Describes collaborative research regarding acetaldhyde uptake and interactions across the blood-brain barrier. Describes experimental design, presents results and concludes acetaldehyde crosses the blood-brain barrier resulting in equal distribution throughout all rat brain regions. States these data, combined with behavioral data, indicate further acetaldehyde studies be pursued.
Page 1: gyc08e00
PHILIP MORRIS U. S. A.
nt,~ RICHMOND, VIRGINIA
~- ERSONAL & CONFIDEi~T~
Dr. Wi 11 i am L. Dunn Date; January 20, 1982
Victor J. DeNoble and Yvonne Dragan
the University of Rochester
The following is areport on research activities per-
formed at the University of Rochester in collaboration with
Dr. Leo Abood. The research was designed to answer basic
questions concerning the transport of acetaldehyde from blood
to brain. In the first study the following questions were
1. Does acetaldehyde cross the blood-brain barrier?
. Is there differential uptake of acetaldehyde by
brain through intravenous or intraarterial injections.?
3. What is the ratio of acetaldehyde
to brain follnwing i"ntravenous or
in blood compared
4. Is there a regional brain distribution' of acetaldehyd'e?-
All tests were performed on male hooded rats weighing =w>"_: i
approximately 350g. The basic preparatilon was the exposure of
an artery (Carotid) or a vein (Femoral) in an anethesized rat.
For the i ntraarteri al preparation an i njecti on of: 10ti,1 of C14
acet.aldehyde (tracer free) with an activity of 0.25pCi/ul was
injected into the carotid artery. For the intravenous prepar-
ation an injection of l~0u1 or 30p1 of C14 acetallidehyde (tracer
free) with an activity of 0.25uCi/p1 was injected into the fem-
oral vein. Five minutes following the injection, a midline in-
cision was made from lower abdomen to clavical, the rib cage
opened, and heart exposed. A blood sample (0.2m1) was colllected
from the left ventricle of the heart. Following this the rat
was perfused with 0.9% saline for 3 minutes. The brain was
quickly removed' dissected into, the following three sections: 0
cortex, mil'dbrain and cerebellum (note that the dissection was . p
done by hand and is subject to some variability). Blood and Q
brain sections were placed' in indiividulal scintillation tubes f+
to which 5.5m1 of scintillation fluid was added. Searle Anal- .~ :
ytical (Model Delta 300)sScintillation Counter was used to ~
analyze the samples. ~
Page 2: gyc08e00
. There is no marked difference between intravenous
. January 20, 1982
1. Acetaldehyde readily permeates the blood-brain.
and intraarterial injections in the amount of acetalde-
hyde.that gets to brain tissue.
5-minutes post injection was approacimately 1 to 10.
. The ratio of acetaldehyde in brain compared to blood -
4. There does not appear to be a gross regional distrib-
ution of acetaldehyde in the brain.
The.-purpose of the second study was to d~etermine if there
was differenti!al uptake of C14 acetaldehyde by nerve endings,
myelin and mitochondria.
Two male hooded rats weighing 300 to 350 grams were util-
ized in a preliminary study designed to test fo.r cellular acet-
aldehyde localization in.brain following an intravenous injec-
tion of the compound. Thirty micrograms (30ug) of C14acetalde-
hyde with an activi-ty of 0.25 microcuries per microliter was
injected into a fermorai.vein preparation. Five minutes post-
infusion the animal was sacrificed by a blunt bl!ow to the l.umba.r
region, and its brain quickly removed. The following method was
performed in duplicate (see Figure 1).
ai- %2,©0o ct's
Se~n 1_kr ~mtcroSam~~ ~raG'~'1o^~
~ . .
tYvc cnc~~^ O
m %4a c.lno n-A +r i a G0
oi frac,l-lonaA7ton rroG¢.cdWt'e. .
Page 3: gyc08e00
Dr. W. L. Dunn -3- January 20, 1982
1) 250pCi /'1 . 0mg
. 25uCi = 1lsg in 1},1
30u1 was injected; therefore,
.2) 30o1 = 7.5uCi = 30pg
3) 100cpm = 4x10ug correctedfor efficienc
of thecounter with a background of 30cpm
The results of expleriment.two show that most of the acet-
counted for radioactivity later. The supernatant was sp~un in
was resuspended (1Rti and 2R1) in 3m1 of 0.32M sucrose to be
as unlysed cells and large fragments of cell membrane and it *
in an analytical centrifuge and spun for 10 minutes at 1000
gravities (G.). The pellet contained large cellular debris suc
and transferred to two centrifuge.tubes. The tubes were pliaced
.The brain was rinsed in 0.9% saline prior to placement in 20m1
of 0.32' molar isotonic sucrose. This mixture was homogenized
,a top layer of 0.8 M sucrose. The non-resuspended synaptosomal -
fractiol n (in 0.32 sucrose) was applied onto these density gradient
tubes. These tubes were spun at 100,000G's for 1 hour. The three
fractions obtained from this spin are myelin, nerve endings, and
mitochondria. To 0.5cc of the initial pellet(non-resuspended),
the second supernate, and the myelin, nerve ending_and mitochon-
drial fractions of the last spin was added 5.5m1 of scintillation
fluid. The C14 activity of these samples was determined ultiliz-
ing a Searle Analytic s Scintillation Counter. The amolunt of
acetaldehyde present in each sample was determined by knowing
- were prepared containing a 5m1 bottom layer of_1.2 M sucrose and
rose. At this point sucrose density gradient centrifuge tubes
aptosomal fraction was resuspended in 2m1 of 0.32M isotonic suc-
tion which was set aside for later counting. The pellet or syn-
an ultracentrifuge at 12,000 G's for 20 minutes to separate the
microsolmal fraction (enidoplasmic reticulum, vesicles and axonal
fragments) from the fraction containing the nerve endings. The
supernate (1S2 and 2S2) from this spin was the microsomal frac-
aldehyde concentrates in the myelin and n.erve endings with a
smaller amount located in the mitochondria.
At present the biochemical data indicate that acetaldehyde p
further investigation of acetaldehyde at both the behavioral
and central nervous system levels. 1 n/ C
readily penetrates the blood-brain barrier and appears to be H
,eq,ually distributed in all brain regions. This data combined .'N'
with the behavioral data collected in our laboratory suggests 0O