194 E
13
animal species, brain regions measured, and the use and perfusate was replaced by [1- C]glucose 99 atom after
type of anesthetic agent. a 30-min equilibration period. Approximately 12 slices
In the present study, we have examined the influence of were prepared and incubated simultaneously. At selected
development on the difference in glucose oxidative metab- times 0, 15, 30, 60, 90, and 120 min after the additions of
olism in the cerebral cortex of 10–12- and 28–30-day-old the labeled glucose, two to three slices were quickly
rats in vivo and in brain slices prepared in vitro. These removed from the incubation chamber directly into liquid
postnatal ages border the major increases observed in N . Thus, a single animal yielded sufficient cortical tissue
2
glucose metabolism and function in the rat cortex. We to permit generation of a single turnover curve for gluta-
hypothesized that if the difference in the rates of glucose mate.
oxidation between the in vivo cortex and the brain slice is dependent on intact, functional interactions present in vivo,
2.3. Preparation of tissue and plasma extracts but absent in the slice, then a comparison of the cortices at
these two postnatal ages in vivo and in vitro would shed Frozen brain tissue was extracted in 0.1 M methanol
light on the fraction of total glucose oxidation related to HCl and 3 M perchloric acid with minor modifications
function as the brain matures. We have found that during from published methods [23]. Following centrifugation the
postnatal development the maturational increase in the supernatant was neutralized with 10 N KOH, centrifuged
basal rate of cortical glucose oxidative metabolism is again to remove perchlorate salts, and lyophilized. The
.2-fold greater in vivo than in vitro. Our results suggest powder was dissolved in 0.5 ml D O and trimethylsilyl-
2 2
that cortical metabolism associated with intact synaptic 2,2- H-propionic acid TSP was added as a chemical shift
1
inputs contributes a large fraction of the basal energy reference for
H NMR spectroscopy. Total protein was expenditure during this period of development.
measured in the acid extracted pellets after solubilization with 1 N NaOH using the ‘enhanced protocol’ of the
Pierce BCA Protein Assay.
2. Materials and methods Frozen blood plasma was extracted in 0.3 M perchloric
acid, centrifuged, and the supernatant was neutralized with 2.1. Animal preparation
3 N KOH. After lyophilization, the powder was dissolved in 135 ml of 1 mM TSP in D O and 20 ml 40 mM
2
Sprague–Dawley rats of both sexes; 10–12 days old 20 potassium fluoride and placed in a micro-NMR tube for
g, n517 and 28–30 days old 150 g, n512 were analysis.
anesthetized with methoxyflurane. The left femoral artery and right jugular vein were cannulated to permit arterial
2.4. NMR spectroscopy
13
blood sampling and intravenous infusion of [1- C]glu-
13 13
cose. The enrichment of [ C]glucose was raised rapidly C enrichments of metabolites in acid extracts of brain
13
and maintained at a constant level using a protocol adapted tissue were determined using indirect detection of
C in
13 1
from Fitzpatrick et al. [13]. The [ C]glucose was infused the
H NMR spectrum at 360.13 MHz AM-360 WB for periods of 15, 30, 60, and 90 min for the 10-day-old
Bruker NMR spectrometer as described previously [13]. rats and 7.5, 15, 30, and 60 min for the 30-day-old rats
All spectra were acquired fully relaxed TR520 s with 16 using three to five rats per time point. The different time
K data points and a sweep width of 6 kHz. The residual periods were chosen to adequately sample the enrichment
water HDO peak was suppressed with the application of curves based on the slower and faster rates expected in 10-
a low power pre-saturation pulse 3 s. and 30-day-old rat cortex, respectively. Arterial blood 100
ml was withdrawn at these time points, immediately 2.5. Quantitation of spectra
centrifuged, and the plasma was frozen in liquid nitrogen.
1
A portion of the plasma was used to measure glucose H spectra were processed by zero-filling to 32 K data
13
concentration Beckman Glucose Analyzer II. At the end points.
C enrichments were measured from spectra of the assigned infusion period, the cranium was frozen in
processed with a mild Lorentzian-to-Gaussian filter LB5 situ with liquid nitrogen and stored at 2908C for the
20.75, GB50.1 and zero and first order baseline correc- subsequent removal and extraction of the brain. Frozen
tions. The fractional enrichments FE of glutamate-C
4
brain tissue |100 mg was chipped from the fronto- 2.35 ppm, glutamate-C 2.13 ppm and lactate-C 1.32
3 3
parietal cortex. ppm were determined from the ratio of the peak heights of
13 12
13
the C-labeled resonances to the total C1 C am-
2.2. Preparation of cortical slices in vitro plitude of the respective metabolites. The concentration of
cortical glutamate was determined for the in vivo group by Brain slices of 300-mm thickness were prepared from
integration relative to a known concentration of TSP and
21
rats of 10- and 30-days postnatal age according to previ- expressed as mmol g
wet weight. The enrichment of ously described procedures [18]. Unlabeled glucose in the
glucose-C in the plasma extract was determined at 320 K
1
E .J. Novotny et al. Brain Research 888 2001 193 –202
195
13
from the ratio of the C coupled satellites of glucose-C
value of 1 2 FE of plasma glucose. Such dilution has no
1
to the total intensity of the triplet centered at 5.2 ppm [5]. effect on the calculated value of V
but would have a
TCA,
small effect on the calculated rate of oxygen consumption 2.6. Calculation of fluxes in the neocortex in vivo
to the extent that such substrates replace glucose. Metabol-
21 21
ic fluxes were expressed as mmol g wet weight min
. Cortical TCA cycle and glucose oxidation rates were
determined from the isotopic labeling of glutamate and 2.7. Kinetic parameters assumed in the calculation of
lactate using a steady state metabolic modeling analysis metabolic rates
[26,27]. TCA cycle V and a-ketoglutarate glutamate
TCA
exchange V were determined from a fit of the metabolic The transport rate constants for glucose and lactate
x
model shown in Fig. 1A to the group-averaged time between blood and brain were taken from the available
courses of the glutamate-C and C enrichments, respec- literature for immature and mature rats. These rate con-
4 3
tively. A Runge-Kutta method was used to solve the stants have not been reported for rats less than |14 days
differential equations, and best fits were determined by old. Therefore, the effects of uncertainties in the parameter
iteration of parameters V , V using a simplex algo-
estimates on the calculated fluxes were assessed by a
TCA x
rithm. A physiologic and metabolic steady-state was sensitivity analysis Appendix A. Brain glucose concen-
assumed with respect to the pathways of glucose metabo- tration and fractional enrichment were calculated from the
lism. Lactate enrichments in the plasma were fitted to a concentration and FE of plasma glucose using values for
monoexponential equation and the resultant best-fit values glucose transport parameters most appropriate for the
were used as input to the model. The additional flux term, particular ages studied. The Michaelis-Menten kinetic
V , represents the flow of any unlabeled carbon substrates parameters for cortical glucose and lactate transport re-
dil
into the TCA cycle through acetyl-CoA e.g. ketone ported by Cremer [8] for |14–19-day-old brain were
bodies. The presence of such flows results in the steady- assumed in the analysis of the 10-day-old cortex glucose:
21 21
state dilution of glutamate-C labeling and a reduction in V
50.52 mmol g min
; K 57.59 mM; K 50.021
4 max
m D
21 21
21
the fractional enrichment FE from the maximum possible min
; lactate: V 51.95 mmol g
min ; K 59.70
max m
Fig. 1. Schematic representation of the metabolic models used to calculate TCA cycle flux from glutamate turnover in vivo A and in vitro B. Plasma glucose G and the intracellular glucose pool G exchange according to the Michaelis-Menten kinetic constants, K
and V . Glucose carbon flows
p i
m max
21 21
through the intermediates of the glycolytic pathway at the rate 2 CMR mmol g
min to pyruvate. Brain pyruvate and lactate are assumed to be in
GLU
isotopic equilibrium and are treated in the analysis as a single pool. Lactate exchange between blood and brain was determined from the Michaelis-Menten
13
kinetic constants K and V
and the plasma enrichment time course data not shown. C label enters the TCA cycle as acetyl-CoA and labels
m max
a-ketoglutarate at carbon 4 a-KG , which undergoes isotopic exchange with glutamate at carbon 4 Glu in both the cytosolic and mitochondrial pools.
4 4
The exchange among cytosolic and mitochondrial pools of a-KG and glutamate are reduced to a single exchange rate V between one grouped pool of
x
a-KG and one group pool of glutamate [27]. Glutamine is synthesized from glutamate at the rate V . The flux represented by V reflects the cyclic flow
gln gln
through glutamine synthetase glu →
gln and glutaminase gln →
glu. Unlabeled carbon enters the acetyl-CoA pool at the rate V . Continued flow of the
dil 13
13
C label through the TCA cycle results in labeling of carbon 3 also C and C of a-KG and glutamate [26,27]. For the analysis of the brain slice C
2 1
enrichments the model B was modified to include a large, essentially infinite, medium in exchange with the tissue. The following assumptions were made: intracellular glucose-C labeling reaches a steady state enrichment of 99 at t50 i.e. step function, lactate leaving the tissue V
to the medium
1 efflux
does not return, i.e. V 50, and V 50. See text for details.
influx gln
196 E
21
mM; K 50.079 min . For 30-day-old rats, glucose
random noise with the same Gaussian distribution as was
D
transport parameters were assumed to be equal to adult observed in the original data. Each of the simulated sets
values of the same species measured in a previous study was analyzed using the same fitting procedure as was
[24] where V CMR
55.8 and K 513.9 mM. Lactate performed with the original data, and the 2000 sets of fitted
max GLU
m
transport kinetic parameters assumed for the 30-day-old parameters were recorded. The Monte-Carlo procedure
cortex were based on those reported for adult rats by provided an estimate of the uncertainties in the fitted
21 21
Pardridge [39] V 50.12 mmol g
min ; K 51.9 mM;
parameters if the experiment were repeated 2000 times. To
max m
21
evaluate the significance of differences in the fitted param- K 50.028 min
.
D
eters between the 10- and 30-day-old groups, the 2000-row arrays of Monte-Carlo generated parameters were sub-
2.8. Calculation of fluxes in brain slices tracted one from the other, generating a 2000-row differ-
ence array. The P-value was calculated as the fraction of Glycolytic and TCA cycle fluxes were determined using
the elements that lay above or below zero for each fitted the same steady state metabolic model but modified to
parameter. The sensitivity of the calculated value of V
TCA
permit free communication between perfusate and the to assumed parameters is given in Appendix A.
extracellular space due to the absence of a blood–brain The statistical significance of comparisons between
barrier. Therefore glucose and lactate transport terms were cortical metabolic rates in vivo and in slices were evalu-
not included in the analysis. Injured cells at the surface of ated by a pair-wise Z-test.
the slice may have impaired their ability to aerobically
14
metabolize glucose. However, studies using C radio-
isotope labeled substrates [15], have estimated that these
3. Results
