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PHILIP MORRIS U. S. A. INTER-OFFlCE CORRESPONDENCE nt,~ RICHMOND, VIRGINIA ~- ERSONAL & CONFIDEi~T~ Dr. Wi 11 i am L. Dunn Date; January 20, 1982 Victor J. DeNoble and Yvonne Dragan ~ Research at the University of Rochester The following is a•report 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 addressed: 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 jections? - in blood compared intraarterial in- 4. Is there a regional brain distribution' of acetaldehyd'e?- PROCEDURE 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. ~ ~

. There is no marked difference between intravenous -2- . January 20, 1982 1. Acetaldehyde readily permeates the blood-brain. barrier. . Dunn 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. PROCEDURE 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). S~croS~~ R-rfSU~?=r,~:."~ v~`<<t RL W1u1aY dLbyIS, Sdc.ns S.~c, c rad~G M 5 I 1 SPtA Zpr,n ai- %2,©0o ct's tn Se~n 1_kr ~mtcroSam~~ ~raG'~'1o^~ ' ~ . . a~ OQ+OO~J ~ 5 ~ a 53 S tYvc cnc~~^ O ~ ~n N m %4a c.lno n-A +r i a G0 IPA~ ~ oi frac,l-lonaA7ton rroG¢.cdWt'e. .

Dr. W. L. Dunn -3- January 20, 1982 1) 250pCi /'1 . 0mg 25011Cil/1000ug . 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 .that - 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. CONCLUSIONS 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 /iw

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