Stereospecific Permeability of Rat
Blood-Brain Barrier to Lactic Acid
BY EDWIN M. NEMOTO, PH.D., AND JOHN W. SEVERINGHAUS, M.D.
Abstract:
Stereospecific
Permeability
of Rat BloodBrain
Barrier
to Lactic
Acid
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• Blood and whole brain 14C and 32P activities were determined in hepatectomized rats one,
two, five and ten minutes after intravenous (I.V.) injection of u C-labeled L-lactate or D-lactate
and 32P-labeled rat red blood cells. Whole brain homogenate 14C was corrected for blood "C
and chemically partitioned into "C-lactate, 14CO2 and other "C compounds. In controls, lactate
was replaced with 14C-D-glucose and 125I-antipyrine. At one minute postinjection, whole brain
14
C expressed as percent of total injected 14C activity and as percent of the antipyrine value
were: antipyrine 1.78% (100%); D-glucose 1.45% (81%); L-lactate 0.36% (20%); and D-lactate
0.13% (7%). One minute after L-lactate injection, brain l4C was 74% lactate, 5% CO 2 and 21%
other compounds. Preloading rats with cold racemic Na-lactate reduced L-lactate uptake to
0.14% of the injectate (8% of antipyrine), and reduced D-lactate uptake to 0.09% (= 5% of antipyrine). At two, five and ten minutes, brain contained more 14C with larger fractions
metabolized to CO 2 and other compounds from both L-lactate and D-lactate. The blood-brain
barrier appears to contain a saturable lactate carrier exhibiting threefold L-stereospecificity to
D-stereospecificity, but resulting in far less net transport than the comparable glucose carrier.
Lactate transport may be limited by the scarcity of neutral lactic acid at normal blood pH.
Additional Key Words
carrier transport
Introduction
• Cerebrospinal fluid (CSF) pH plays an important
role in the regulation of respiration1 and cerebral
blood flow.2 During hypoxia,1 ischemia3 and hypocapnic alkalosis4 CSF pH changes are moderated by
alterations in lactic acid (LA) concentration, which is
a function of: (1) brain LA production and
metabolism, (2) blood-brain and blood-CSF LA
permeability, and (3) CSF pH itself. Factors contributing to the importance of LA in determining CSF
pH are: (1) high metabolic rate of brain5 with low oxygen stores8 and, therefore, higher sensitivity to oxygen lack, and (2) low permeability of the blood-CSF
and blood-brain barrier (BBB) to ions7 (at a pH of 7.4
only one out of 2,700 LA molecules exists in the nonionized form). In this study we have attempted to
determine LA permeability and mechanism of
transport across the BBB.
Several studies have demonstrated bidirectional
net transport of LA across the BBB. McGinty8 and
Wortis9 reported net uptake or excretion of LA by
brain. The method of LA assay in these experiments
were of questionable accuracy and specificity.10' u
O'Neal and Koeppe12 and Sacks13 showed LA exFrom the Cardiovascular Research Institute, University of California, San Francisco Medical Center, San Francisco, California
94122.
Present address for Dr. Nemoto: Department of
Anesthesiology, University of Pittsburgh, 1081 Scaife Hall,
Pittsburgh, Pennsylvania 15261.
Strode, Vol. 5, January-February 1974
lactate permeability
facilitated transport
brain lactate uptake
saturable transport
change between blood and brain in 14C-labeled LA
studies. Sacks also reported more rapid brain
metabolism of L-LA than of D-LA. These steady state
tracer studies were complicated by the possibility that
labeled LA could be resynthesized to labeled glucose
and metabolized by the brain. During hypoxia, Cohen
et al.M obtained evidence suggesting that 25% of the
glucose consumed by the brain was converted to LA.
S^rensen et al.16> 16 confirmed these findings with
hypoxia of similar magnitude at high altitude but CSF
LA was not markedly elevated,1 suggesting rapid excretion of LA by brain.
Some recent studies have refuted the studies
demonstrating significant permeability of LA across
the BBB. Crone and S^rensen,'7 using double indicator cerebral venous dilution curves, were unable to
detect measurable exchange of labeled LA across the
BBB. Alexander18 and Leusen19 found brain and CSF
LA unchanged after 15 to 30 minutes of I.V. infusion
of NaLA.
We have attempted to quantify BBB permeability
of LA compared to that for glucose and antipyrine
and to test its stereospecificity and saturability.
Methods
The trachea, right jugular vein and left common carotid
artery were cannulated in pentobarbital-anesthetized (60 mg
per kilogram intraperitoneally) female Sprague-Dawley rats
(200 to 400 gm body weight). The hepatic artery and hepatic
portal veins were looped with a suture (at the hepatic
trigone) for ligation ten seconds before the I.V. injection of
81
NEMOTO, SEVERINGHAUS
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the isotopic mixture. Blood was drawn from the carotid
artery immediately before the injection of the isotope for enzymatic 20 determination of arterial LA, and for P o ,, PCo, and
pH determinations.
The following labeled compounds were used: sodium DLA-14C, s.a., 9.1 mCi/mmole; sodium L-LA-"C, s.a., 56
mCi/mmole; D-glucose-14C, s.a., 3.0 mCi/mmole; and
antipyrine- 125 I (Amersham/Searle). Ascending paper
chromatography of the "C-L-LA and D-LA (nbutanol:water:acetic acid; 12:5:3) and D-glucose (nbutanol:ethanol:water; 52:33:15) showed essentially 100% of
the " C activity as the labeled molecule. Rat erythrocytes
were labeled by the incubation of 10 cc of blood, with approximately 60 /id of carrier-free H 3 a2 PO 4 . After a twohour incubation period, the cells were washed with saline
four times and suspended in saline at a hematocrit of approximately 40%. The injected solution contained about 2
nC\ of the test substance and about 6 /tCi of 32P-tagged cells
in a total volume of approximately 1.5 ml. The 82P-labeled
cells permitted correction of whole brain homogenate " C
activity for trapped blood " C activity. The rats were
decapitated at one, two, five and ten minutes postinjection.
Immediately following decapitation, blood was
collected from the neck, and the entire brain was rapidly
removed and frozen in liquid N 2 (see DISCUSSION for LA
metabolism prior to freezing). One-half of each brain and
blood sample were homogenized in 0.1N KOH and the other
half in 0.1N HC1 (tissue:KOH or HC1 ratios of 1:4 for brain
and 1:1 for blood), the difference between them indicating
the activity of 14CO2. HC1 homogenized samples were
vacuum (20 cm H2O) extracted with stirring for ten minutes.
Brain (0.2 ml) and blood (0.2 ml) homogenates were then
solubilized at room temperature with 1.2 and 0.6 ml NCS
solubilizer (Nuclear-Chicago), respectively. Blood samples
were bleached by the addition of 0.07 ml of benzoyl peroxide
(6 gm/30 ml) in toluene. 14C, 126I and 32P activities were
determined by liquid scintillation counting (Packard TriCarb) in a PPO/toluene (6 gm/L) mixture. Channel efficiencies were determined by means of internal standards.
Brain and blood 14C metabolites (i.e., glucose, pyruvate,
malate, etc.) other than 14C-LA were determined by treatment of 0.1 N HC1 homogenized samples with the BarkerSummerson copper-lime precipitation technique21 and
counted in Bray's solution. In test extractions, 99% of "Clabeled D-glucose was removed, while 95% to 98% of L-LA
was retained.
Regional concentrations of 14C (L-LA) in cortex, midbrain, cerebellum and medulla also were determined in two
rats killed at one minute.
Four rats were loaded with racemic NaLA (0.7 mmole
per kilogram, I.V.) ten minutes before 14C injection to test
for saturability of LA transport.
arterial LA values were significantly elevated in both
groups (table 2), which probably is a consequence of
the functional hepatectomy.
Whole brain 14C activity in 0.1N KOH
homogenized samples (except for one-minute groups
which were 0.1N HC1 homogenized) is shown in table
2. At one, two and five minutes, significantly more
14
C-L-LA than "C-D-LA had entered the brain
(P < 0.05). Whole brain "C-L-LA to "C-D-LA ratio
at one minute was 2.9.
Metabolism to 14CO2 and other 14C compounds
was detectable for L-LA at one minute and was
significantly faster for L-LA than for D-LA at two
and five minutes (table 2).
The distribution of 14C activity in HC1
homogenized brain of two rats studied at one minute
was relatively uniform: cortex, 0.172; midbrain, 0.131;
cerebellum, 0.150; and medulla, 0.142 (percent of total
injected activity per gram tissue).
Rats preloaded with racemic NaLA ten minutes
TABLE 1
Mean Arterial pH and Blood Gas Values in L-Lactate-'4
C(U) and D-Lactate-'4 C(U) I.V. Injected, Pentobarbital
(60 mg per Kilogram Intraperitoneally) Anesthetized and
Hepatectomized Rats
I4
1
X
n
2
SE
X
5
SE
X
n
n
10
SE
X
n
1
X
n
2
SE
X
5
SE
X
10
SE
X
n
Results
Arterial pH, P o , and PCo, w e r e similar in both M C-LLA and "C-D-LA injected rats (table 1). Ten-minute
Paoz
(mm Hg)
7.288
6
0.054
7.338
4
0.027
7.377
4
0.027
7.361
4
0.034
46.1
6
2.05
41.1
4
2.47
39.2
4
2.53
36.4
4
6.66
71.4
6
6.6
61.6
4
9.2
73.2
4
7.2
68.1
4
7.9
7.331
5
0.023
7.364
5
0.020
7.360
4
0.018
7.343
3
0.027
44.0
5
3.68
39.1
5
2.04
40.8
4
1.06
41.2
3
3.32
62.4
5
3.4
71.2
5
4.9
73.1
4
3.1
64.5
3
8.5
SE
»C(U)-D-Lactate Rats:
12S
Whole brain C or I activity (G) as percent of injected
activity (F) was calculated as: G = (C - DA/B). (100 E/F),
where A = brain " P , dpm per gram; B = blood 82 P, dpm
per milliliter; C = brain " C , dpm per gram; D = blood 14C,
dpm per milliliter; and E = brain weight, gram.
Paco2
(mm Hg)
C(U)-L-Lactate Rats:
Calculations
14
pHa
Mln postinjection*
n
n
SE
"Minutes postinjection refers to time of decapitation after
injection of the labeled lactate.
Stroke, Vol. 5, January-February 1974
BRAIN BARRIER LACTATI PIKMIABILITY
TABLE 2
Brain Uptake of Labeled L-Lactate and D-Lactaie After Intravenous Injection in Anesthetized Rats
•«rrltlon*4 "C act. (% of wboU brain act.)
n
5
Orh*r compounds n
L
D
5
5
179 ± 0.24
1.18 ± 0.14
0.360 * 0.049
0.126 * 0.017*
L
4
2.83 ± 0.59
0.141 =t 0.013
D
4
3.29 ± 0.56
0.089 =t 0.014*
2
L
0
4
5
1.42 ± 0.36
1.42 ± 0.38
0.546 =<= 0.144J:
0.192 =t 0.014*t
31
66
27
13
3
5
42
21
5
L
D
4
4
1.58 ± 0.15
1.60 ± 0.04
0.540 =<= 0.134
0.180 =t 0.042*
24
72
27
15
4
4
49
13
10
L
D
4
3
2.49 ± 0.17
2.61 ± 0.27
0.484 =•= 0.122
0.277 =t 0.107
45
35
15
24
4
3
40
41
1
(lactate
loaded)
74
88
"CO,
iot
:
21
2t
:
_
ro ro
Lact«t*uC
ro ro
n
What* brain »C
activity (% of total
act. ln|*ctod)
CM CM
1
lunv
Moos' [LA]
Cn Cn
Tim.
(min)
CM CN
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Column 4 is the nonlabeled blood lactate concentration, which was elevated by preloading in four animals. Column 5 is the
percentage of the total injected 14 C which was recovered in brain after correcting for blood 14C trapped in the brain. The
CO 2 fraction is the difference in homogenate radioactivity between KOH and HC1 homogenation. Other compounds are
those removed by copper-lime precipitation, including glucose, pyruvate, malate and other citric acid cycle intermediates.
*D < L.
tn.s. with a significance limit of P = 0.05.
tTwo minutes > one minute.
before injection of label showed less brain uptake of
label at one minute for both L-LA and D-LA
(P < 0.05), even though loading increased arterial lactate levels only two to two and one-half times normal.
Two rats were given u C-D-glucose and two given
1M
I antipyrine, both with "P-labeled erythrocytes, to
determine the percent of injectate found in whole
brain at one minute for a earner-mediated and a freely
permeable, simple diffusion substance. Of the total injected activity, 1.78% of the antipyrine and 1.45% of
the glucose were recovered in the brain. By comparison with antipyrine as 100%, at one minute the
brain concentrations of the other substances were:
glucose, 81%; L-LA, 20%; and D-LA, 7%.
Discussion
The results suggest that rat BBB contains a saturable
carrier for LA with a threefold stereospecificity for the
normal L-isomer as compared to the D-isomer. L-LA
transport (20%) is substantially less than for Dglucose (81%), where transport across the BBB also is
carrier-mediated." The difference is probably even
larger than indicated, since a greater percentage of
glucose than LA taken up by the brain during peak
blood concentration would diffuse back to blood
(because of its greater BBB permeability) after
passage of the injected bolus. Also, glucose is more
rapidly and readily metabolized by the brain.
Therefore, we conclude that far less LA will cross the
BBB per unit concentration gradient than for glucose.
The difference between BBB permeability for glucose
Sfroka, Vol. 5, Jonuary-Ftbruary 1974
and LA may be due to fewer carrier sites for LA or
scarcity of the ionized species of LA at pH 7.4.
Why were three groups of investigators17'19 unable to show BBB permeability to LA? In two
studies,18-19 neither brain nor CSF LA was elevated
after 15 to 30 minutes of I.V. infusion of LA. The lack
of increase in brain or CSF LA after LA infusion may
be due to a limited influx of LA; brain metabolism of
inwardly diffusing LA," or slow turnover of CSF.
In a subsequent study, we found that in dogs the CSF
LA can be gradually elevated after one to three hours
if blood LA is raised to 5 to 10 jimoles per milliliter.
Crone and S^rensen17 reported no extraction of
D-LA or L-LA from a single bolus of "C-LA passing
through the brain as compared to 'H-mannitol, an intravascular reference tracer. S^rensen has examined
the problem involved in his experiment (personal communication) and concluded that two factors may have
obscured the small LA uptake by brain, which has
been reported by OldendorP* and by us. First, mannitol, which was used as their reference tracer, is
slightly more permeable, at least through the choroid
plexus," than inulin,17 and inulin, according to Crone,
shows an extraction of 2.5% compared to albuminbound Evan's blue dye. 16 Second, LA enters
erythrocytes more rapidly than mannitol, with
kinetics which make estimation of plasma activity
during transit difficult.
It is of interest that brain uptake of D-LA was
significantly depressed by loading with racemic lactate, although not as dramatically as that of L-LA.
83
NEMOTO, SEVERINGHAUS
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This would suggest that a carrier is functioning for
both. Whether it is the same carrier remains to be established by studying the effect of loading with one
isomer on transport of the other.
OldendorPs method,22 in which the test and
permeable indicators are injected into an artery
supplying the brain, rather than intravenously, offers
advantages over our technique in terms of the correction for residual label in the blood at the time of
sacrifice. Unfortunately, with his technique (decapitation at 15 seconds), it is difficult to obtain a relevant
blood sample to demonstrate the absence of label
from intracerebral blood. However, the two
techniques have yielded comparable results.
A large fraction of the 14C-LA which entered the
brain was converted to other compounds and CO 2 in
each group of rats except those studied at one minute.
We presume this to be aerobic metabolism, terminated by anoxia shortly after decapitation. Insertion of a thermistor probe in whole rat brain followed
by complete immersion in liquid N 2 showed that the
brain completely froze in about 20 seconds. During
the 20 to 50 seconds between decapitation and freezing, brain glycolysis generates LA with little further
modification of the chemical nature of the 14C-labeled
compounds caught in transit from LA to CO 2 .
Therefore, freezing probably is not essential in these
experiments.
While the LA loading experiments showed
significant suppression of 14C-LA uptake, they were
incomplete in that the entire saturation curve remains
to be defined. Such information might be relevant to
the ability of the brain to excrete LA during and after
hypoxic episodes, the effectiveness of LA as a
moderator of CSF pH, and therefore of respiration
and CBF during hypoxia and hypocapnic alkalosis.
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Stroke, Vol. 5, January-February 1974
Stereospecific Permeability of Rat Blood-Brain Barrier to Lactic Acid
EDWIN M. NEMATO and JOHN W. SEVERINGHAUS
Stroke. 1974;5:81-84
doi: 10.1161/01.STR.5.1.81
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