Pharmacology Biochemistry & Behavior, Vol. 9, pp. 1-6. Printed in the U.S.A.
Al cohol Withdrawal and Magnesi um Def i ci ency
in Mi ce I J. K. BELKNAP, J. H. BERG AND R. R. COLEMAN Department of Psychology, University of Texas at Austin, Austin, TX 78712 (Recei ved 16 De ce mbe r 1977) BELKNAP, J. K. , J. H. BERG AND R. R. COLEMAN. Alcohol withdrawal and magnesium deficiency in mice. PHAR- MAC. BI OCHEM. BEHAV. 9(1) 1--6, 1978. --DBA/ 2J mi ce exposed t o chr oni c al cohol (et hanol ) i nt oxi cat i on wer e f ound t o have l ower whol e br ai n magnesi um (Mg) concent r at i ons t han cont r ol ani mal s. The sympt oms of al cohol with- drawal wer e found t o be strikingly si mi l ar t o t hose seen in Mg defi ci ent mice exposed t o a low Mg di et wi t hout al cohol exposure. These fi ndi ngs suggest t hat CNS bi g deficits pr oduced by al cohol exposur e coul d cont r i but e t o t he obser ved al cohol wi t hdrawal syndrome. Serum Mg concent r at i ons wer e al so det er mi ned, and low correl at i ons (~<0.3) wer e found wi t h brai n Mg concent r at i ons. Alcohol withdrawal Magnesium deficiency DBA/ 2J mice THE SYMPTOMS of alcohol (ethanol) withdrawal in physi- cally dependent persons include coarse tremors, spasms, convulsions, delirium (confusion, disorientation), hallucina- tions, delusions, shock, hyperreflexia, and hyperreactivity. The symptoms of magnesium deficiency in man (not involv- ing alcohol) are strikingly similar, which has led to the sug- gestion that Mg deficiency may be involved in producing many of the symptoms of alcohol withdrawal [5]. Magnesium deficiency is usually assessed clinically by the blood serum Mg concentrations. Normal serum levels typically fall in the relatively narrow range of 1.5-2.0 mEqA e in man. However, about a dozen studies reviewed by Flink [5] have indicated a very high incidence of hypomagnesemia (levels below 1.5 mEq/O among alcoholic patients, most of whom were studied during withdrawal. While Mg deficien- cies do not occur in all of the alcoholic patients studied, the very high incidence of hypomagnesemia is quite remarkable across the over 300 cases reported in the literature. In addi- tion, several studies have shown that the lowest serum Mg levels are found in those patients who eventually manifest the most severe withdrawal symptoms [17, 18, 19, 20, 22, 23]. Hypomagnesemia in alcoholics would be expected for several reasons. Inadequate food intake is well known among alcoholics, and this could lead to inadequate inges- tion of minerals, including Mg. Vomiting and diarrhea would greatly increase the rate of Mg loss [5]. Thirdly, the ingestion of alcohol has been shown to lead to a marked increase in urinary magnesium loss in both alcoholic and nonalcoholic subjects [12, 13, 15, 16, 17]. While most investigators agree that Mg deficiency is a common occurrence in chronic alcoholics, it is not clear what role this deficiency plays in the manifestations of the alcohol withdrawal syndrome [6]. This problem is compli- cated by the fact that some alcoholics have been observed to show delirium tremens or convulsions with essentially nor- mat serum Mg levels. Conversely, abnormally low levels have been observed in some patients showing minimal with- drawal symptoms [21]. These findings can be interpreted to mean either that (1) magnesium deficiency contributes little to the symptoms of alcohol withdrawal, or that (2) mag- nesium deficiency contributes importantly, but serum Mg levels are inadequate for assessing Mg deficiency in the cen- tral nervous system. A major obstacle in resolving this issue is the paucity of Mg determinations in the CNS of alcoholic patients. Glickman [7] found no significant differences in cerebrospinal fluid (CSF) Mg levels between alcoholics and nonalcoholics, although the difference was in the direction of reduced levels in the alcoholic patients. However, Mg is primarily an intracellular cation, so CSF levels would be expected to represent only a small percentage of the total brain Mg content (<5%) or the total brain Mg concentration (< 15%). One reason for the lack of a perfect correlation between serum Mg levels and alcohol withdrawal severity may be that serum Mg levels may not be a sufficiently accurate predictor of Mg deficiency. Several investigators have reported a low correlation between serum Mg levels and intraceUular red blood cell or whole muscle Mg levels [14, 20, 21]. One reason for this is that only a small fraction (less than 3%) of the total body Mg exists in the extracellular fluid space, and the ex- change of Mg between intraceUular and extracellular com- partments is very slow. This is especially true in the brain, where the levels of brain Mg are highly buffered relative to the rest of the body. It is widely believed that an energy dependent process is involved at the blood-brain interface 1This work was supported by NIDA Grant DA 01800 and a BRSG grant awarded to J. K. Belknap, Department of Psychology. Animals were maintained and utilized in accordance with NIH ethical standards. Reprint requests should be sent to J.K.B., Department of Pharmacology, School of Medicine, University of North Dakota, Grand Forks, ND 58202. Copyr i ght 1978 ANKHO I nt er nat i onal Inc. --0091-3057/ 78/ 0901-0001500. 75/ 0 BELKNAP, BERG AND COLEMAN TABLE 1 MEAN (-+ SD) BRAIN AND SERUM MG CONCENTRATIONS IN EXPERIMENT 1 (VAPOR INHALATION METHOD) Run No. 1 Run No. 2 Run No. 3 Brain Mg (mEq/kg) Control, ad lib - - 13.31 _+ .61 (9) 13.14 _+.31 (4) Control, pair fed 13.90 _ 1.85 (8) 13.59 -+ 1.06 (9) 13.74 _+.62 (8) Alcohol-intox 12.30 -+ 1.42 (9) - - - - Alcohol-withdrawn 12.64 _+ 1.89 (10) 12.82 +- .52 (I0) 13.08 -+ .69 (7) Serum Mg (mEq/l) Control, ad lib - - 1.67 _+ .37 (9) !.80 _+.33 (4) Control, pair fed 1.63 -+ .13 (8) 1.67 +- .39 (9) 1.76 _+.49 (8) Alcohol-intox 1.57 _+ .13 (9) - - - - Alcohol-withdrawn 1.56 _+ .21 (10) 1.50 + .25 (10) 1.55 -+ .57 (7) Sample Sizes (N) are in Parentheses. whi ch is responsi bl e for the rel at i vel y st abl e Mg levels in br ai n despi t e wildly fl uct uat i ng pl asma or serum l evel s [3,14]. Thus, it becomes ext r emel y i mpor t ant to measur e brai n Mg concent r at i ons in order to assess properl y the ex- t ent of behavi or al l y r el evant Mg defi ci ency and the role this may pl ay in the sympt oms of alcohol wi t hdrawal . The pur pose of t he pr esent report was to utilize ani mal model s of al cohol physi cal dependence to answer basi c quest i ons whi ch woul d be ext remel y difficult or i mpossi bl e to pur sue in human alcoholics. These quest i ons are: (1) is t here a Mg defi ci ency i n br ai n t i ssue resul t i ng from chroni c al cohol i nt oxi cat i on, and (2) to what ext ent can serum Mg concent r at i ons be used to predi ct t hose i n brai n. METHOD In Exper i ment 1, physi cal dependence on alcohol was produced by the vapor i nhal at i on met hod of Gol dst ei n and Pal [9]. DBA/2J male mice (Jackson Labor at or i es, Bar Har- bor, ME), 12-17 weeks of age, were chroni cal l y exposed to an i nt oxi cat i ng alcohol vapor for 4 days at 24 _+ IC. Four 17 liter jar s with tight fitting lids (Eco-Jar, Nal ge Co.) served as living chamber s with a maxi mum of 8 mi ce housed per jar. Because of the limited capaci t y of the chamber s, it was nec- essar y to conduct t hree separat e r uns under identical condi - t i ons. The groups and N' s i nvol ved are shown in Tabl e 1. The flow rat e was 35 to 50 ml / mi n of air per mouse deliv- ered from a regulated compr essed air cyl i nder. For the al cohol -exposed groups, this air flow was di vi ded into two parts: the first (1/3 of the total) passed t hrough the head space of a 1000 ml flask cont ai ni ng 250 ml of 95% al cohol , and the second (pure air) passed di rect l y to the chambers. The ratio of t hese two air flows i nt o the chamber s (cont rol l ed by two Gi l mont fl owmet ers with needl e val ves) det er mi ned the alcohol vapor concent r at i ons. Each day an exhaust sampl e from each chamber was collected by a gas sampl i ng val ve in a Carle Model 211 gas chromat ograph (FI D, Porapak Q col umn at 130C) to moni t or the alcohol concent - rat i ons in each of the alcohol vapor chamber s. Thi s concent - rat i on was al ways mai nt ai ned bet ween 9 and 10 mg/ (. Tail vei n blood sampl es (10/xl) were assayed for al cohol by gas chr omat ogr aphy [1]. Fi ve mice were sampl ed at r andom from the al cohol chamber s on Days 2 and 4 duri ng Runs 1 and 2. Two cont rol groups were r un concur r ent l y with the al cohol -exposed mi ce; one was allowed ad lib access to food (Puri na Lab Chow), while t he ot her (pair fed cont rol ) was gi ven the same amount of food (as a group) as was consumed ad lib by the al cohol -exposed groups. The cont rol groups were housed and t reat ed i dent i cal l y to the al cohol -exposed groups except for the i nt r oduct i on of alcohol vapor i nt o the chamber s. Wat er was avai l abl e ad lib for all groups. The Mg cont ent of the diet was 125 mEq per kg as det er mi ned by at omi c absor pt i on spect r oscopy (see below). At the same t i me each day (3-4 hr after light onset on a 12L: 12D cycle), all of the ani mal s and uneat en food were weighed, and new food (preweighed), beddi ng (pine shavings), and fresh wat er provi ded. All groups also recei ved daily i nject i ons (IP) of pyrazol e (68 mg/kg), an al cohol dehydr ogenase i nhi bi t or, to st abi l i ze the bl ood al cohol l evel s as r ecommended by Gol ds- t ei n [8]. The first i nject i on was gi ven j us t pri or to pl aci ng the mi ce in the chamber s; the last (fourth) was gi ven 1 day pri or to wi t hdrawal . For the al cohol -exposed groups, t he initial pyr azol e i nject i on was gi ven al ong with a 2 g/kg pri mi ng dose of alcohol. Aft er four days of al cohol exposur e, wi t hdrawal was accompl i shed by r emovi ng the mice from the chamber s at 3 hr after light onset . The al cohol -exposed groups were ei t her sacrificed for Mg assay on the last day of i nt oxi cat i on (Run 1 onl y, 16 hr pri or to withdrawal) or 24 hr later at the appr oxi mat e t i me of max- imal wi t hdrawal severi t y (7-9 hr after wi t hdrawal ). Thi s was done so that all ani mal s coul d be sacrificed at about the same t i me of day (just before light offset). The cont rol s were sac- rificed concur r ent l y. Magnesi um levels were det er mi ned by at omi c absor pt i on spect r oscopy (Per ki n- El mer Model 303). The mice were de- capi t at ed and the whole br ai n r emoved, weighed, and di- gested in 15 vol umes of a mi xt ure of 81Y~ concent r at ed ni t ri c, 15% concent r at ed sulfuric, and 5% perchl ori c acid (70%). Blood sampl es (.2 ml) were also t aken and di l ut ed 6 fold in 0.~}% saline, al l owed to st and for about 60 mi n, and cen- trifuged at 3000xg for 10 mi n to r emove cells and coagul um. Fol l owi ng appropri at e di l ut i ons, the br ai n and bl ood sampl es were aspirated i nt o the at omi c absor pt i on spect rophot ome- ALCOHOL WI T HDRAWAL I N MI CE 3 TABLE 2 MEAN (_+ SD) BRAIN WT., % BRAIN WATER, BODY WEIGHTS AND FOOD CONSUMPTION DATA FROM EXPERIMENT 1 (VAPOR INHALATION METHOD) Alcohol Pair Fed Ad Lib Runs Brain Wt (mg) 365 17 (36) 369 --- 18 (25) 371 --- 15 (13) 1-3 ' (at sacrifice) Brain % water 78.6 --- .8 (10) 78.4 .5 (9) 78.3 - .5 (9) 2 Body Wt 28.6 2.0 (36) 27.8 - 1.9 (25) 28.2 1.7 (13) 1-3 (initial) Body Wt 25.7 2.6 (36) 25.6 1.7 (25) 27.6 _+ 1.4 (13) 1-3 (at sacrifice) Food Consumption 1.7 1.6 3.0 1-3 (g/day/mouse) Blood Alcohol 2.36 .40 (20) - - - - 1-2 Sample Sizes (N) are in Parentheses. t er accor di ng t o t he Per ki n- El mer pr oce dur e s manual . Lant hanum oxi de (. 5% fi nal concent r at i on) was pr es ent in all sampl es. Al l Mg det er mi nat i ons wer e done on a bl i nd basi s. Br ai n wat er cont ent was det er mi ned in s ome of t he ani mal s by compar i ng wet wei ght (fresh) wi t h dr y wei ght (af t er 20 hr at 150C). The per cent age wa t e r cont ent was vi r t ual l y i dent i - cal in al l gr oups (Tabl e 2). The mor t al i t y r at e dur i ng i nt oxi cat i on (Runs 1-3) aver - aged 11%: t hes e a ppe a r e d t o be due t o over dos es . Thes e mi ce wer e excl uded f r om t he da t a anal ysi s. A f our t h run was conduct ed as des cr i bed above, except t hat t hes e mi ce (N = 19) wer e al l owed t o under go wi t hdr awal f or t he pur pos e s of moni t or i ng t he wi t hdr awal s ymp- t oma t ol ogy f ol l owi ng f our days of al cohol expos ur e. At 2 hr i nt er val s, each ani mal was s us pended by t he t ai l for 10 sec, r et ur ned t o i t s home cage, and obs e r ve d f or t he next 1 mi n. Si x s ympt om cat egor i es wer e chos en on t he basi s of (1) ease of obj ect i ve eval uat i on and (2) di s t i nct nes s f r om any behav- i or seen in t he cont r ol mi ce. Thes e s ympt om cat egor i es wer e: convul s i ons on handl i ng ( CH) - - my o cl o n i c spasms of t he f ace, usual l y ext endi ng t o t he r est of t he body; t r emor (TR)- - - mar ked t r emul ous nes s of t he ent i r e body; St r aub t ai l (ST)- - a r chi ng of t he t ai l over t he back, a sign of mus cul ar r i gi di t y; hyper r eact i vi t y ( HR) - - p r o n o u n ce d j umpi nes s when t ouched r esul t i ng in l eaps in exces s of t he ani mal ' s body l engt h; sei zur es ( SZ) - - cl oni c or t oni c- cl oni c sei zur es (non- l et hal ) i nvol vi ng most of t he body; l et hal sei zur es (LSZ)- - - t oni c e xt e ns or sei zur es r esul t i ng in r es pi r at or y par al ys i s . Convul si ons on handl i ng was s cor ed whi l e t he mouse was s us pended by t he t ai l ; all of t he ot her s ympt om cat egor i es wer e scor ed whi l e t he ani mal was in i t s home cage. Convul - si ons on handl i ng have been f ur t her des cr i bed by Gol ds t ei n [8,10]. I n Expe r i me nt 2, ni ne DBA/ 2J mal e mi ce, 11 weeks ol d, wer e r ender ed phys i cal l y de pe nde nt on al cohol by t he l i qui d di et met hod of Gol ds t ei n and Ar nol d [11]. Thi s exper i ment was cons i der ed des i r abl e be ca us e i t i nvol ves a ver y di f f er ent met hod of phys i cal de pe nde nce pr oduct i on compa r e d t o t he va por i nhal at i on met hod, and pyr a zol e i nject i ons wer e not empl oyed. I n t he pr et r eat ment phas e, t he al cohol - expos ed mi ce wer e gi ven a 3.9% (w/v) al cohol sol ut i on in wat er as t hei r sol e fl ui d s our ce f or 5 days . Thi s was done t o mi ni mi ze t he devel op- ment of a condi t i oned aver s i on t o t he al cohol sol ut i ons dur - i ng t he s ubs equent t r eat ment phase. Pur i na l ab chow was avai l abl e ad l i b. On Days 6 t hr ough 12, t he t r eat ment phas e cons i s t ed of t he ad lib pr es ent at i on of a l i qui d di et (Car nat i on Sl ender , chocol at e f l avor ) cont ai ni ng 3. 9% w/v al cohol (final concent r at i on) as t he sol e fl ui d and f ood sour ce. Thi s was accompl i s hed by addi ng 17 ml of 95% al cohol and 30 ml of wat er t o each can (300 ml) of liquid di et . A cont r ol gr oup (N=9) was t r eat ed i dent i cal l y t o t he al cohol - expos ed gr oup except t hat i s ocal or i cal l y equi val ent amount s of s ucr os e wer e subst i t ut ed f or t he al cohol dur i ng t he pr e t r e a t me nt and t r eat ment phas es . The cont r ol gr oup was pai r - f ed (as a gr oup) t o i nsur e t hat bot h gr oups r ecei ved equi val ent cal or i e and nut r i t i onal i nt ake t hr oughout t he exper i ment . Bl ood al cohol det er mi nat i ons wer e made at 2 hr af t er light onset on Days 3 and 6 of t he t r eat ment phas e as des cr i bed in Exper i - ment 1. Veni punct ur e was al so done on t he cont r ol s, but no as s ays wer e per f or med. Bot h t he al cohol - expos ed and cont r ol gr oups wer e sac- r i f i ced j us t bef or e l i ght of f set at t he end of t he si xt h day of t he t r eat ment phase. The pr ocedur e f or magnes i um a s s a y was t he same as in Exper i ment 1. I n Expe r i me nt 3, an addi t i onal gr oup of mi ce (N=9) wer e pl aced on di st i l l ed wat er and a l ow Mg di et (Nut r i t i onal Bi ochemi cal s) cont ai ni ng 0.08 mEq/ kg di et f or 21 days (no al cohol ). Begi nni ng on Day 13, when t he s ympt oms of Mg def i ci ency wer e evi dent in all of t he mi ce, a 1.5 g/kg I P dos e of al cohol was gi ven t o t hr ee of t he ani mal s chos en at r an- dom. Anot he r t hr ee mi ce r ecei ved 0.75 g/ kg of al cohol and t he l ast t hr ee r ecei ved onl y sal i ne. The i nject i on vol ume was 10 ml / kg in 0. 9% sal i ne. The s ympt oms of Mg def i ci ency wer e t hen moni t or ed as des cr i bed above (Exper i ment 1) at 15 mi n i nt er val s f or 75 min. Ther e wer e t hus a t ot al of fi ve obs er vat i ons made pe r mous e af t er each i nject i on. Thi s pr o- cedur e was r epeat ed on Days 15, 17, 19 and 21, wi t h each ani mal assi gned t o each of t he t hr ee t r eat ment gr oups in a count er bal anced f ashi on. RESULTS The Mg a s s a y da t a f or al l t hr ee r uns in Exper i ment 1 (vapor i nhal at i on) ar e shown in Tabl e 1. Combi ni ng br ai n Mg concent r at i on dat a f or all al cohol - expos ed (N=36), pai r f ed 4 BELKNAP, BERG AND COLEMAN 6 0 4 0 2 0 I I I t i 80 l HYPERREACTI Vl TY CO l -- J ILl --3 nn Z) O0 U _ 0 F-- z UJ n" LU n 6 0 4 0 2 0 0 _ TREMOR 6 0 4 0 2 0 TONI C- CLONI C SEI ZURES STRAUB TAI L A I - - B _LETHAL SEI ZURES ~ l I t I u / ~ k ~ I 5 1 0 1 5 2 0 2 5 5 1 0 1 5 2 0 2 5 HOURS AFTER WI T H D R A WA L FIG. 1. The relative frequencies for the six symptom categories used to assess alcohol withdrawal in DBA/2J male mice (N= 19) as a function of time (hours) after withdrawal (Experiment 1). (N =25) and ad lib cont rol ani mal s (N = 13) over all t hree r uns resul t ed in an F ratio of 6.2 (p<0. 003, df=2/72). Compari - sons of t hese t hree groups combi ned across all t hree r uns showed that the al cohol -exposed mi ce had si gni fi cant l y l ower brai n Mg concent r at i ons t han ei t her pai r fed (t =3. 2, p<0. 002) or ad lib cont rol s (t =2. 2, p<0. 03). An unexpect ed t endency for pai r fed mice to have hi gher brai n Mg concen- t rat i ons t han ad lib cont rol s was also obser ved ( t =l . 6, p<0. 10). A si mi l ar anal ysi s was performed on serum Mg concent r at i ons. While the mean serum Mg concent r at i ons followed the same pat t er n as the br ai n Mg concent r at i ons, the F ratio was nonsi gni f i cant (F=1. 68, p<. 20, df=2/72). When pai r fed and ad lib cont rol s, whi ch did not differ (t--0. 21, p<0. 6), were pool ed and compar ed with the al cohol -exposed ani mal s, significance was near l y achi eved (F=3. 36, p<0. 07, dr= 1/73). Serum Mg concent r at i ons proved to be a rat her poor predi ct or of br ai n Mg concent r at i ons. Combi ni ng over all r uns, the obser ved correl at i ons bet ween brai n and serum l evel s were, for the al cohol -exposed ani mal s, r =. 17; for pai r fed cont rol s, r =. 02; and for ad lib cont rol s, r =. 06. Combi n- ing over all groups wi t hi n each r un yi el ded correl at i ons of . 30 (Run 1), .09 (Run 2) and .16 (Run 3). Fi gure 1 di spl ays the t i me cour se for the six sympt om cat egori es used to moni t or wi t hdrawal in Exper i ment I. The sympt oms began at about 3 hr aft er wi t hdrawal and were ALCOHOL WI THDRAWAL I N MICE 5 essentially ended at about 30 hr. The vapor inhalation tech- nique produced a marked degree of physical dependence as evidenced by the withdrawal syndrome. The data for Experiment 2 (liquid diet method) are shown in Table 3. Once again, the alcohol-exposed group exhibited significantly lower brain Mg concentrations than did the pair fed controls (t=4.30, p<0.001). The serum Mg concentra- tions were lower in the alcohol-exposed group, but significance was not achieved (t = 1.26, p<0. 25). The corre- lation coefficient between brain and serum Mg concentra- tions was .23 (N=IS). In our experience, the withdrawal severity produced by this method is equal to or slightly greater than that produced by the vapor inhalation method. The withdrawal symptomatology and time course are very similar for both methods of physical dependence production (unpublished). The alcohol consumption for individual mice can be readily calculated with the liquid diet method. The correlation coefficient between total alcohol consumed (cor- rected for body weight) in the treatment phase and brain Mg concentrations was - . 85 (N=9). The corresponding correla- tions for serum Mg concentrations was - . 20 (N=9). Thus, animals consuming the most alcohol tended to show the low- est Mg concentrations. TABLE 3 MEAN ( - SD) VALUES I N EXPERI MENT 2 (LI QUI D DI ET METHOD) DURI NG THE TREATMENT PHASE Alcohol Control Br a i n Mg ( mEq/ k g ) 13. 10 --- . 43 13. 86 - .31 S e r u m Mg ( mEq/ l ) 1. 53 --- . 26 1. 66 --- . 17 Bo d y Wt (i ni t i a l ) 23. 0 --- 3. 4 22. 9 --- 3. 6 Body Wt (at sacrifice) 19.6 -+ 2.7 19.8 - 2.6 Diet Consumption 9.9 -+ 1.2 10.4 +_ .2 (ml/day/mouse) Alcohol Consumption 19.0 -+ 4.3 - - (g/kg/day) Blood Alcohol 2.02 -+ 1.0 - - (mg/ml), days 3 and 6 N=9 per group. In Experiment 3 (low Mg diet), the saline injected mice exhibited the following relative frequencies (in percent of total observat i ons)--CH, 67%; TR, 27%; ST, 13%; HR, 15%; and SZ, 15%. The 1.5 g/kg alcohol injected mice showed no symptoms whatsoever (p<0.05 for each symptom category, Fisher Exact Probability Test), while the 0.75 g/kg injected mice showed no symptoms except CH, 40% and HR, 11%. This suppression of Mg deficiency symptoms occurred at doses of alcohol which produced no overt ataxia or other signs of gross intoxication. The Mg deficiency symptoms essentially returned to control (saline) levels at about 3 hr after injection. DI SCUSSI ON The results represent the first demonstration that chronic alcohol exposure can produce a state of Mg deficiency in brain tissue. The alcohol-exposed mice averaged 7.7% lower Mg concentrations than the pair fed controls and 4.3% lower than the ad lib controls (Experiment 1). In Experiment 2, the alcohol-exposed group averaged 5.5% lower brain Mg con- centrations vs. pair fed controls. But is this Mg deficit large enough to be behaviorally significant? Some insight can be gained from studies where a Mg deficiency was induced by means of a low Mg diet (no alcohol). Belknap, e t al . [2] exposed DBA/2J mice to a low Mg diet for 14 days, at which time a 10.8% reduction in brain Mg concentration was ob- served. Unfortunately, brain Mg determinations were made at only one point in time (Day 14) while the symptoms began much earlier on the fifth day of diet exposure. Using a low Mg diet with weanling rats, Chutkow and Grabow [4] first observed audiogenic seizures in over half of the subjects at a time (Day 4) when the brain Mg concentrations had declined by only 4.2% compared to pair fed controls. In a similar study, Chutkow [3] reported spontaneous and audiogenic tonic-clonic seizures in the majority of rats after 8 days of low Mg diet exposure. At that time, an 8.2% decline in brain Mg concentrations was noted vs. pair fed controls. Thus, it is possible that the brain Mg deficiency seen in the alcohol- exposed mice might be large enough to contribute to the withdrawal symptomatology; however, more work is needed to determine the extent of the contribution. One drawback of the vapor inhalation method is that it calls for the use of pyrazole to stabilize the blood alcohol levels [11]. However, the results from Experiment 2 (liquid diet method) are closely similar to those seen in Experiment 1 (vapor inhalation method). This strongly suggests that the use of pyrazole in both the control and alcohol-exposed groups in Experiment 1 did not appreciably affect the overall I~a~t~ern of the results. ?The factors responsible for the Mg loss in brain tissue are not known, but it could be due to either a greater rate of renal loss relative to dietary intake, or a redistribution of Mg among body pools. The latter possibility has been suggested by some studies with human alcoholics [17, 22, 23]. During withdrawal, serum Mg levels are closely correlated with (respiratory) alkalosis, and these pH imbalances have been hypothesized to produce shifts in Mg pools [17, 22, 23]. De- finitive evidence is lacking, however, and more work is needed to determine the mechanisms responsible for the low Mg concentrations in brain and serum. The results from Experiment 3 clearly demonstrate that moderate doses of alcohol can suppress the symptoms of Mg deficiency produced by a low Mg diet. This finding probably explains why the intoxicated animals from Experiment 1 (3 1/3 days of alcohol intoxication) did not exhibit any symp- tomatology in spite of the low brain Mg concentrations found in these animals. These results are consistent with the hy- pothesis that alcohol masks the latent hyperexcitability due to CNS Mg deficiency: when the alcohol is withdrawn, the Mg deficiency may contribute to the manifest hyperexcita- bility characteristic of the alcohol withdrawal syndrome. The low correlations between serum and brain Mg con- centrations are consistent with the low correlations reported by others between serum and intracellular fluids such as blood cells or whole muscle in man [14, 20, 21]. The use of serum concentrations to assess CNS Mg deficiency is open to considerable error, and this consideration is very impor- tant in interpreting the data from human alcoholics where serum values are typically the only ones available. Finally, the symptoms of Mg deficiency in mice (Experi- ment 3, low Mg diet) were seen to be strikingly similar to those seen in alcohol withdrawal (Experiments 1 and 2). Thus, the present findings suggest that alcohol-induced Mg deficiency may contribute, along with other factors, to the etiology of the withdrawal syndrome. 6 BELKNAP , BERG AND COLEMAN REF ERENCES 1. Bel knap, J. K. , N. D. Bel knap, J. H. Berg and R. Col eman. Pr eabsor pt i ve vs. post absor pt i ve cont rol of et hanol i nt ake in C57BL/ 6J and DBA/ 2J mice. Behav. Genet. 7: 413-425, 1977. 2. Bel knap, J. K. , J. H. Berg, R. Cocke and A. N. Cl ancy. Induc- t i on and reversal of t he magnesi um defi ci ency syndr ome in in- br ed mice. Expl Neurol. 57: 506-515, 1977. 3. 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