(13)C-labelled microdialysis studies of cerebral metabolism in TBI patients

Abstract

Human brain chemistry is incompletely understood and better methodologies are needed. Traumatic brain injury (TBI) causes metabolic perturbations, one result of which includes increased brain lactate levels. Attention has largely focussed on glycolysis, whereby glucose is converted to pyruvate and lactate, and is proposed to act as an energy source by feeding into neurons' tricarboxylic acid (TCA) cycle, generating ATP. Also reportedly upregulated by TBI is the pentose phosphate pathway (PPP) that does not generate ATP but produces various molecules that are putatively neuroprotective, antioxidant and reparative, in addition to lactate among the end products. We have developed a novel combination of (13)C-labelled cerebral microdialysis both to deliver (13)C-labelled substrates into brains of TBI patients and recover the (13)C-labelled metabolites, with high-resolution (13)C NMR analysis of the microdialysates. This methodology has enabled us to achieve the first direct demonstration in humans that the brain can utilise lactate via the TCA cycle. We are currently using this methodology to make the first direct comparison of glycolysis and the PPP in human brain. In this article, we consider the application of (13)C-labelled cerebral microdialysis for studying brain energy metabolism in patients. We set this methodology within the context of metabolic pathways in the brain, and (13)C research modalities addressing them.

Keywords: (13)C-labelling; Brain metabolism; Human; Microdialysis; NMR; Traumatic brain injury.

Graphical abstract

Fig. 1

Simplified schematic of major energy pathways in the brain include glycolysis, which takes places in the cytosol and produces pyruvate, which enters mitochondria and is converted into acetyl CoA that enters the TCA cycle. Alternatively, pyruvate can stay in the cytosol and is converted into lactate that is exported out of the cell. The pentose phosphate pathway (PPP) takes place in the cytosol and is an alternative energy pathway that can be up-regulated after injury; it is an important source of NADPH used to produce the reduced form of glutathione (GSH) for preventing oxidative stress.

Fig. 2

Glucose (Glc) from the vasculature is metabolised to lactate (Lac) in astrocytes, exported into the extra-cellular fluid, taken up by neurons and processed (via pyruvate (Pyr) and acetate (Ac)) by the TCA cycle. This spins off glutamate (Glt), which is released and then taken up by astrocytes, which convert it to glutamine (Gln), which is released into the extra-cellular fluid and taken up by neurons, which re-convert it to glutamate (Gallagher et al., 2009). Figure originally published in Brain (Gallagher et al., 2009).

Fig. 3

Some examples of ¹³C-labelling patterns in brain metabolism, based on results with singly labelled substrates (Gallagher et al., 2009; Tyson et al., 2003). Turquoise fills indicate ¹³C atoms. Yellow highlight indicates the metabolic pathway by which ¹³C-labelled lactate is processed via the TCA cycle, emerging as ¹³C-labelled glutamate or ¹³C-labelled glutamine that can be recovered in brain microdialysates. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Upper panel: (a) Example of ¹³C NMR spectrum of brain microdialysate from a TBI patient, who received perfusion with 2-¹³C acetate (4 mM) by a microdialysis catheter via a craniotomy (CTO); red stars indicate ¹³C signals for glutamine C4, C3 and C2 indicating metabolism via TCA cycle. (b) ¹³C NMR spectrum of the 2-¹³C acetate substrate solution prior to perfusing. (c) ¹³C NMR spectrum of brain microdialysate from an unlabelled patient whose microdialysis catheter was perfused with plain perfusion fluid without labelled substrate. Lower panel: (a) and (b) Examples of ¹³C NMR spectra of brain microdialysates from a TBI patient, who received perfusion with 3-¹³C lactate (4 mM) by microdialysis catheters via a craniotomy (CTO); red stars indicate ¹³C signals for glutamine C4, C3 and C2 indicating metabolism via TCA cycle. (c) ¹³C NMR spectrum of the 3-¹³C lactate substrate solution prior to perfusing. (d) ¹³C NMR spectrum of brain microdialysate from an unlabelled patient (as in Upper panel (c)). Abbreviations: see Fig. 2 legend. For further details, see (Gallagher et al., 2009). Figures originally published in Brain (Gallagher et al., 2009).

Fig. 5

Simplified schematic of steps in glycolysis and the pentose phosphate pathway (PPP), showing ¹³C labelling patterns resulting from 1,2-¹³C₂ glucose substrate. Red fills indicate ¹³C atoms. Abbreviations: Glc-6-P, glucose-6-phosphate; 6PGL, 6-phosphogluconolactone; F6P, fructose-6-phosphate; G3P, glyceraldehyde-3-phosphate; PYR, pyruvate.

Fig. 6

Left panel: Magnetic resonance imaging (MRI) scan, using 3D MP-RAGE sequence, at 3 Tesla, showing the brain of a TBI patient with a microdialysis catheter (via a cranial access device) in position, the catheter tip indicated by an arrow (yellow). Scale bar (green) shows centimetre divisions. Image is courtesy of the Wolfson Brain Imaging Centre, Dept. of Clinical Neurosciences, University of Cambridge. Right panel: Schematic of ¹³C-labelled microdialysis. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 7

Examples of ¹³C NMR spectra of brain microdialysates from patients who received 1,2-¹³C₂ glucose (4 mM) perfused via the microdialysis catheter (Jalloh et al., unpublished data). Uninjured brain is normal-appearing brain in a patient operated on for a benign tumour elsewhere in the brain. TBI brain is from a traumatic brain injury patient with diffuse injury. The part of the spectrum illustrated in each case is for the C3 carbon of lactate. Also present in this part of the spectrum is one of the signals due to the internal standard DSS (2,2-dimethyl-2-silapentane-5-sulfonate sodium salt). The remainder of the spectrum, including the main DSS signal (at 0 ppm) is not shown. The C3 doublet indicated by red stars represents lactate doubly labelled with ¹³C, produced by glycolysis; the C3 signal for ¹³C is split into 2 peaks by coupling to ¹³C also present at the neighbouring C2 position within the same molecule. The C3 singlet indicated by green stars represents lactate singly labelled with ¹³C, produced via the PPP; the singlet also contains a contribution from endogenous lactate due to the natural abundance background of ¹³C (1.1% of all carbon atoms).