ABSTRACT
The lipid composition of the brain is of great importance to its
metabolism and function. Although much research has been done on
regional brain lipid composition, studies usually suffer from limited
brain regions or from limited lipids analyzed. We modified a previously
described method for the separation of brain phospholipids and
glycolipids, improving the separation and sensitivity of the method.
Using this modified method, we measured the lipid composition of
the frontal and entorhinal cortices, the hippocampus, basal ganglia,
cerebellum, and medulla oblongata of five rats under nitrous oxide
analgesia. Total lipid content was highest (p<0.05) in the medulla
oblongata (111.0±6.0 mg/g wet brain, X±SD) followed by the hippo-
campus (72.6±2.8), cerebellum (62.7±4.6), basal ganglia (62.6±1.5),
frontal cortex (57.7±2.1), and entorhinal cortex (53.3±1.9). The areas
with higher total lipid content (p < 0.05) also had higher percentages
of cerebrosides (18.6±2.2 in the medulla oblongata vs 8.3±1.2 in the
frontal cortex) and 40 to 50% lower levels of phosphatidylcholine and
phosphatidylinositol. The relation between the ratio of cerebrosides
plus sulfatides to phosphatidyicholine and the total lipid content in-
dicates that differences in brain lipid composition between regions
are attributable to their relative gray/white matter content.
Index Entries: Cerebrosides; sulfatides; phospholipids; brain
regions.
INTRODUCTION
The lipid composition of the brain is of great functional and metabolic
importance. For example, phospholipids, a major constituent of biologi-
cal membranes, are involved in the regulation of membrane properties
related to structure, permeability, enzyme activities, and receptor affini-
ties (Cullis and Hope, 1985). Cholesterol, another major membrane con-
stituent, increases membrane stability and decreases permeability (Bloch,
1985). In neurons, whose functional activity is critically dependent on
membrane properties, functional differences might be reflected in dif-
ferences in membrane lipid composition. However, relatively few studies
have attempted to characterize the lipid composition of different brain
regions (O'Brien and Sampson, 1965; Svennerholm and Vannier, 1972;
Hoshi et al., 1973; Rao, 1977; Nonaka and Kishimoto, 1979; Ishibe and
Yamamoto, 1979; Macala et al., 1983; Okazaki et al., 1990; Soderberg et
al., 1990), partly because of the difficulty in quantitating lipids in small
amounts (20-30 mg) of brain tissue. In addition, most of the published
data provide information on only one or two lipid classes.
In this paper, our aim is to characterize the distribution of phospho-
lipids, cholesterol, and glycolipids in the cerebral cortex, hippocampus,
basal ganglia, cerebellum, and brain stem by a combination of column
chromatography, high performance thin layer chromatography (HPTLC),
and densitometry using a modification of a method previously described
by Macala et al. (1983).
Surgical Procedures
Male Wistar rats (350-450 g) were intubated under 4% halothane
anesthesia, immobilized with 0.3 mg pancuronium bromide (Elkin Sinns,
Column Chromatography
The columns (Bond-Elute NH 2 ) were equilibrated with 20 mL chloro-
form/methanol/0.8M sodium acetate (30:60:8 [v/v]) and washed with 50
mL chloroform/methanol/water (30:60:8 [v/v]). The brain samples were
applied to the column and the neutral lipids eluted with 15 mL chloro-
form/methanol/water (30:60:8). Acidic lipids were eluted with 15 mL
chloroform/methanol/0.8M sodium acetate (30:60:8). Recovery of neutral
and acidic lipids from the columns was estimated at about 95% using
phosphatidylcholine (PC) and phosphatidylserine (PS) standards.
HPTLC
One to six micrograms of each lipid and four different standard mix-
tures were spotted on HPTLC plates prewashed with chloroform/metha-
nol/water (60:35:8 [v/v]) activated at 115°C for 15 min, and cooled in a
1
B s ss ¤ V
Al
® d i^ SU
_
"^-^
—
ps
^— — oe ® .. — — P1
1
Fig. 1. Representative chromatograms from densitometric quantitation
of (A) neutral and (B) acidic lipids from rat brain cortex. In A and B, the two out-
side lanes are standards 1-4 and the other lanes are rat brain samples.
Densitometry
TLC plates were scanned using a Bio-Rad-620 reflectance densitometer
(Bio-Rad, Richmond, CA) and areas computed using the Bio-Rad 1-D
Analyst software. Standard calibration curves were generated and curvi-
linear regression equations constructed. The amount of lipid in each
sample was calculated from the regression equation.
Statistical Analysis
Statistical significance was determined by one-way analysis of
variance and t-test. All data are expressed as X± SD.
RESULTS
The total lipid content of the medulla oblongata was 1.5 to 2 times
higher than that of the other regions examined (Table 1). The total lipid
content in other brain regions varied from 73 to 53 mg/g wet brain in the
Table 1
Lipid Composition of Rat Brain Regionsa
TL SPM PC PE CER CH PI PS SU
F. Cortex 57.7 1.54 16.08 14.92 4.82 12.46 1.20 4.62 2.08
±2.1 ±0.21 ±1.54 ±1.30 ±0.76 ±0.96 ±0.18 ±0.61 ±0.61
E. Cortex 53.3 1.78 15.98 13.64 3.96 10.60 1.12 4.56 1.64
±1.90 ±0.25 ±1.09 ±0.30 ±0.82 ±0.84 ±0.13 ±0.30 ±0.38
Hippocampus 72.6 2.08 17.72 17.10 9.50 16.84 0.90 5.34 3.16
±2.8 ±0.38 ±0.82 ±1.06 ±1.94 ±1.79 ±0.16 ±0.80 ±0.51
Basal 62.6 1.94 16.86 14.86 7.24 13.50 1.08 4.76 2.36
ganglia ±1.5 ±0.32 ±0.69 ±0.77 ±0.78 ±0.75 ±0.08 ±0.25 ±0.55
Cerebellum 62.7 2.40 17.34 13.96 8.16 12.84 1.14 4.26 2.56
±4.6 ±0.48 ±1.08 ±0.38 ±1.95 ±0.70 ±0.11 ±050 ±0.74
Medulla 111.0 3.90 20.22 25.48 20.72 24.48 1.24 6.66 8.24
oblongata ±6.0 ±0.53 ±1.09 ±1.75 ±3.42 ±1.67 ±0.18 ±0.52 ±0.79
Abbreviations: TL=total lipid, SPM sphingomyelin, PC= phosphatidylcholine,
PE = phosphatidylethanolamine, CE = cerebrosides, CH = cholesterol, PI = phosphatidyl-
inositol, PS = phosphatidylserine, SU = sulfatides.
a Results are expressed in mg/g wet brain X±SD (n=5).
DISCUSSION
Although degradation of membrane lipids is believed to be negligible
even hours after death (O'Brien and Sampson, 1965), rapid accumulation
of brain free fatty acids (FFA) after decapitation ischemia (Bazan, 1970;
Shiu et al., 1981) indicates that membrane lipid degradation occurs rapidly
postmortem. It contradicts the suggestion that hydrolysis products of
lipids, including FFA, were similar in postmortem and surgical biopsy
specimens (O'Brien and Sampson, 1965). The quantity of FFA accumulat-
ing 1 h postmortem is a small fraction of the total available FFA pool.
Nevertheless, its accumulation indicates rapid hydrolysis of membrane
lipids, which may be important compared with levels reported for differ-
ent human brain regions from postmortem tissue (Soderberg et al., 1990).
Our chromatography and densitometry methods combined two mod-
ifications of the separation and quantitation techniques developed by
Macala et al. (1983). First, we separated neutral from acidic lipids using
prepacked Bond-Elute NH, columns instead of DEAE-Sephadex columns,
thus eliminating the time-consuming need to prepare the columns. The
recovery of neutral and acidic lipids and their separation on the column
was estimated with a mixture of PC and PS. No detectable cross-contam-
ination of either fraction occurred, and recovery was about 95%. Macala
et al. (1983) and Wood et al. (1989) reported some loss of acidic phospho-
lipids during separation on the DEAE-columns and during a partitioning
step to remove sodium acetate. Despite this potential loss, the PS levels
they reported were about two times higher than those reported by Norton
and Poduslo (1973).
ACKNOWLEDGMENTS
This work was supported in part by the American Heart Association
(Western Pennsylvania Affiliate), the University Anesthesiology and Crit-
cal Care Medicine Foundation, and the U.S. Public Health Service, Grant
No. 5 RO1 H L27208-09. The authors gratefully acknowledge the editorial
assistance of Lisa Cohn and the technical assistance of Zachery Pantazes.
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