-invasive tool capable of sub-cellular resolution, the potential of which has been proven for studying and differentiating between the biochemical states of biological systems. Since, vibrational spectroscopy does not necessarily require the system to be in a steady state, as it does not rely on external labels and is non-invasive such that it can acquire biochemical insights non-destructively, it is hypothesised that the approach can potentially be used for real-time, sub-cellular, label-free, kinetic analysis of a system as a function of time. Raman spectroscopy is furthermore suited to the purpose since the most abundant water molecules in any biological system tend to have relatively lower signal due to the weak polarisability of the molecule.

In this project, the kinetics of the cellular glycolysis pathway were monitored as a function of time, using the conventional kinetic extracellular acidification assay for commercially available human lung (A549), liver (HepG2) and monkey kidney (LLCMK2) cell lines. Since the primary contributor to the extracellular acidification is lactate, the byproduct of cellular glycolysis pathway, the rate of extracellular acidification as a function of time can directly be correlated with the cellular glycolysis pathway kinetics. The glycolysis pathway was modulated using two well known drugs 2-deoxyglucose (inhibitor) and oligomycin (stimulator) and their effect on the pathway kinetics were elucidated. As the kinetic assay is limited to the end-point of the pathway, a simplistic numerical model was developed which can simulate the pathway end-point kinetics and shed light on the cascading processes leading to the phenotype. The pathway model can predict the cascading processes leading to the end-point kinetics and provides a quantitative numerical basis for comparing the observed differential pathway kinetics.

Further, to elucidate the sensitivity of Raman spectroscopy, standard glucose and lactate spectra ranging from high to low (biologically relevant) concentrations were acquired; extended multiplicative signal corrected (EMSC) and used to optimise the optimal wavenumber region for analysis. Since, the biologically relevant concentrations were not distinguishable visually the spectra were regressed using partial least squares (PLS) to elucidate the limits of detection and quantification (LOD and LOQ). The results demonstrated the suitability of the Raman spectroscopic approach for monitoring low concentrations of analytes with confidence.

The metabolites of interest (glucose and lactate) were mixed in varying concentrations according to the expected biological turnover of the metabolites over time in the extracellular medium and assigned to a simulated polynomial timescale. The spectra were analysed using the multivariate curve resolution- alternating least squares (MCR-ALS) toolbox which, as the name suggests, identified the principal components (glucose and lactate) in the spectra (MCR). Then, a using combinatorial hard and soft modelling approach, the components of each spectrum were quantitatively resolved, constrained by their kinetic evolution over the simulated timescale (ALS). The toolbox showcased the potential of kinetic Raman spectroscopy coupled with MCR-ALS for elucidating the glycolysis pathway kinetics through the extracellular medium with a greater confidence compared to the extracellular acidification-based assay.

In the future work, towards the completion of the PhD, kinetic Raman spectroscopy coupled with MCR-ALS to elucidate the kinetics of cellular glycolysis pathway through the extracellular medium is proposed. The kinetic rate equations from the numerical model developed for conventional kinetic assay can be used in the combinatorial hard-soft modelling part of ALS for accurate estimate of pathway rates. The approach is further proposed for sub-cellular kinetic analysis, thereby enriching the insight into the intracellular metabolic kinetics which can be extended to several metabolic biomarker components.

DOI

10.13140/RG.2.2.21732.28803

Funder

Science Foundation Ireland

Creative Commons License

Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License
This work is licensed under a Creative Commons Attribution-NonCommercial-Share Alike 4.0 International License.

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