doi:10.1093/brain/awp202
Brain 2009: 132; 2839–2849
| 2839
BRAIN
A JOURNAL OF NEUROLOGY
The human brain utilizes lactate via the
tricarboxylic acid cycle: a 13C-labelled
microdialysis and high-resolution nuclear magnetic
resonance study
Clare N. Gallagher,1,2,* Keri L.H. Carpenter,2,3,* Peter Grice,4 Duncan J. Howe,4
Andrew Mason,4 Ivan Timofeev,2 David K. Menon,3,5 Peter J. Kirkpatrick,2 John D. Pickard,2,3
Garnette R. Sutherland1 and Peter J. Hutchinson2,3
*These authors contributed equally to this work.
Correspondence to: K.L.H. Carpenter, PhD,
Division of Neurosurgery,
Department of Clinical Neurosciences,
University of Cambridge,
Box 167, Addenbrooke’s Hospital,
Hills Road,
Cambridge CB2 0QQ,
UK
E-mail: [email protected]
Energy metabolism in the human brain is not fully understood. Classically, glucose is regarded as the major energy substrate.
However, lactate (conventionally a product of anaerobic metabolism) has been proposed to act as an energy source, yet whether
this occurs in man is not known. Here we show that the human brain can indeed utilize lactate as an energy source via the
tricarboxylic acid cycle. We used a novel combination of 13C-labelled cerebral microdialysis both to deliver 13C substrates into
the brain and recover 13C metabolites from the brain, and high-resolution 13C nuclear magnetic resonance. Microdialysis
catheters were placed in the vicinity of focal lesions and in relatively less injured regions of brain, in patients with traumatic
brain injury. Infusion with 2-13C-acetate or 3-13C-lactate produced 13C signals for glutamine C4, C3 and C2, indicating tricarboxylic acid cycle operation followed by conversion of glutamate to glutamine. This is the first direct demonstration of brain
utilization of lactate as an energy source in humans.
Keywords: brain metabolism;
13
C-labelling; microdialysis; NMR; traumatic brain injury (human)
Received April 5, 2009. Revised June 12, 2009. Accepted June 21, 2009. Advance Access publication August 20, 2009
ß The Author (2009). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved.
For Permissions, please email: [email protected]
Downloaded from brain.oxfordjournals.org by guest on May 17, 2011
1 Division of Neurosurgery, Department of Clinical Neurosciences, University of Calgary, 1403, 29th Street NW, Calgary, Alberta T2N 2T9,
Canada
2 Division of Neurosurgery, Department of Clinical Neurosciences, University of Cambridge, Box 167, Addenbrooke’s Hospital, Hills Road,
Cambridge CB2 0QQ, UK
3 Wolfson Brain Imaging Centre, Department of Clinical Neurosciences, University of Cambridge, Box 65, Addenbrooke’s Hospital, Hills Road,
Cambridge CB2 0QQ, UK
4 Department of Chemistry, University of Cambridge, University Chemical Laboratory, Lensfield Road, Cambridge CB2 1EW, UK
5 Division of Anaesthesia, Department of Medicine, University of Cambridge, Box 93, Addenbrooke’s Hospital, Hills Road, Cambridge CB2
0QQ, UK
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| Brain 2009: 132; 2839–2849
C. N. Gallagher et al.
Abbreviations: Ac = acetate; ATP = adenosine triphosphate; C = carbon; CAD = cranial access device; CNS = central nervous system;
CoA = coenzyme A; CTO = craniotomy; D2O = deuterium oxide; Glc = glucose; Gln = glutamine; Glt = glutamate; Lac = lactate;
NMR = nuclear magnetic resonance; p.p.m. = parts per million; PPP = pentose phosphate pathway; Pyr = pyruvate; ROI = region of
interest; TBI = traumatic brain injury; TCA = tricarboxylic acid
Introduction
Figure 1 Schematic of the proposed mechanism of metabolic
trafficking in the brain. Glucose from the vasculature is
metabolized by astrocytes to lactate in the cytosol. Lactate is
then exported into the extracellular fluid, and taken up by
neurons, which utilize it via the TCA cycle. This first involves
conversion (in the neuron’s cytosol) of lactate to pyruvate,
which is transported into the mitochondrion, and converted
into acetyl-CoA, which then feeds into the TCA cycle. A
portion of the TCA cycle intermediate a-ketoglutarate is
converted into the spin-off product glutamate. The neuron’s
glutamate is predominantly stored intracellularly within vesicles.
Glutamate molecules are released from the pre-synaptic neuron
into the synaptic cleft where they can stimulate glutamate
receptors on the post-synaptic neuron. Within the synaptic
cleft, ‘resting’ glutamate concentrations are low (1–10 mM),
and reach 100–1000 mM briefly during neurotransmission
(McKenna, 2007). Leftover extracellular glutamate is taken
up by astrocytes, converted into glutamine (by glutamine
synthetase in the cytosol and endoplasmic reticulum)
(Norenberg and Martinez-Hernandez, 1979) and exported into
the extracellular fluid. Glutamine in the extracellular fluid can
then be taken up by neurons and converted back into
glutamate, by glutaminase in the mitochondria and cytosol
(Aoki et al., 1991). Outside the synaptic cleft, as measured by
microdialysis, extracellular concentrations of glutamate are
typically low (1–20 mM), rising to 100–200 mM in ischaemia,
while glutamine concentrations are typically 400–1000 mM
(Hutchinson et al., 2002; Samuelsson et al., 2007).
Downloaded from brain.oxfordjournals.org by guest on May 17, 2011
Controversy exists over how metabolism is coupled between
neurons and glia, and how brain metabolism responds to injury
or disease. Moreover, much of our current knowledge of brain
chemistry is based on animal models or cell cultures and more
in vivo evidence in humans is required.
Originally, glucose taken up from the bloodstream was regarded
as a universal fuel for brain cells, creating lactate as a waste
product. However, more recently, the idea of metabolic trafficking
between cell types has evolved (Fig. 1). Notably, Magistretti
and Pellerin have described a model in which glucose from the
vasculature is metabolized by glial cells to lactate, which is then
transported to neurons for use in the tricarboxylic acid (TCA) cycle
(Pellerin and Magistretti, 1994; Pellerin et al., 1998; Magistretti
et al., 1999; Pellerin et al., 2007). Glutamate, produced by neurons, is taken up by glial cells and converted to glutamine and
cycled back to neurons, termed the glutamine–glutamate cycle.
Recent results using 13C-labelled acetate, lactate and glucose in
rats without brain injury, have suggested that all three substrates
can be taken up from the vasculature and used as energy
sources—lactate primarily by neurons and acetate by glial
cells, while glucose can be utilized by both cell types (Tyson
et al., 2003).
The specific high-affinity glial Na+-dependent glutamate transporters GLAST and GLT-1 mediate uptake of glutamate from the
synaptic cleft into astrocytes (Danbolt, 2001). Sodium ions (Na+)
are co-transported with glutamate. Though the precise stoichiometry is debated, it appears that one molecule of glutamate is
imported into the cell along with two or three Na+ and one H+,
in exchange for export of one K+ and one OH (or one HCO3 )
(Pellerin and Magistretti, 1994; Danbolt, 2001). This influx of Na+
increases the activity of Na+, K+ ATPase (sodium pump), which
in turn stimulates glycolysis, i.e. glucose utilization and lactate
production. The Na+, K+ ATPase family consists of many
isoenzymes. In rat cerebral cortex, the Na+, K+ ATPase a2 subunit
is almost entirely co-localized with GLAST and GLT-1 (Cholet
et al., 2002). Glutamate uptake by astrocytes is essential to
terminate its effect as a neurotransmitter and to prevent
extracellular glutamate from reaching excitotoxic levels (Pellerin
and Magistretti, 1994).
Microdialysis is already established in neurocritical care for monitoring patients’ brain chemistry by measurement of endogenous
(unlabelled) energy-related molecules (Bhatia and Gupta, 2007;
Belli et al., 2008). The microdialysis catheter, whose outer wall
at the distal end consists of dialysis membrane, is inserted into
the cerebral parenchyma (Supplementary Fig. 1). Molecular
exchange by diffusion takes place bi-directionally across the
membrane, between the perfusion fluid inside the catheter and
the cerebral extracellular space. In conventional microdialysis, a
‘physiological’ artificial solution of salts, termed CNS perfusion
fluid, is employed to collect endogenous molecules that diffuse
into the catheter from the extracellular space (Tisdall and Smith,
2006). Contrastingly, in the present study, the CNS perfusion fluid
was supplemented with a 13C-labelled substrate (lactate, acetate
or glucose) that diffused through the microdialysis membrane
into the brain’s extracellular space, where it was available for
metabolism by cells. Labelled metabolites from the extracellular
space then diffused across the microdialysis membrane into the
catheter, and the fluid emerging (termed microdialysate) from
the catheter was collected in a vial, allowing us to sample brain
chemistry and evaluate cerebral metabolism. Nuclear magnetic
13C-labelling
and microdialysis in TBI
resonance (NMR) spectroscopy of the microdialysates enabled
identification of metabolites and determination of the positions
of the 13C-label within the metabolite molecules, thereby shedding
light on biochemical pathways.
One aspect of brain chemistry requiring better understanding
is that occurring after traumatic brain injury (TBI), a devastating
condition affecting thousands of individuals of all ages each year,
and the largest single cause of mortality in the age group under
40 years in developed countries. Survivors of severe TBI are left
with varying degrees of disability. The primary physical injury
caused by impact is followed over the ensuing hours and days
by secondary injury mediated by a complex series of biochemical
and electrophysiological responses such as spreading depolarizations. The latter are in turn associated with marked changes in
glucose and lactate levels in the extracellular fluid of the human
brain (Parkin et al., 2005).
By devising the present novel combination of cerebral microdialysis with 13C metabolic labelling and NMR spectroscopy
of the microdialysates, we developed a methodology by which
local brain metabolism could be continuously investigated in
patients.
Patients
The study was approved by the Cambridge Local Research Ethics
Committee and assent obtained from each patient’s next of kin.
Patients over 16 years with TBI requiring ventilation and intracranial
pressure monitoring were eligible for the study. The major exclusion
criteria were deranged clotting and/or low platelets, which precluded
placement of a microdialysis catheter. All patients were treated in the
Neurocritical Care Unit at Addenbrooke’s Hospital, Cambridge, during
the period 2007–08. The primary aim of the study was to compare
different 13C-substrates, for which we recruited 14 TBI patients, who
we divided into three groups to receive 2-13C-acetate (five patients),
3-13C-lactate (five patients), or 1-13C-glucose (four patients), by infusion via microdialysis catheters. The patients (12 males and 2 females)
were aged 17–56 years (average 31.9 years). One further TBI patient
(male, 20 years) was recruited and received non-labelled perfusion
fluid as per our standard clinical practice, to assess the natural background 13C spectrum. No non-TBI subjects were included in the study.
Patient demography details are presented in Supplementary Table 1.
Patients were treated according to the Brain Trauma Foundation
guidelines for TBI and were mechanically ventilated, paralysed and
sedated. CT scans were used to evaluate the nature of injury and to
decide placement of microdialysis catheters.
Microdialysis
Microdialysis catheters were CMA70 or CMA71 (CMA Microdialysis
AB, Solna, Sweden) with molecular weight cutoffs of 20 kDa or
100 kDa, respectively, which have previously been shown to perform
equally well in measuring small-molecule energy metabolites
(Hutchinson et al., 2005). Catheters were placed via a triple lumen
cranial access device (CAD) (Technicam, Newton Abbott, UK) in sites
not close to a focal lesion or via the bone flap at craniotomy (CTO)
with direct visualization of the injured brain and placement in
| 2841
the vicinity of injury (Supplementary Table 1). Cranial access device
catheters were oriented perpendicularly into the brain, targeting white
matter. Craniotomy catheters were oriented at a shallow angle
(i.e. tangential) relative to the brain’s surface, to target grey matter.
Catheters were perfused using CMA106 pumps at 0.3 ml/min, with
CNS perfusion fluid (CMA Microdialysis AB) composed of NaCl
(147 mM), KCl (2.7 mM), CaCl2 (1.2 mM), MgCl2 (0.85 mM) in
water, supplemented with 4 mM sodium acetate (2-13C), or 4 mM
sodium L-lactate (3-13C), or 2 mM D-glucose (1-13C). Concentrations
of 13C-labelled substrates were chosen to represent typical endogenous levels seen in brain microdialysates in unlabelled studies, to
minimize perturbation (Reinstrup et al., 2000; Hutchinson et al.,
2005; Tisdall and Smith, 2006; Hutchinson et al., 2007, 2009).
All the 13C-substrates (isotopic enrichment 5 98%, chemical
purity 5 98%) were obtained from Cambridge Isotope Laboratories
(Andover, MA) and were formulated in CNS perfusion fluid by the
Manufacturing Unit, Department of Pharmacy, Ipswich Hospital
NHS Trust (Ipswich, UK), who then tested them to verify purity,
sterility and absence of pyrogenicity, to comply with current
regulations, before releasing them for use in patients. Microdialysate
collection vials were changed hourly. The samples were then stored at
4 C (or at –20 C if storage over a weekend was necessary) prior
to NMR analysis. Unless stated otherwise, 1 day’s microdialysates
(the contents of 24 vials collected from one catheter) were pooled
for NMR spectroscopy. To determine the natural 13C background
spectrum, brain microdialysate (24 h pool, from Patient 15) obtained
by perfusion with plain CNS perfusion fluid without labelled substrates,
was also analysed by NMR. Catheter placements are coded as follows:
CAD A, cranial access device in the opposite hemisphere to the main
focal lesion; CAD B, cranial access device in the same hemisphere as
the main focal lesion but not in the immediate vicinity; CTO, craniotomy (i.e. in the vicinity of the main focal lesion). In some patients,
two catheters were placed in one craniotomy and are coded CTO 1
and CTO 2, the latter being closer to the focal lesion, though not
within the lesion itself.
NMR spectroscopy
The samples were pooled from the microdialysate collection vials into
3, 4 or 5 mm NMR tubes depending on the volume available and
made up to the required depth of solution with deuterium oxide
(D2O). 13C spectra were obtained using a Bruker Avance 500 MHz
Spectrometer (Bruker BioSpin GmbH, Karlsruhe, Germany) equipped
with a dual 1H/13C cryoprobe (CP DUL500 C/H, Bruker BioSpin
GmbH). The 13C spectra were acquired at 125.75 MHz (quarter of
the base 1H frequency) using 4096 (4k) scans with a d1 (relaxation
delay) of 3 s. Metabolite signals in the brain microdialysates were
identified by comparison of their chemical shifts (p.p.m.) to values
from NMR databases (BMRB—Biological Magnetic Resonance Bank,
University of Wisconsin; NMR Metabolomics Database, University of
Linköping; NMRShiftDB) and the literature (Jung et al., 1972; Quirt
et al., 1974; London et al., 1978; Cistola et al., 1982), and to those
of our own standards and substrates. Unlabelled L-glutamate and
L-glutamine standards (from Sigma-Aldrich, Poole, Dorset, UK)
were individually dissolved at 10 mM in CNS perfusion fluid with
and without the 13C-substrates, and run on our NMR spectrometer.
We also ran spectra for the 13C-substrates in CNS perfusion fluid, prior
to infusion into patients. These verified the positional specificity of
labelling and purity of the 13C-substrates. Signals for 13C at carbon
positions other than the nominal position of label within the molecules
were proportionally very small, consistent with the natural abundance
of 13C in the non-enriched state, which is 1.1% of all carbon atoms.
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Materials and Methods
Brain 2009: 132; 2839–2849
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| Brain 2009: 132; 2839–2849
The plain CNS perfusion fluid itself (without
infusion) gave no detectable 13C-signals.
C. N. Gallagher et al.
13
C-substrates, prior to
Results
Acetate infusion produces glutamine
Acetate is a well-described substrate for glial cells. Infusion of
2-13C-acetate was performed in a total of 10 catheters, comprising
five patients, each with two microdialysis catheters, inserted via a
cranial access device or craniotomy. Catheter placements and the
13
C-metabolite results are presented in Table 1. The predominant
signal in these spectra was that of the substrate acetate labelled at
C2 (23 p.p.m.). The presence of label in glutamine was detected
at positions C4, C3 and C2. In one case (Patient 3, CAD A) a small
peak was found at 33.46 p.p.m., ascribed to labelling of glutamate
at C4. No glutamine or glutamate signals were detected in the
2-13C-acetate substrate solution (Fig. 2B) or in the unlabelled brain
microdialysate (Patient 15, Fig. 2C). Other 13C signals in
the microdialysates resulting from 2-13C-acetate infusion included
lactate labelled at C3 and C2 for all of the catheters (Table 1), and
glucose C1-6 (see footnote of Table 1), for the majority of the
catheters. 13C-glucose and 13C-lactate were not contaminants of
the 2-13C-acetate substrate solution (Fig. 2B). However, the
13
C spectrum of the unlabelled microdialysate (Patient 15)
contained signals for glucose and lactate (see below and
Fig. 2C), so in the microdialysates resulting from infusion with
labelled substrates, glucose and lactate probably contain a
Table 1 13C-Labelled key metabolites in microdialysates and their chemical shifts (p.p.m.), resulting from perfusion with
2-13C-acetate
Identity
Lac C2
Gln C2a
Glt C4b
Gln C4
Glt C4c
Gln C3d
Ac C2
Lac C3
Standard
(p.p.m.)
Patient 1 (p.p.m.)
Patient 3 (p.p.m.)
Patient 4 (p.p.m.)
Patient 5 (p.p.m.)
Patient 6 (p.p.m.)
CTO 1
CTO 2
CAD A
CAD B
CAD A
CAD B
CAD A
CTO
CAD A
CTO
68.52
54.17
33.25
30.80
30.25
26.21
23.20
20.08
68.53
54.70
–
31.12
30.25, 29.66
27.73
23.27e
20.10
68.51
–
–
31.00
30.23
–
23.25e
20.07
68.51
–
33.46
–
–
–
23.25e
20.07
68.53
54.53
–
31.01
29.70
–
23.26e
20.09
68.53
54.48
–
30.99
29.70
27.00
23.26e
20.10
68.52
–
–
31.07
–
–
23.26e
20.09
68.48
–
–
–
–
–
23.23e
20.04
68.50
54.67
–
31.12
29.65
27.82
23.25e
20.06
68.53
54.61
–
31.08
29.66
27.45
23.27e
20.10
68.54
54.51
–
31.00
29.67
–
23.27e
20.11
CAD A and CAD B were contralateral and ipsilateral to the focal lesion, respectively. CTO 2 was closer to the focal lesion than CTO 1, in the same craniotomy. See
‘Materials and methods’ section for more placement details. Dash denotes no signal above background. Standard p.p.m. values were determined in CNS perfusion fluid
on our own NMR spectrometer. Given the known dominance of Gln over Glt in brain extracellular concentrations, the 27 and 54 p.p.m. signals in the microdialysates
above were therefore assigned as Gln C3 and Gln C2, respectively (rather than Glt C3 and Glt C2), particularly as none of them was accompanied by a clear Glt C4
signal at 33–34 p.p.m. Patient 3 (CAD A) showed a clear Glt C4 signal at 33.46 p.p.m., but none at 26–27 p.p.m. or 54 p.p.m. For further explanation, see Discussion.
13
C signals detected but not tabulated included glucose C1 b and a (96 and 92 p.p.m.), glucose C2–5 (multiple signals 69–76 p.p.m.), and glucose C6 (61 p.p.m.) for all
the above catheters, except for Patient 1 (CTO 2) and Patient 3 (CAD A) whose glucose signatures were negligible.
a Standard chemical shifts for C2 of Gln and Glt (glutamic acid monosodium salt and glutamic acid) were all at around 54 p.p.m.
b Glt C4 signal was at 33.25 p.p.m. for standard glutamic acid monosodium salt.
c Glt C4 was at 30.25 p.p.m. for standard glutamic acid. Small signals at 29.65–30.25 p.p.m., in 7 of the 10 catheters, were not clearly identified but may be due to
C4 for differing ionisation states of glutamate or glutamine.
d Standard chemical shifts for C3 of Gln and Glt (glutamic acid monosodium salt and glutamic acid) were all at around 26–27 p.p.m.
e Indicates 13C-substrate remaining in the microdialysates.
Downloaded from brain.oxfordjournals.org by guest on May 17, 2011
Microdialysates from TBI patients’ brains that had been infused
via the microdialysis catheter with perfusion fluid containing
13
C-labelled substrates underwent NMR spectroscopy to analyse
the 13C-containing metabolites. Supplementary Table 1 shows
patient demography and sampling details. A total of 24 catheters
were studied with labelled substrate in 14 TBI patients, plus one
unlabelled TBI patient whose catheter was perfused without
labelled substrate. NMR results are summarized in Tables 1
and 2 and illustrative examples of spectra are shown in Figs 2
and 3, clearly showing incorporation of label into metabolites.
There were also signals corresponding to the 13C-labelled substrate
in each case, at the expected chemical shift (p.p.m.). Spectra
obtained prior to infusion into patients for the 13C-substrates in
CNS perfusion fluid demonstrated that background signals from
contaminants were negligible. Chemical shift values (p.p.m.) for
identifying metabolites were obtained from standards run on
our own spectrometer in CNS perfusion fluid (see ‘Materials and
methods’ section), as well as from NMR databases (BMRB—
Biological Magnetic Resonance Bank, University of Wisconsin;
NMR Metabolomics Database, University of Linköping;
NMRShiftDB) and from the literature (Jung et al., 1972; Quirt
et al., 1974; London et al., 1978; Cistola et al., 1982).
Our analysis focussed on key metabolites in cerebral metabolism, and assignments of the relevant signals above background in
the microdialysates are presented in Tables 1 and 2. Several signals
not included in Tables 1 and 2 were common to the majority of
the catheters, as follows. Signals at 182.4–182.9 p.p.m. for all
catheters (except Patient 5 CAD A) were assigned as lactate C1
(standard 182.9 p.p.m.) or other carboxylate carbon. Signals
at 160.3–161.4 p.p.m. for all catheters were assigned as either
pyruvate C1 (database 161.4 p.p.m.), or, more likely, bicarbonate
(HCO3 ) (literature 161.2 p.p.m.).
13C-labelling
and microdialysis in TBI
Brain 2009: 132; 2839–2849
| 2843
Table 2 13C-Labelled key metabolites in microdialysates and their chemical shifts (p.p.m.), resulting from perfusion
with 3-13C-lactate
Identity
Lac C2
Gln C2a
Glt C4b
Gln C4
Glt C4c
Gln C3d
Ac C2
Lac C3
Standard
(p.p.m.)
Patient 2 (p.p.m.)
Patient 7 (p.p.m.)
Patient 8 (p.p.m.)
Patient 9 (p.p.m.)
Patient 10 (p.p.m.)
CAD A
CAD B
CAD A
CAD A
CTO
CAD A
CTO
CTO 1
CTO 2
68.52
54.17
33.25
30.80
30.25
26.21
23.20
20.08
68.52
–
–
–
30.24
26.43
23.25
20.09e
68.52
–
–
–
–
26.43
23.26
20.09e
68.53
–
–
–
–
26.43
23.26
20.09e
68.54
54.67
–
–
–
–
–
20.11e
68.49
54.74f
34.15, 33.46
–
–
27.12f
23.24
20.05e
68.49
54.55
–
31.04
–
26.41
23.24
20.06e
68.51
54.40
–
30.95
–
26.43
23.26
20.08e
68.50
54.49
–
31.02
–
26.41
23.24
20.06e
68.52
54.54
–
31.03
29.65
26.42
23.25
20.08e
See Table 1 footnote for explanation of superscript letters a–e, dash and standards. The signals at 33.46 p.p.m. and at 34.15 p.p.m. (Patient 8, CTO) were small but
clearly present, and may be due to C4 for differing ionization states of glutamate. Small signals at 29.65 p.p.m. (Patient 10, CTO 2) and 30.24 p.p.m. (Patient 2, CAD A)
were not clearly identified, but may be due to C4 for differing ionization states of glutamate or glutamine. fSignals at 27.12 p.p.m. and 54.74 p.p.m. in Patient 8 (CTO)
may be due to Glt C3 and Glt C2, as they were accompanied by a clear Glt C4 signal at 33-34 p.p.m. Judged by the same criteria as in Table 1, the remaining
microdialysates’ 26 and 54 p.p.m. signals in Table 2 were assigned as Gln C3 and Gln C2, respectively (rather than Glt C3 and Glt C2) as they were not accompanied by
a clear Glt C4 33-34 p.p.m. signal. For further explanation, see the ‘Discussion’ section. 13C signals detected but not tabulated included glucose (see footnote of
Table 1), for all the above catheters. In Patient 10 (CTO 2), a 27.27 p.p.m. signal (smaller than its accompanying 26.42 p.p.m. signal assigned as Gln C3 in the table)
may be due to C3 for a different ionization state of glutamine or glutamate. A small signal at 23.23–23.26 p.p.m. assigned as acetate C2 was present in all but one
(Patient 8, CAD A) of the catheters. Unidentified signals at 36–51 p.p.m. were sporadic amongst the above catheters.
Downloaded from brain.oxfordjournals.org by guest on May 17, 2011
Figure 2 Acetate is metabolized to glutamine in the human brain. (A)
13
C NMR spectrum of microdialysate from Patient 1 (CTO
catheter 1) infused with 2- C-acetate (4 mM). Plotted in parallel on the same x and y axis scale are: (B) the substrate 2-13C-acetate
(4 mM) in CNS perfusion fluid, prior to infusing, and (C) microdialysate from an unlabelled subject (Patient 15), whose microdialysis
catheter was perfused with plain CNS-perfusion fluid without labelled substrate. For numerical p.p.m. values of signals see Table 1. The
remainder (data not shown) of the spectrum, run as far as 250 p.p.m., showed signals only at 182.5 p.p.m. (lactate C1 or other
carboxylate) and 160.9 p.p.m. (bicarbonate, or pyruvate C1).
13
proportion of endogenous material. There were no striking differences in labelling results between the sites of catheter placement.
Lactate infusion produces glutamine
To investigate lactate utilization by neurons and glial cells,
3-13C-lactate infusion was performed in a total of nine catheters,
comprising five patients, four of whom had two microdialysis
catheters each, and one had a single catheter. Catheter placements (via CAD or CTO) and the 13C-metabolite results are
presented in Table 2, and illustrative examples of spectra are
shown in Fig. 3A and B. The spectra showed the substrate lactate
C3 (20 p.p.m.) as the major 13C signal. As seen above with
2-13C-acetate infusion, the 13C metabolite labelling pattern arising
from 3-13C-lactate showed glutamine C4, C3 and C2 signals,
indicative of lactate utilization via the TCA cycle (see above).
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| Brain 2009: 132; 2839–2849
C. N. Gallagher et al.
13
C NMR spectra of microdialysates from Patient 10,
CTO catheters 1 and 2, respectively, both infused with 3-13C-lactate (4 mM). Plotted in parallel on the same x- and y-axis scale are:
(C) the substrate 3-13C-lactate (4 mM) in CNS perfusion fluid, prior to infusing, and (D) microdialysate from an unlabelled subject
(Patient 15), whose microdialysis catheter was perfused with plain CNS-perfusion fluid without labelled substrate. For numerical p.p.m.
values of signals, see Table 2. The remainder (data not shown) of the spectrum, run as far as 250 p.p.m., showed signals only at
182.5 p.p.m. (lactate C1 or other carboxylate) and 160.9 p.p.m. (bicarbonate, or pyruvate C1).
Figure 3 Lactate is metabolized to glutamine in the human brain. (A and B)
Glucose infusion produces lactate
Infusion of 1-13C-glucose (2 mM) was performed in a total of
five catheters (all via CAD), comprising four patients, three of
whom had a single catheter each, and one patient who had two
catheters. Large 13C signals in these microdialysates’ spectra
corresponded to the substrate glucose C1. Also detected were
13
C signals for lactate C2 and C3, and glucose C2–C6. In contrast,
13
C signals for glutamine and glutamate were negligible.
Unlabelled microdialysate shows
endogenous lactate and glucose
One subject (Patient 15) received standard CNS perfusion fluid,
without labelled substrate. This patient, who had a diffuse injury,
and no apparent focal lesion, had a single cranial access
device catheter. The plain CNS perfusion fluid prior to infusion
gave no detectable 13C-signals. The 13C spectrum of Patient
15’s microdialysate (24-h pool) showed signals for lactate C2
(68.53 p.p.m.) and C3 (20.09 p.p.m.) as the strongest peaks.
Other 13C-signals detected were lactate C1 (182.46 p.p.m.),
bicarbonate (160.45 p.p.m.), glucose C1 b and a (95.93 p.p.m.
and a weak 92.12 p.p.m.), glucose C2–C5 (multiple signals
in the range 69–76 p.p.m.), glucose C6 (60.79 p.p.m.,
60.63 p.p.m.), and an unidentified small signal at 28.27 p.p.m.
The 13C spectrum of this unlabelled microdialysate is illustrated
alongside the labelled spectra in Fig. 2 and 3.
Unlike Patients 1–14 (who were infused with labelled substrates), the microdialysates collected for NMR from the unlabelled
Patient 15 were first analysed every hour on a bedside analyser
(ISCUS; CMA Microdialysis AB, Solna, Sweden). Average concentrations (SD) for these vials, which were subsequently pooled
for NMR, were glucose 0.67 0.26 mM, lactate 1.77 0.72 mM,
pyruvate 73.6 34.4 mM, glutamate 6.7 25.0 mM and glycerol
70.0 189.6 mM.
Discussion
This in vivo 13C-labelled microdialysis technique, with laboratory
analysis by NMR, for studying cerebral metabolism in humans has
proved to be both safe and feasible. The patients received detailed
continuous clinical monitoring throughout the 13C-labelled
Downloaded from brain.oxfordjournals.org by guest on May 17, 2011
Also, in one case (Patient 8, CTO) small signals were found at
33.46 and 34.15 p.p.m., ascribed to labelling of glutamate at C4.
No glutamine or glutamate signals were present in the substrate
solution (3-13C-lactate in CNS perfusion fluid) or in the unlabelled
microdialysate (Patient 15). Other 13C signals detected in the
microdialysates resulting from 3-13C-lactate infusion included
glucose C1-6, and lactate C2 and C1. There appeared to be a
preference for glutamine (or glutamate) C4 labelling in the
craniotomy sites compared with the cranial access device sites
(Table 2), albeit in this small group of patients.
13C-labelling
and microdialysis in TBI
microdialysis period and beyond, as part of their standard
Neurosciences Critical Care Unit routine, and there were no
adverse events, nor serious adverse events, related to the
infusions. 13C is a stable isotope and not radioactive. The presence
of 13C-labelled metabolites in the microdialysate emerging from
the brain indicates that the 13C-labelled substrates entered the
brain and were taken up by cells and metabolized. Our results
from 2-13C-acetate and 3-13C-lactate infusions are consistent
with those (from the same substrates) in animals and cell cultures,
where glutamine produced via glutamate emerging from the first
turn of the TCA cycle is labelled at C4, with C3 and C2 becoming
labelled at subsequent turns (Tyson et al., 2003; Bouzier-Sore
et al., 2006). The results of the present study reveal novel
evidence in humans for uptake of lactate from the extracellular
space by brain cells and its metabolism through the TCA cycle.
Labelling of glutamine indicates
operation of the TCA cycle
| 2845
(labelled at C4, C3 and C2) from 3-13C-lactate substrate thus
suggests entry of label, via pyruvate and acetate as intermediates, into mitochondria. Moreover, the results of 2-13C-acetate
infusion demonstrated feasibility of achieving transport of this
molecule from the catheter into the extracellular fluid, across
the cell surface membrane, into the cytosol, and then across
the mitochondrial membrane into the mitochondrial matrix
where it entered the TCA cycle. The finding of 13C signals
for lactate in the microdialysates resulting from infusion of
1-13C-glucose is consistent with glycolysis as an energy pathway
in injured brains.
Although labelled glutamine was a product of 2-13C-acetate
and 3-13C-lactate substrates, glutamate production was less
evident in the present microdialysis study. In contrast, rats that
received intravenous infusions of the above 13C-substrates
showed labelling in both glutamate and glutamine, in neocortex
tissue extracts (Tyson et al., 2003). The key NMR signal for
glutamate synthesis from the above substrates is the appearance
of label at C4. Glutamate (and hence its product glutamine)
emerging from the first turn of the TCA cycle is labelled at C4,
with C3 and C2 becoming labelled at subsequent turns (Tyson
et al., 2003; Bouzier-Sore et al., 2006). The NMR behaviour of
glutamate C4 is influenced by its proximity to the C5 carboxylate
(pKa 4.5). The C4 chemical shift can range from 30 to 35 p.p.m.
depending upon glutamate’s ionization state, and under
‘physiological’ conditions C4 is typically 33–34 p.p.m. In contrast,
glutamine C4 is around 31 p.p.m. and is less variable as there is
no carboxylate at C5. The C3 chemical shifts for glutamate and
glutamine (26–27 p.p.m.) show little difference from each other,
as evidenced by standards, and analogously for C2 of glutamate
and glutamine (54 p.p.m.). Thus the only clear differentially
diagnostic signal for glutamate (as opposed to glutamine) is
the glutamate C4 signal with a chemical shift of 33–34 p.p.m.,
if present. Given the known dominance of glutamine over glutamate in brain extracellular concentrations (see below), we have
therefore assigned the C3 and C2 signals observed in the labelled
microdialysates as glutamine rather than glutamate, except
where accompanied, as was only the case for Patient 8 (CTO),
by a clear glutamate C4 signal at 33–34 p.p.m., in which case the
C3 and C2 may be glutamate, as indicated in the footnote
of Table 2.
Glutamate and glutamine are enzymatically inter-convertible.
Endogenous glutamine concentrations in human brain microdialysates are substantially higher than those of glutamate (Hutchinson
et al., 2002; Samuelsson et al., 2007) (see below and Fig. 1).
The majority of glutamate molecules are packaged within intracellular vesicles (Ni et al., 2007). Glutamate is toxic, so extracellular
concentrations of glutamate need to be kept low, by means of
uptake by astrocytes (Pellerin and Magistretti, 1994; Schousboe
and Waagepetersen, 2005). Even so, we originally expected
that at least some label would appear in glutamate if glutamineglutamate cycling were intact, but although microdialysate
was pooled from 24 h of sampling in each case, labelling of
glutamate was lacking, judged by the absence of a clear C4
signal (33–34 p.p.m.) for all but two of the catheters. This might
be interpreted as deficiency of glutamine–glutamate cycling in the
ROIs of the catheters, or as domination of the cell population
Downloaded from brain.oxfordjournals.org by guest on May 17, 2011
Qualitative similarity is seen for the 13C-metabolites of
2-13C-acetate and 3-13C-lactate substrates in our study. These
microdialysates’ 13C signals for glutamine (C4, C3 and C2) suggest
that the TCA cycle is operating in the regions of interest (ROIs) of
the catheters, since glutamine is a product of glutamate that
is a spin-off of the TCA cycle. 3-13C-lactate substrate can be
metabolized by lactate dehydrogenase to pyruvate labelled at
C3, and then, by pyruvate dehydrogenase, to acetate labelled at
C2, which can enter mitochondria. Incorporation of acetate
labelled at C2 into citrate will label a–ketoglutarate (a TCA cycle
intermediate), and hence the ensuing spin-off glutamate and
glutamine, at C4 on the first turn of the cycle, and at C4 plus
C2 or C3 on the second turn (Tyson et al., 2003; Bouzier-Sore
et al., 2006). Another route (termed anaplerosis) into the TCA
pathway, whereby pyruvate carboxylase converts pyruvate into
oxaloacetate, a TCA cycle intermediate, will label glutamate and
therefore glutamine at C2 on the first turn of the TCA cycle, and
at C2 plus C1 on the second turn (Tyson et al., 2003; Bouzier-Sore
et al., 2006). The negligible 13C signals for glutamine and
glutamate in microdialysates resulting from 1-13C-glucose infusion
might be at least partly due to loss of 13C via the pentose
phosphate pathway (PPP). The PPP is upregulated by cellular
stress, e.g. TBI (Bartnik et al., 2007; Dusick et al., 2007).
In the first step of the PPP, C1 of glucose is removed as carbon
dioxide (Kaibara et al., 1999; Bartnik et al., 2007; Dusick et al.,
2007).
We did not quantify the percentage enrichments of 13C label in
each position. However, there were no signals for glutamine in the
13
C spectra of either the substrate solutions or the unlabelled brain
microdialysate, suggesting that the 13C-glutamine detected is
a metabolite of the 13C-labelled substrates acetate and lactate
rather than being endogenous material or a contaminant of
the substrates. 13C signals for glutamine in the microdialysates
resulting from infusion of 1-13C-glucose were negligible compared
with those resulting from 2-13C-acetate or 3-13C-lactate. We are
unaware of any alternative pathway of glutamine synthesis,
other than via the TCA cycle. The production of glutamine
Brain 2009: 132; 2839–2849
2846
| Brain 2009: 132; 2839–2849
by astrocytes/glia rather than neurons. The prevailing view of
glutamine-glutamate cycling involves trafficking between astrocytes and neurons (Fig. 1). Glutaminase and glutamine synthetase
enzymes occur within neurons (Aoki et al., 1991) and astrocytes
(Norenberg and Martinez-Hernandez, 1979), respectively.
However, glutaminase is not exclusively neuronal and also
occurs in glia (Aoki et al., 1991). Astrocytes can inter-convert
glutamine and glutamate (Schousboe et al., 1993). Moreover, in
some circumstances, astrocytes/glia can release glutamate (Bezzi
et al., 1998; Parpura and Haydon, 2000; Angulo et al., 2004;
Ni et al., 2007). So, deficiency of 13C labelling in microdialysate
glutamate C4 cannot solely be judged as paucity of neurons in
the ROIs. The glutamine–glutamate cycle is not stoichiometric;
glutamate can be consumed via the TCA cycle, as well as
being metabolized by various other biochemical pathways, in
both neurons and astrocytes/glia (McKenna, 2007). Taken
together, multiple factors may thus be relevant to the apparent
deficiency of clear glutamate C4 signal (33–34 p.p.m.) in the
microdialysates.
Influences on labelling results
The influence of catheter placement in injured versus ‘noninjured’ sites cannot be definitively answered by the present
study. All of the craniotomy catheters, which were inherently in
the vicinity of focal lesions, were tangential, i.e. targeted on grey
matter, whereas all of the cranial access device catheters were
perpendicular, and were therefore targeted on white matter.
In those patients with two cranial access device catheters
(A and B, in different hemispheres), or two craniotomy catheters
(1 and 2, within the same craniotomy), there was little difference
within-patient for the NMR results. This suggests that there may
be too small a pathophysiological differential at the cellular level
between CTO 1 and 2 sites to impact significantly on the chemistry, or likewise between CAD A and B, especially with such a
small number of patients. Future comparison of white matter
chemistry in peri–lesional versus ‘non-injured’ sites would be
achievable by comparing a perpendicularly inserted craniotomy
catheter with a cranial access device catheter. Also, in future
it might be possible to compare grey matter chemistry in perilesional versus ‘non-injured’ sites by placing the catheters
tangentially via decompressive craniectomy, since the extent of
bone removal required to relieve intracranial pressure is potentially
large enough to encompass both injured and relatively
‘non-injured’ brain.
Sedation can lower brain energy metabolism (Archer et al.,
1990). All our patients were under propofol sedation throughout
microdialysis, except Patient 10, who was allowed to wake
and was extubated before removal of the microdialysis catheters.
Even so, there was no clear 13C-glutamate labelling (evidenced
by absence of C4 signal at 33–34 p.p.m.) in this subject’s
microdialysates, although 13C-glutamine C4 was detected.
Microdialysis is selective for the extracellular pool. Brain
extracellular endogenous levels of glutamate are usually very low
(1–20 mM), whereas extracellular glutamine levels are much
higher (400–1000 mM) (Hutchinson et al., 2002; Samuelsson
et al., 2007). Normally, the vast majority of glutamate molecules
are intracellular (Ni et al., 2007). During ischaemia, extracellular
glutamate can rise to 100–200 mM, as the glutamate—glutamine
cycle is energy demanding, but even so, the extracellular
glutamine/glutamate ratio is usually much greater than one
(Hutchinson et al., 2002; Samuelsson et al., 2007). In future,
in vivo magnetic resonance spectroscopy (MRS) could be
teamed with 13C-labelled microdialysis, to measure intra- and
extra-cellular molecules.
Infusion of the 13C-substrates might in theory perturb the endogenous brain chemistry, although concentrations of 13C-substrates
were representative of levels found in previous unlabelled microdialysis studies (Reinstrup et al., 2000; Hutchinson et al., 2005;
Tisdall and Smith, 2006; Hutchinson et al., 2007, 2009). Glucose,
lactate, pyruvate, glutamate and/or glycerol in microdialysates are
routinely measured hourly for severe TBI patients using a bedside
analyser, also performed for this study’s patients after (and, for
the unlabelled patient, during) the 24 h collection period for NMR.
If endogenous levels were measured prior to the labelling period
then the 13C-substrate concentration could be individually tailored
to each patient, although logistically this would be more
complicated.
Downloaded from brain.oxfordjournals.org by guest on May 17, 2011
Microdialysis is a focal technique. Catheters were placed either via
a cranial access device or directly via a craniotomy. As the cranial
access device involves insertion of the microdialysis membrane
perpendicular to the brain surface, microdialysate may be collected
from white matter (rich in glia) proportionally more than from
grey matter (rich in neurons), thus skewing results in favour of
glial metabolism. The human cortex is 2.5 mm thick, varying
from 1 to 4 mm (Fischl and Dale, 2000). To target grey matter,
catheters were placed at craniotomy with the microdialysis
membrane oriented parallel to the cortex in the first few
millimetres. The results for 2-13C-acetate substrate showed no
obvious differences between the two modes of placement (CAD
and CTO). However, for 3-13C-lactate substrate there appeared to
be a preference for glutamine (or glutamate) C4 labelling in the
craniotomy sites compared with cranial access device, consistent
with lactate being a neuronal substrate. Our findings, albeit in
this small group of patients, thus suggest that the tissue
type encountered by the microdialysis catheter may influence
the propensity for lactate utilization via the TCA cycle.
Differences in glutamate signal between patients might be at
least partly due to spreading depolarizations. The latter have been
demonstrated in TBI patients by use of subdural strip electrodes
(Strong et al., 2002). Spreading depolarizations can propagate
from lesion sites to the more healthy surrounding tissue, so
might be able to reach microdialysis catheters even if they are
not in close proximity to a focal lesion. Glutamate release during
spreading depolarizations, even in healthy tissue, has been shown
in rats (Fabricius et al., 1993). Spreading depolarizations were also
associated with marked changes in glucose and lactate levels in
the extracellular fluid of the human brain (Parkin et al., 2005).
Moreover, clusters of prolonged spreading depolarizations were
associated with lesion progression, in patients with subarachnoid
haemorrhage (Dreier et al., 2006). Monitoring with subdural strip
electrodes was not attempted in the present study, but could be a
useful adjunct to 13C-labelled microdialysis studies in the future.
C. N. Gallagher et al.
13C-labelling
and microdialysis in TBI
| 2847
An AJVD study with intravenous 1-13C-lactate infusion in healthy
volunteers demonstrated slight net export of lactate from brain at
rest prior to infusion, shifting to slight net import of lactate into
brain on infusion at rest, and increasing net import with infusion
plus exercise (van Hall et al., 2009). Lactate import into brain was
accompanied by a decrease in net glucose uptake, and 13CO2 was
detected in blood, indicating lactate utilization. An AJVD study
of TBI patients has demonstrated brain uptake of endogenous
blood-borne lactate, with an association between more efficient
lactate uptake and better outcome (Glenn et al., 2003, 2005).
Taken together, evidence thus supports the idea that lactate can
be utilized as an energy substrate in humans by both normal
and injured brain.
To our knowledge, the results of the present study are the first
in vivo demonstration in humans that brain cells can take up
labelled lactate from the extracellular space and process it via
the TCA cycle, evidenced by the finding of labelled glutamine as
a metabolite in the microdialysates. Our results are compatible
with the astrocyte–neuron lactate shuttle hypothesis, whereby
astrocytes metabolize glucose by glycolysis to form lactate,
which is then exported into the extracellular space and taken
up by neurons to use as fuel (Pellerin and Magistretti,
1994; Pellerin et al., 1998; Magistretti et al., 1999; Pellerin
et al., 2007).
The brain’s ability to utilize lactate as an energy source may be
an evolutionary adaptation to minimize the risk of energy shortfall
when demand for rapid energy generation entails abundant
lactate production by glycolysis. The orientation of the catheters
appeared to influence the results of 3-13C-lactate infusion, with
more labelling of glutamine (or glutamate) being apparent for
the catheters targeted on grey matter rather than on white.
Microdialysis is primarily a research technique. It is increasing
our understanding of the pathophysiology of brain injury.
However, we are now using it (specifically the lactate/pyruvate
ratio) to assist in the management of patients on an individual
basis. The findings of the present study, i.e. that the human
brain utilizes lactate via the TCA cycle, contribute to our knowledge and raise the interesting issue as to whether there is an ideal
flux of lactate to optimize astrocyte–neuronal interaction and
energy metabolism. High extracellular lactate levels are generally
associated with poor outcomes following TBI (Goodman et al.,
1999). Low extracellular lactate levels, with better outcomes,
might be because astrocytic glycolysis-derived lactate is being
efficiently taken up by neurons and utilized via the TCA cycle.
Moreover, in an AJVD clinical study of TBI, high endogenous
arterial lactate concentrations were associated with poorer outcome, while better outcome was associated with higher rate of
lactate uptake by brain relative to arterial lactate concentration,
i.e. higher lactate extraction fraction (Glenn et al., 2003, 2005).
Beneficial effects of intravenous lactate infusions post-TBI
have been reported in rats (Holloway et al., 2007) and in a
small-scale patient study (Ichai et al., 2009). This might be
partly due to lactate acting as an energy fuel for neurons, assuming that they have an adequate oxygen supply and functioning
mitochondria. Astrocytes might constitute a potential primary site
of simultaneous uptake of blood-borne lactate and production
of lactate by glycolysis, followed in both instances by release of
Downloaded from brain.oxfordjournals.org by guest on May 17, 2011
Microdialysis is a continuous technique with inherently very low
flow rates. To have sufficient volume for 13C-NMR necessitated
pooling a whole day’s microdialysate collection and topping
up with D2O in the NMR tube. The NMR cryoprobe we used
possesses greater sensitivity than older technology. Very recently,
NMR micro-cryoprobes have become commercially available, for
smaller samples, enabling detailed time-course measurements
of metabolic 13C-labelling in future.
Strengths of NMR are that it identifies the position of labelled
atom within the metabolite molecule, very little sample manipulation is required, it is non-destructive, and it is prospective, not
requiring prior selection of analytes to seek. Our 13C-labelled
microdialysis technique can be used safely for severe TBI patients
without interrupting their standard medical care. The use of
appropriate substrates can potentially separate different metabolic
pools, and other 13C-labelled substrates could be used if desired.
To our knowledge, we are the first to perform 13C-labelled
microdialysis in the human brain. A 1-13C-glucose microdialysis
study has been carried out in human adipose tissue, with
microdialysate analysis by gas chromatography–mass spectrometry
(GC–MS) (Gustafsson et al., 2007). However, sample preparation
for GC–MS was much lengthier than for NMR, and moreover
GC–MS did not reveal the position of 13C within the metabolite
molecules, unlike NMR.
In the present study, quantification was not attempted, other
than noting whether 13C signals were strong or weak. A future
refinement would be to acquire the 13C-spectra quantitatively with
full spin relaxation and without nuclear Overhauser enhancement,
though would take significantly longer. Percentage 13C enrichments at individual positions within the metabolite molecules
could be measured by spin-echo difference NMR spectroscopy
(Tyson et al., 2003). A limitation to measuring low incorporations
of 13C label in metabolites is the natural abundance (nonenriched) background of 13C, which constitutes 1.1% of carbon
atoms. Double labelling (on adjacent atoms) could be used to
track specific bonds within a metabolic pathway. Chemical
shift (p.p.m.) can be affected by variables such as solvent, pH,
ionization, temperature, etc., and by whether the molecule
is free or bound (e.g. in an ester or peptide). Identification by
13
C-chemical shift can be complicated by slight differences in
p.p.m. values. 2D NMR techniques can provide more information
to help resolve ambiguity.
Previous studies have shown that 1-13C-glucose or
2-13C-acetate intravenous infusions resulted in labelled glutamate
and glutamine in the brain tissue of healthy volunteers, demonstrated by in vivo MRS (Bluml et al., 2002; Lebon et al., 2002).
However, no labelled infusions of lactate appear to have been
previously tested in humans with direct measurements in brain,
nor direct infusion into the cerebral parenchyma. In healthy
volunteers, intravenous infusions of unlabelled lactate produced
an increase of lactate levels in human brain tissue, measured by
MRS (Dager et al., 1992), and lowered the cerebral metabolic
rate of glucose, measured by FDG–PET, suggesting preferential
utilization of lactate (Smith et al., 2003). Moreover, the normal
human brain can import endogenous extra-cranial exercisegenerated lactate from the bloodstream, judged by arterial–jugular
venous difference (AJVD) (Ide et al., 2000; Larsen et al., 2008).
Brain 2009: 132; 2839–2849
2848
| Brain 2009: 132; 2839–2849
lactate for neuronal utilization (van Hall et al., 2009). A better
understanding of lactate’s function in human brain may assist in
guiding patient care, and the combination of 13C-labelling with
microdialysis may be able to help address this.
Conclusion
Here we have shown that 13C-labelled microdialysis for studying
human brain chemistry is safe and feasible, and that metabolism in
the injured human brain can be interrogated using 13C-labelled
substrates. There is much scope for future refinement of the
technique and extension to other substrates, including labelled
analogues of naturally occurring molecules or pharmaceutical
agents, to explore brain chemistry further. More research is
needed on human cerebral metabolism before we can understand
the interactions between brain cells after injury. This technique is a
potentially useful tool in that undertaking. The results of the
present study shed light on the biosynthetic pathways involved
in energy metabolism in the human brain, and include the first
direct demonstration that the human brain can utilize lactate
as an energy source via the TCA cycle.
The authors thank Mr A. Helmy for obtaining the unlabelled
patient’s microdialysates. They thank Ms L.B. Maskell for assistance with patient demography. They also thank Drs L. Bluck
and S. Jackson (MRC HNR Centre, Cambridge) for advice on
formulating the 13C substrates and Mr C. Hanson (Ipswich
Hospital NHS Trust) for supervising their formulation.
Funding
The Canadian Institute of Health Research, the Medical Research
Council (Grant Nos. G9439390 ID 65883 and G0600986
ID79068); Academy of Medical Sciences/Health Foundation;
MRC (Acute Brain Injury Programme Grant) and the National
Institute for Health Research Biomedical Research Centre,
Cambridge (to K.L.H.C.); The Evelyn Trust (to I.T.); Academy of
Medical Sciences/Health Foundation Senior Surgical Scientist
Fellowship (to P.J.H.).
Supplementary material
Supplementary material is available at Brain online.
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