J Neurosurg 90:339–347, 1999
Hypothermia: depression of tricarboxylic acid cycle flux
and evidence for pentose phosphate shunt upregulation
TARO KAIBARA, M.SC., M.D., GARNETTE R. SUTHERLAND, M.D., F.R.C.S.(C),
FRED COLBOURNE, PH.D., AND RANDY L. TYSON, PH.D.
Departments of Neurological Sciences (Division of Neurosurgery) and Pathology, University of
Calgary, Calgary, Alberta, Canada
Object. Hypothermia is used in neurosurgery and other surgical disciplines to reduce tissue injury, but the mechanism of such protection remains elusive. The authors have endeavored to delineate the mechanism of neural protection afforded by hypothermia through a study of glucose metabolism.
Methods. Nuclear magnetic resonance spectroscopy was used to follow the carbon-13 label from [1-13C]glucose
as it was metabolized through the glycolytic and tricarboxylic acid pathways. Male Sprague–Dawley rats were
maintained at either 37.5˚C or 31˚C and infused with labeled glucose for 10, 30, 60, 100, or 200 minutes (five rats
were used for each time point and for each temperature). At the end of the infusion period, the rats’ brains were subjected to rapid freeze-funnel fixation. Water-soluble metabolites were extracted from samples of the neocortex and
hippocampus by using perchloric acid extraction. The fractional enrichment of these metabolites was used to calculate the reaction rate constant of formation and steady-state enrichment for a number of metabolites.
Hypothermia resulted in a 30 to 40% depression of metabolism (p , 0.0001) in both the neocortex and hippocampus. Steady-state fractional enrichment of metabolites was also decreased by 20 to 25% with hypothermia (p ,
0.0001), implying a loss of label during metabolism.
Conclusions. The results of this study suggest that an increased fraction of glucose metabolism was shunted
through the pentose phosphate pathway in the presence of hypothermia.
KEY WORDS • glucose metabolism • pentose phosphate pathway • hypothermia •
tricarboxylic acid cycle • nuclear magnetic resonance spectroscopy
tissue-protecting effect of lowering temperature
has interested scientists and clinicians for many
years. This property has been applied to the management of selected clinical disorders, including intra- and
extracranial vascular disease, traumatic brain injury, and
cardiac surgery.16,18,30,31,46 As an adjunct in tissue transplantation, the cooling of donor organs allows safe preservation for up to 48 hours.45 In laboratory studies, investigators have found hypothermia to be an efficacious
protectant against both global10,11,14,15,23,35 and focal3,13,52
brain ischemia. Mild hypothermia (30–36˚C), even when
instituted following an ischemic insult,15,52 provides significant behavioral14,15 and histopathological protection.
Conversely, slight increases in temperature result in exacerbation of injury compared with normothermia in animals.15 There is little doubt that hypothermia protects tissues and much has been published in studies in which
ischemic neuronal injury models are used regarding the
neurobiology of hypothermia-induced changes. However,
the actual mechanism by which hypothermia protects tissue against injury remains elusive.
It has been traditionally regarded that hypothermic neu-
T
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J. Neurosurg. / Volume 90 / February, 1999
roprotection arises through a global depression of metabolism. The principles of thermodynamics and enzyme reactions predict that decreases in temperature will reduce
molecular kinetic energy and enzymatic reaction velocities. It is presumed that as the global metabolic rate slows,
oxygen and energy demands decrease, raising the thresholds for injury during ischemia. Indirect measures of
metabolism provide evidence for this. Hypothermia uniformly results in the depression of the cerebral metabolic
rate of oxygen consumption (CMRO2).6,7,24,41 Barbiturate
medications can alter the CMRO2 to a similar degree;36
however, barbiturate neuroprotection during ischemia is
inconsistent and certainly less impressive than that provided by induced hypothermia.9,25,36 Thus, although barbiturate agents are capable of depressing oxygen metabolism to a level similar to that induced by hypothermia, it
appears that these agents do not provide comparable protection for neural tissue. Therefore, it is thought that the
depression of cellular metabolism does not fully account
for the protective effect of hypothermia.
Proton (1H) nuclear magnetic resonance (NMR) spectroscopy not only can identify compounds in a complex
339
T. Kaibara, et al.
mixture, but also distinguishes between magnetically inequivalent protons (such as protons bonded to different
carbons in organic molecules) in a single compound
based on their unique resonance frequencies. Combining
1
H NMR spectroscopy with an infusion of 13C-labeled glucose, 1H[13C] spin-echo difference NMR (1H[13C] SED
NMR) spectroscopy allows the measurement of the fractional enrichment of the various metabolites of glucose
into which carbon-13 becomes incorporated.1,19,42 In this
study, 1H[13C] SED NMR spectroscopy was used to monitor the effects of mild hypothermia on the flux of glucose
through the tricarboxylic acid (TCA) cycle in the brain
and, thus, to measure cerebral metabolism directly.
Materials and Methods
Fifty male Sprague–Dawley rats, each weighing between 250 and
350 g, were food restricted overnight prior to experimentation. On
the day of the experiment, the rats were intubated and mechanically ventilated with 0.7% halothane and a 2:1 mixture of N2O/O2.
Each rat’s femoral artery and vein were cannulated with a No. 24
Teflon catheter to permit blood pressure monitoring and sampling
for blood gas and serum glucose levels. The cranium was exposed
and covered with wet gauze for later freeze-funnel fixation. Middle
ear temperature, which correlates with brain temperature,47 was
monitored using a thermocouple probe. Temperature control was
accomplished by placing a temperature-controlled water blanket beneath the animal and by warming the rat with an infrared lamp or
cooling it by spraying a water/alcohol solution supplemented with
fanned air. Temperature was maintained in the ranges of 37.5 6
0.2˚C in the normothermia group and 31.0 6 0.2˚C in the hypothermia group for 20 minutes before subjecting the animal to [113
C]glucose infusion. A 1-hour cooling period was allowed for the
hypothermia group and the normothermia group was maintained at
normal temperatures for the same period. Middle ear temperatures
were recorded at 5-minute intervals before and during experimentation. Each animal received a bolus (0.08 ml/100 g) of a 10% [113
C]glucose solution (Catalog No. 29,704-6; Sigma Chemical Co.,
Ontario, Canada) followed by infusion of the same solution via a
syringe pump to raise and maintain the blood glucose concentration
by approximately 3 mM at 10, 30, 60, 100, or 200 minutes. The end
of the infusion period was immediately followed by freeze-funnel
brain fixation for which liquid nitrogen was used.40 Five animals
were used for each time point for each temperature group. There
were no deaths during these experiments.
Perchloric acid extraction of brain samples was performed as
previously described.38,39 Excised, frozen samples of neocortex and
hippocampus were thoroughly homogenized in chilled 0.3-M perchloric acid (10:1 volume/weight). The homogenate was centrifuged and the supernatant was collected, after which the pellet
was reextracted and the homogenate was again centrifuged. The
supernatants were pooled, neutralized with 1.5-M KOH, and centrifuged to remove precipitated KClO4. Samples were stored
at 280˚C until analysis. The resulting samples were dissolved in 0.6
ml D2O containing 5 ml of 30-mM sodium 3(trimethylsilyl)propionate as a chemical shift reference. The pH was adjusted to 6.9 to
7.1 by using DCl or NaOD, and the sample was placed in a 5-mm
MR tube for analysis.
All 1H[13C] SED NMR spectroscopy studies were performed
using a spectrometer (AMX 500; Bruker Spectrospin Ltd., Ontario,
Canada) set at 298K, with the following spectral parameters: 8 k
complex data points in the free-induction decay, 500-Hz spectral
width (centered on the residual H2O resonance), 0.41-second acquisition time, 6.13-second relaxation delay, and a hydrogen-1 90˚pulse length lasting 10.8 6 0.4 msec. The spin-echo interval was set
to 1/JHC = 7.2 msec, in which JHC is the mean 1H-13C spin–spin coupling constant (140 Hz). Composite-pulse decoupling was used
with a carbon-13 90˚-pulse length of 20.0 msec. The residual H2O
resonance was diminished using an on-resonance presaturation
pulse. At least 512 scans were obtained for each subspectrum.
340
Before the Fourier transformation of the free-induction decay, the
time-domain data were zero-filled to 32 k data points and multiplied
with a line-broadening parameter of 0.5 Hz. All chemical shifts are
shown in parts per million (ppm) relative to the 3-(trimethylsilyl)propionate resonance, which is defined to be 0 ppm.
The 1H[13C] SED NMR spectra were analyzed using a commercially available NMR analysis software package (NUTS; Acorn
NMR Inc., Fremont, CA) to determine the fractional enrichment of
the label in glutamate (C4 and C3 complement positions), g-aminobutyric acid (GABA) (C2 and C4), glutamine (C4 and C3), lactate (C3), aspartate (C3), alanine (C3), and succinate (C2,3). One
advantage of using 1H[13C] SED NMR spectroscopy over direct
detection of the label using 13C-NMR spectroscopy, aside from the
increased sensitivity, is that the fractionated enrichment can be
directly obtained from the difference spectrum, containing only signals from protons attached to 13C nuclei, and the total proton subspectrum. Peak heights were used in lieu of peak areas because of a
high degree of overlapping resonances. The relative accuracy of
using peak heights and peak areas was determined by calculating
the fractional enrichment of glutamate C4 by using both methods. It
was found that similar results were obtained, but higher precision
was achieved using peak heights. Any differences in linewidth between the resonance from protons bonded to 12C and 13C nuclei are
assumed to be small compared with the applied line-broadening
parameter.2
The percentages of fractional enrichment data were compared
between groups for each metabolite by using pooled or separate ttests, depending on the absence or presence of heterogeneity of variance. Enzyme kinetics for label incorporation into the metabolite of
interest were estimated by fitting the parameters in the function used
to describe the incorporation of label into the metabolite of interest
x, that is:
to the time course data by using a nonlinear regression analysis software package (NLREG; Phillip H. Sherrod, Nashville, TN). Here,
A represents the steady-state fractional enrichment, t represents time
in minutes, kx21 is the first-order rate constant approximating the
increase in label in the metabolic precursor x21 to x, and 1.11 represents the natural abundance of carbon-13. For several sites of
incorporation the kx21 was too large to be calculated, and only one
exponential function was used. For incorporation into sites labeled
on the second turn of the TCA cycle (glutamate and glutamine C3
and GABA C4), the value of kx calculated for the first turn label
incorporation was used, rather than determined from the fit.
Although the value of kx determined on the first and second turns of
the TCA cycle should be identical, the value of kx calculated from
the first turn is more precise. This yields better values for kx21 on the
second turn of the TCA cycle.
Results
The preinfusion blood glucose level was similar in both
groups (4.8 6 0.2 mM for the normothermia group and
4.7 6 0.2 mM for the hypothermia group, mean 6 standard error of the mean [SEM]). No significant difference
in blood glucose increase was observed between the normothermia and hypothermia groups. Figure 1 shows the
mean blood glucose increase for animals infused for 200
minutes.
When [1-13C]glucose was used as the substrate, labeling
of a number of metabolites was observed in the 1H[13C]
SED NMR spectrum (Fig. 2). End products of glycolysis,
lactate, and alanine were labeled in the C3 positions. The
TCA cycle intermediate succinate C2,3 was strongly labeled, as were aspartate C2 and C3, glutamate C4, GABA
C2, and glutamine C4 by precursors from the TCA cycle.
J. Neurosurg. / Volume 90 / February, 1999
Hypothermia upregulates pentose phosphate pathway activity
FIG. 1. Graph showing changes in blood glucose levels (mean
6 SEM) during infusion of a 10% (w/v) solution of [1-13C]glucose
in normo- and hypothermic rats.
Curves showing isotopic enrichment of the C4 position of
glutamate, the C2 position of GABA, and the C4 position
of glutamine are given in Fig. 3. The label was also able
to travel the TCA cycle a second turn, labeling glutamate
and glutamine C2 and C3, and GABA C3 and C4. The
slower increase in fractional enrichment of the 13C label in
C3 of glutamate, C4 of GABA, and C3 of glutamine corresponds to the time required for the label to pass through
to the second rotation of the TCA cycle. A comparison of
the rate constants for second-turn labeling of glutamate,
GABA, and glutamine (Table 1) provides a relative measure of TCA cycle activity.
The rate constants kx and kx21 for the formation of the
various compounds were similar in both the hippocampus
and neocortex. Both neocortex and hippocampus values
for the steady-state fractional enrichment of metabolites A
reached the same value within each temperature group.
Mild hypothermia resulted in both a significantly slower rate of incorporation of the 13C label into TCA metabolites and a significant decrease in steady-state levels of
fractional enrichment of the 13C label (the values of A in
Table 1) in all metabolites.
Mild hypothermia resulted in a 30 to 40% depression of
the rate of overall TCA cycle activity. The data in Table 1
show the percentage of depression of the rate constants
and steady-state levels of fractional enrichment for the
various metabolites at 37.5˚C and 31˚C. There was also a
significant decrease (20–25%) in the steady-state fractional enrichment with hypothermia.
Discussion
Limitations of the Data
The calculation of parameters in the equation assumes
that the incorporation of label has reached steady state
within the longest infusion time used. Although this may
FIG. 2. A 500-MHz 1H[13C] spin-echo difference NMR spectrum of rat neocortex extract showing the major resonances observable and the labeling pattern. A: The 12C+13C subspectrum containing resonances from all protons.
B: Difference spectrum showing signals only from protons attached to 13C nuclei. Ala = alanine; Asp = aspartate; Cho =
choline; Cr = creatine; Gln = glutamine; Glu = glutamate; Ino = myo-inositol; Lac = lactate; NAA = N-acetylaspartate;
Suc = succinate; Tau = taurine.
J. Neurosurg. / Volume 90 / February, 1999
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T. Kaibara, et al.
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Hypothermia upregulates pentose phosphate pathway activity
TABLE 1
Results of fitting the parameters in the equation with the time-course data for [1-13C]glucose infusion in hypothermic
and normothermic rats*
Neocortex
Metabolites & Parameters
31˚C
Hippocampus
37.5˚C
31˚C
37.5˚C
GABA C2
A
21.0 6 0.9
27.3 6 0.8
21.7 6 1.3
27.2 6 0.9
kx–1
0.15 6 0.10
—
0.41 6 1.6
0.43 6 1.3
kx
0.019 6 0.002
0.022 6 0.002
0.010 6 0.001
0.016 6 0.001
GABA C4
A
14.8 6 1.0
19.1 6 1.1
12.2 6 0.5
19.0 6 0.5
kx21
0.016 6 0.003
0.025 6 0.005
0.043 6 0.013
0.024 6 0.002
glutamine C4
A
19.0 6 1.9
22.8 6 1.2
25.9 6 6.8
20.4 6 1.1
kx21
0.098 6 0.072
0.16 6 0.12
0.28 6 0.90
0.030 6 0.019
kx
0.0091 6 0.014
0.014 6 0.002
0.0038 6 0.001
0.017 6 0.009
glutamine C3
A
17.3 6 0.5
21.4 6 0.6
22.2 6 0.6
18.1 6 0.4
kx21
0.025 6 0.003
0.033 6 0.005
0.054 6 0.018
0.020 6 0.002
glutamate C4
A
20.6 6 0.8
25.6 6 0.9
20.4 6 0.9
25.6 6 0.7
kx21
0.19 6 0.12
—
0.21 6 0.17
—
kx
0.021 6 0.002
0.024 6 0.002
0.013 6 0.001
0.018 6 0.001
glutamate C3
A
15.5 6 1.0
19.2 6 0.1
13.5 6 0.5
17.7 6 0.4
0.013 6 0.002
0.019 6 0.002
0.013 6 0.001
0.018 6 0.001
kx21
succinate C2,3
A
16.6 6 0.8
21.5 6 0.8
16.5 6 1.1
20.7 6 0.8
kx21
0.15 6 0.11
—
0.15 6 0.12
0.17 6 0.12
kx
0.015 6 0.002
0.018 6 0.002
0.0010 6 0.001
0.015 6 0.001
alanine C3
A
21.8 6 1.1
21.5 6 0.8
16.5 6 1.1
20.7 6 0.8
kx21
0.37 6 0.93
—
0.19 6 0.10
—
kx
0.026 6 0.004
0.042 6 0.007
0.034 6 0.004
0.029 6 0.003
aspartate C3
A
21.6 6 1.3
25.8 6 1.2
21.2 6 2.6
26.3 6 1.8
kx21
0.088 6 0.046
—
0.058 6 0.039
—
kx
0.014 6 0.002
0.015 6 0.002
0.011 6 0.003
0.013 6 0.002
* Values are expressed as the mean 6 SEM. The parameter A is given in units of percentage of fractional enrichment, whereas kx21
and kx are given in minutes21. For the values of kx21 not given (—), only a single exponential function was used in the curve fit.
Definitions of the parameters are given in the text.
be true for some of the sites of label incorporation, others,
such as glutamine C4 and C3 and GABA C4, had not
reached steady state. For this reason, the calculated rate
constants appear to show an increase with hypothermia.
This could be corrected through increasing the infusion
period to longer than 200 minutes. This was not necessary
because the data for many of the metabolites showed a
significant decrease in metabolism. It is assumed that the
blood glucose level remains constant throughout the infusion period. Although the term containing kx21 (the rate
constant for the increase of label in the precursor compound) attempts to compensate for the effect of deviation
from a square-wave character (it includes processes such
as glucose transport and all reactions before the precursor
is labeled), it is only an approximation of its true shape. In
several cases in which kx21 was not given in Table 1, the
value of kx21 was too high for the curve fit to obtain an
accurate value. The upper limit for the determination of
kx21 by using the time points in this experimental design is
probably approximately 0.2 minutes21.
The deviation of the precursor labeling profile from
a square-wave function, which the term in the equation
containing kx21 attempts to address, is due to a number
of processes and has been previously modeled.32 These
processes include glucose transport from the blood to the
brain, glycolysis, and formation of intermediates in the
TCA cycle up to and including the metabolite used to
form the compound of interest.
A second important limitation of the methodology used
here is the possibility of artifacts from anaerobic metabo-
FIG. 3. Graphs showing fractional enrichment of glutamate C4 (upper), GABA C2 (center), and glutamine C4 (lower) over time for neocortex and hippocampus showing a significant decrease in the rate of incorporation for all time points except for the 10-minute time point in
the case of glutamine C4. Values are expressed as the mean 6 SEM. Near steady state (200 minutes) there is a decreased level of carbon-13
incorporated into glutamate, GABA, and glutamine, respectively, at 31˚C compared with that at 37.5˚C (neocortex: p , 0.0001; hippocampus: p , 0.0001). *p , 0.005; **p , 0.002; †p , 0.001; ††p , 0.0001.
J. Neurosurg. / Volume 90 / February, 1999
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T. Kaibara, et al.
FIG. 4. Schematic drawing demonstrating the predicted pattern of labeling due to the metabolism of [1-13C]glucose
through the glycolytic and TCA pathways. Solid black ovals represent labeling positions via glycolysis and through the
first turn of the TCA cycle, solid gray ovals represent labeled positions on the second turn of the TCA cycle, and hatched
patterned ovals represent labeled positions on subsequent turns of the TCA cycle. For clarity, pathways such as pyruvate
carboxylase and the GABA and malate shunts have been omitted. CoA = coenzyme A.
lism due to freeze-funnel fixation. This method involves
the application of liquid N2 over the exposed cranium,
resulting in a freezing front that moves radially from the
surface. In this manner the tissue underlying the freezing
front remains perfused with oxygenated blood. The rate of
freezing is 5 seconds/mm for the first 4 mm and 11.5 seconds/mm for the deeper regions.40 This is of particular
importance in the measurement of alanine because it is
known that, under anaerobic conditions, the glycolytic
rate increases by approximately a single order of magnitude. Although Fig. 2 shows that the lactate was observable, it remained very low relative to the other metabolites
and did not differ between the two brain regions studied
or between normothermic and hypothermic animals. Previously experiments have shown that tissue lactate levels
are higher in animals subjected to high-frequency microwave fixation.26,37 Although lower tissue lactate levels
may be found in tissue fixed with the freeze-blowing technique,34 that method precludes regional assessment of metabolite levels.
Tricarboxylic Acid Cycle in Neurons and Glia
Glucose is taken up from the microcirculation by astrocytes and converted through glycolysis into lactate (see
344
Fig. 4 for labeling patterns of compounds by metabolism
of [1-13C]glucose). It is this lactate that is released and
then taken up by neurons for conversion to pyruvate and
entry into the TCA cycle.48 From NMR spectroscopic
studies of the metabolism of [1-13C]glucose in cultured
neurons and glia, it has been found that glutamine is primarily glial in nature, whereas GABA is largely produced
in neurons.44
A comparison of the values of steady-state first and second turns of TCA cycle labeling in Table 1 shows that a
significant amount of label is lost during one cycle in both
normo- and hypothermic animals. However, the loss of label in glia (given by comparing glutamine C4 and C3
labeling) is much less than the loss in neurons (GABA C2
and C4 labeling). Although hypothermia-induced alterations in glucose transport are expected to alter the kinetics
of glucose metabolism, this cannot explain the observed
decrease in steady-state labeling. This is determined solely by the ratio of labeled glucose to all glucose. Before
infusion of labeled glucose the animals have the same
blood glucose levels and during the infusion the increase
in blood glucose is the same for both groups, so that this
ratio should be the same. Thus, the apparent loss of label
can only result in one of two ways: a relative increase in
J. Neurosurg. / Volume 90 / February, 1999
Hypothermia upregulates pentose phosphate pathway activity
an unlabeled source of carbon or a relative increase in the
pentose phosphate pathway (PPP).
Hypothermia-Induced Metabolic Depression
We have clearly demonstrated that mild hypothermia
results in a significant (30–40%) depression of TCA cycle
activity and, thus, cellular metabolism. This depression is
consistent with that seen using previous indirect measures
of metabolism. In the nonshivering rat, the CMRO2 decreases linearly from 37 to 22˚C, falling by approximately 5% per degree of Celsius, which is mirrored by a smaller decrease in cerebral blood flow.24
Described by its discoverer as “the main energy-yielding process in all of nature,”29 the ubiquitous nature of the
TCA cycle would make the control of its activity an ideal
mechanism of hypothermic neuroprotection. Despite our
findings, the role of hypometabolism in hypothermic neuroprotection remains unclear and difficult to interpret.
This is partly a result of the fact that barbiturate agents
depress metabolism with little neuroprotection. At electroencephalographic burst-suppressing doses, barbiturates
effect large (up to 50%) reductions in the CMRO236 and we
have observed that barbiturate anesthesia at normothermia
depresses TCA cycle activity to a level similar to that in
this study (unpublished data). The efficacy of neuroprotection afforded by barbiturates is not conclusive and,
although some have shown reduced infarction volumes
using barbiturate medications,25 the inconsistent nature33
and failure of randomized clinical trials9,50 raise doubt concerning their value. The differences in the mechanisms by
which barbiturate agents and hypothermia suppress neuronal activity may also contribute in part to the contrast in
neuroprotection.
Hypothermia-Induced Upregulation of the PPP
In addition to a depression of metabolism, mild hypothermia resulted in an apparent decreased steady-state
fractional enrichment of 13C label. This implies that a significant amount of glucose has been metabolized through
the PPP rather than through the glycolytic and tricarboxylic acid pathways. In the oxidative branch of the PPP, the
13
C-labeled first carbon of glucose is lost as 13CO2 during
the formation of ribulose-5-phosphate (Ru5P), corresponding to the observed loss of 13C label. Both glycolysis and the PPP produce lactate as an end product. However, glycolysis produces one molecule each of labeled
and unlabeled lactate per molecule of [1-13C]glucose,
whereas the PPP produces only unlabeled lactate. Thus,
the ratio of labeled lactate produced to labeled glucose
administered is less than the expected 1:1. As the activity
of the PPP increases, this ratio lowers. In this study we
observed that the ratio of labeled lactate to labeled glucose
was less than 1:1, indicating that an increase in the PPP
had occurred.
In the normothermic rat, the PPP constitutes 2.3% of
the metabolism of brain glucose.20 Composed of two reaction sequences, one forming CO2, Ru5P, and the reduced
form of nicotinamide-adenine dinucleotide phosphate
(NADPH) and the other regenerating hexose phosphates
from Ru5P, it has been suggested that the separate control
of these two pathways would allow for fine regulation of
NADPH compared with Ru5P formation.5 Ribulose-5phosphate is a precursor for nucleic acid synthesis whereJ. Neurosurg. / Volume 90 / February, 1999
as NADPH serves a number of cell-protective functions.
The NADPH is involved in a variety of reductive reactions, including neurotransmitter synthesis and degradation, cholesterol and fatty acid synthesis, hydrogen
peroxide detoxification, and maintenance of reduced glutathione.4 The maintenance of membrane-bound sulfhydryl groups in reduced form by NADPH and glutathione
reductase is vital to the maintenance of membrane potential, ion transport, and mitochondrial permeability. The
NADPH also assists the glutathione peroxidase removal
of the highly membrane-toxic hydrogen peroxide.4
Upregulation of PPP in conditions of cellular stress,
such as hypothermia or ischemia, may play a vital role in
maintaining cellular integrity and function. The PPP appears to possess a large reserve capacity and it has been
suggested that it may have a specific role in brain metabolism, perhaps being intermittently turned on at a high
level of activity to provide large amounts of product as
required.4 Such a hypothermia-induced increase in PPP
has been observed in isolated pancreatic islets, in which
lowering the temperature to 27˚C results in a greater than
twofold increase in PPP glucose utilization.17 Artificial
stimulation of the PPP by diethyl maleate or buthionine
sulfoximine, which act through glutathione depletion, increases the survival time of animals suffering from ischemia.49
Relationship to Cerebral Ischemia
Many investigators have attempted to characterize and
explain the mechanism of hypothermic neuroprotection
by describing hypothermia-induced cellular changes
associated with ischemic injury in the central nervous system. Neurochemical modulation such as synaptic activation and neurotransmitter release,5,11,21,43 free radical formation,22,27,28 and protein kinase C activity12,51 have been
characterized in studies of hypothermic ischemia. Despite
such evidence, several key issues suggest that these
changes alone do not account for the effect of hypothermia. Likewise, upregulation of PPP activity may not solely account for the beneficial effects of hypothermia. However, further study of this intriguing possibility is certainly
warranted. Furthermore, it would be useful to determine
whether barbiturate medications affect PPP activity. Given that they provide minimal benefit, it is expected that
they will not.
The neuroprotective effect of hypothermia in ischemia
has been demonstrated in many different species and is
highly reproducible. Hypothermia also provides protection to tissues other than the central nervous system. The
harvested human heart when cooled (4–5˚C) is able to restore normal function when warmed and transplanted after
4 to 6 hours of ischemia.45 This period can be extended to
24 hours for the kidney and the pancreas.45 Furthermore,
reports of humans achieving complete recovery after prolonged cardiac arrest in cold environments are not uncommon.8 Thus, the diversity of tissues, in addition to their
profound and consistent natures, suggest that the underlying mechanism by which hypothermia provides protection
is of a fundamental nature.
Conclusions
Mild hypothermia in this study resulted in decreased
345
T. Kaibara, et al.
metabolism and provided evidence consistent with upregulation of the PPP. The various membrane-stabilizing and
cell-protective effects of NADPH, such as fatty acid and
cholesterol production and peroxide detoxification, suggest that an upregulation of this pathway might be an
important mechanism of hypothermic neuroprotection.
Common to all tissues, the combination of decreased metabolism together with upregulation of the PPP in hypothermia could account for the tissue-protective effects
observed. If this is the case, hypothermic neuroprotection
could be largely under glial control because in the brain
glucose metabolism both by glycolysis and the PPP occurs in astrocytes.
Acknowledgments
The authors thank Dr. Fang Wei Yang for performing the animal
surgery and Dr. Hans Vogel at the Department of Biological Sciences, University of Calgary, for access to the AMX 500 NMR
spectrometer.
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Manuscript received March 19, 1998.
Accepted in final form September 28, 1998.
This study was supported by a grant from the Heart and Stroke
Foundation of Canada to Dr. Sutherland.
Address reprint requests to: Randy Tyson, Ph.D., Seaman Family MR Research Centre, Foothills Provincial Hospital, 1403–29
Street NW, Calgary, Alberta, T2N 2T9 Canada.
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