Abstract
Real-time detection of the rates of metabolic flux, or exchange rates of endogenous enzymatic reactions, is now feasible in biological systems using Dynamic Nuclear Polarization Magnetic Resonance. Derivation of reaction rate kinetics from this technique typically requires multi-compartmental modeling of dynamic data, and results are therefore model-dependent and prone to misinterpretation. We present a model-free formulism based on the ratio of total areas under the curve (AUC) of the injected and product metabolite, for example pyruvate and lactate. A theoretical framework to support this novel analysis approach is described, and demonstrates that the AUC ratio is proportional to the forward rate constant k. We show that the model-free approach strongly correlates with k for whole cell in vitro experiments across a range of cancer cell lines, and detects response in cells treated with the pan-class I PI3K inhibitor GDC-0941 with comparable or greater sensitivity. The same result is seen in vivo with tumor xenograft-bearing mice, in control tumors and following drug treatment with dichloroacetate. An important finding is that the area under the curve is independent of both the input function and of any other metabolic pathways arising from the injected metabolite. This model-free approach provides a robust and clinically relevant alternative to kinetic model-based rate measurements in the clinical translation of hyperpolarized 13C metabolic imaging in humans, where measurement of the input function can be problematic.
Highlights
Magnetic resonance spectroscopy (MRS) is capable of distinguishing endogenous chemical metabolites together with xenobiotics in whole cells in vitro, in perfused organs ex vivo as well as providing non-invasive measurements in vivo
Therapy-induced changes in the forward apparent reaction rate constant have been identified as a potential early metabolic biomarker of treatment response to inhibitors of cell signaling such as phosphoinositide 3-kinase (PI3K) [8], and to chemotherapy treatment, which induced apoptosis [9]
We present a comparison of the areas under the curve (AUC) ratio method with kPL values derived from both 2-site and 3-site kinetic models for in vitro data performed across a wide variety of cancer cell types (CHL-1, HCT116 Bax-KO, HT29, SF188, SW1222, WM266-4, PC3) and for PC3 prostate cancer cells in response to the PI3K inhibitor, GDC-0941
Summary
Magnetic resonance spectroscopy (MRS) is capable of distinguishing endogenous chemical metabolites together with xenobiotics in whole cells in vitro, in perfused organs ex vivo as well as providing non-invasive measurements in vivo. [1-13C]pyruvate can be shuttled into the mitochondria and metabolized in the TCA cycle (demonstrated by the formation of [1-13C]bicarbonate, which is in equilibrium with carbon dioxide, under the action of the enzyme carbonic anhydrase (CA)), or converted to [1-13C]alanine in the cytosol by alanine transaminase (ALT) [10]. These apparent reaction rate constants are typically determined by kinetic modeling of the dynamic data using the McConnell equations (modified Bloch equations) [11]
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