Role of Tissue Lactate and Substrate Availability in 1,3-Butanediol-Enhanced Hypoxic Survival in the Mouse*

Transcription

1 Rle f Tissue Lactate and Substrate Availability in 1,3-Butanedil-Enhanced Hypxic Survival in the Muse* J. R. KlRSCH AND L. G. D'ALECY SUMMARY Previusly we fund that 1,3-butanedil-treated mice live lnger during hypxia. We hypthesized that 1,3-butanedil culd reduce the brain's accumulatin f ptentially cyttxic lactate and/r elevate brain substrate availability (ketnes r glucse) and thus maintain the brain's energy prducing capability even during redued xygen availability. T test these hyptheses, whle brain metablites frm nrmxic and hypxic mice, pretreated with 1,3-butanedil r insulin, were cmpared t saline cntrls. During hypxia bth pretreated grups had lwer brain lactate than cntrls. If lactate accumulatin was the sle factr respnsible fr hypxic tlerance, insulin shuld have increased brain lactate since insulin has been shwn previusly t reduce hypxic tlerance. In nrmxic mice the rati f lactate t pyruvate and the level f malate and fumarate were nt changed by 1,3-butanedil as is fund with ther agents knwn t prtect the hypxic animal. When substrate availability was directly elevated by beta-hydrxybutyrate and glucse administratin hypxic survival time increased thus implicating substrate availability as an imprtant factr in hypxic tlerance. We cnclude that reduced brain lactate and augmented substrate availability bth cntribute t 1,3-butanedil-enhanced hypxic tlerance in this animal mdel. Strke Vl 14, N 6, 1983 IN PREVIOUS STUDIES frm ur labratry ketsis induced by 1,3-butanedil (BD) pretreatment was shwn t be cincident with a dramatic increase in the tlerance f mice t hypxia. 1 Myles 2 reprts increased hypxic tlerance in fasted, and presumably kettic animals, expsed t simulated high altitude. Others have shwn that a fasting individual's brain uses ketnes fr as much as 60% f their energy requirement. 3 ' 7 In cnditins ther than fasting, e.g. in newbrn animals ketnes are nrmally used fr brain metablism 5-6 and newbrn animals als have increased tlerance t hypxia. 7-8 Such changes in hypxic tlerance during these kettic cnditins culd, f curse, be prduced by sme mechanism ttally distant frm ketsis. We have identified tw general hyptheses that initially ffered plausible explanatins fr the increased hypxic tlerance bserved in animals rendered kettic by BD. The first hypthesis cntends that tissue lactate is the key cyttxic factr in that lactic acid accumulatin has been crrelated with autlysis f neurnal tissue An alternative and less traditinal hypthesis is that ketsis enhances hypxic tlerance by directly r indirectly maintaining the brain's energy prducing capability. Directly the ketnes culd enter the tricarbxylic acid cycle thrugh cnversin t acetyl CA and indirectly they culd, in additin, have a permissive effect n glyclytic flux by, fr example, cnsuming reducing equivalents during the frmatin Dwnladed frm by n September 27, 2018 Frm the Department f Physilgy, The University f Michigan Medical Schl, Ann Arbr, Michigan *Supprted by: American and Michigan Heart Assciatins, U.S. Public Health Service Grants NS 12386, HL20399, T35-NS07197 and the University f Michigan Diabetes Research and Training Center's U.S. Public Health Service Grant AM An abstract f part f this bk was presented at the Tenth Internatinal Sympsium n Cerebral Bld Flw and Metablism, St. Luis. Address crrespndence t: Dr. Luis G. D'Alecy, Department f Physilgy, The University f Michigan, 7799 Medical Science Building II, Ann Arbr, Michigan Received February 28, 1983; revisin accepted May 13, f fatty acids. It shuld be stressed that either f these ketne based mechanisms require xygen and hence wuld nly be functinal during hypxia and nt during anxia. T test the hypthesis which is cncerned with the rle f tissue lactate we pretreated mice with either BD r insulin and measured brain metablites after varius times f expsure t hypxia. T determine if elevated substrate availability wuld enhance hypxic tlerance, the survival time f mice pretreated with either saline, BHB and dextrse, BHB alne r dextrse alne was cmpared. The results suggest that lactate accumulatin and cntinued substrate supply are imprtant factrs in determining the utcme f a hypxic challenge in BD pretreated animals. Methds General Prcedure The animal mdel used was described by Wilhjelm and Arnfred 12 and Steen and Michenfelder 13 and used in ur labratry. 1 I4-15 Adult male Sprague-Dawley albin mice (HA-ICR) weighing apprximately 30 g were pretreated and then thirty minutes later expsed t either a nrmxic gas mixture (rm air) r a hypxic gas mixture (apprximately 4.5% 0 2, 95.5% N 2 ). All IV injectins were made with a 26 gauge needle int a lateral tail vein. Brain Metablite Experiments Fr each trial tw cntrl (saline IV r IP) and three treated animals were used. The grup f five mice was placed in an airtight 5.5 liter flw-thrugh Dewar flask which was cntinuusly flushed with the gas mixture at rm temperature (22-24 C). The imprtance f simultaneusly testing cntrl and treated mice was recently restated in a critical assessment f this animal mdel. 16 The diurnal variatin in hypxic tlerance reprted by Stupfel et al 17 als fcuses n the need fr simultaneus testing f cntrl and treated animals. Further, t assure the cmparability f separate runs

2 Dwnladed frm by n September 27, 2018 the cmpsitin f the gas was cntinuusly mnitred with an xygen analyzer (Beckman OM-14). Thirty minutes prir t hypxia animals were pretreated by ne f the fllwing: 1.4 mmles f BD in 0.25 ml sterile water IV; 0.25 ml f sterile saline (0.9% sdium chlride) IV; 2U Of regular insulin in 0.5 ml f sterile saline IP; 0.5 ml f sterile saline IP. Hypxia was induced by first flushing the flask at abut 10 liters/min with an 8% 0 2 and 92% N 2 gas mixture. After secnds the chamber xygen was reduced t abut 4.5% at a rate f 10 liters/min. In the nrmxic grup the chamber was flushed at a rate f 10 liters/min with cmpressed rm air (20.95% 0 2 ). These prcedures were used t mimic, as clsely as pssible, the timing, pretreatments, and gas expsures used in ur previus studies thus permitting direct cmparisns with previus data. After 30, 60, 90, r 120 sec f expsure t hypxia the mice were submerged in apprximately 4 liters f liquid N 2. Brain Metablite Analysis The brains were dissected ut f the skulls, grund in liquid N 2 with a mrtar and pestle and pured int a preweighed vial cntaining 1 ml f 3M HC10 4. The dissectin, grinding and deprteinizatin were dne in a cld rm at - 20 C. After each vial was re weighed, 2 ml f deinized distilled water was added t the HC10 4 -brain mixture. Each sample was hmgenized fr 30 secnds at maximum speed (Brinkman Plytrn) at 4 C and centrifuged at 7710g fr 20 minutes. The supernatant (2.7 ml) was withdrawn and neutralized slwly by mixing it with 2N KHC0 3 prir t analysis fr the brain metablite. Lactate was measured by spectrphtmetry (Gilfrd 3500 Cmputer Directed Analyzer) using the lactate dehydrgenase methd. 18 Recvery curves were generated fr each grup f samples assayed and recvery averaged 99.7 ± 0.4% fr lactate. Pyruvate, 19 malate, and fumarate 20 were measured flurmetrically (Farrand Rati Flurmeter-2). Recvery was ± 2.9, ± 3.6, and 98.6 ± 6.8 percent, respectively, fr each metablite. Hypxic Survival Time Experiments Fr the reasns described abve, 2 cntrl and 3 experimental animals were placed in five individual airtight, 110 ml flw-thrugh chambers munted in parallel and cntinuusly flushed with a mnitred (Beckman OM-14) gas mixture at rm temperature. When the animals were first placed in the chambers cmpressed rm air was delivered t the chambers. Thirty minutes prir t the initiatin f hypxia animals were given bth an IV and IP injectin. The IV injectin was f either 62.5 mg dextrse in 0.25 ml sterile water r 0.25 ml f sterile nrmal saline. IP injectins included 60 mg DL-BHB delivered in 0.5 ml sterile water r 0.5 ml f sterile nrmal saline. Hypxia was induced by first flushing the system at 2 1/ min with an 8% xygen, balance nitrgen, gas mixture. After sec the chamber xygen was reduced t abut 4.5% withut changing the flw rate. Hypxic survival time (HST) as described by Wilhjelm and Arnfred 12 and Steen and Michenfelder 13 and currently used in ur labratry is the time frm the nset f hypxia t the cessatin f spntaneus ventilatin. Althugh the exact relatinship between HST and brain dysfunctin has nt been defined in this muse mdel it is mre clearly described in dgs. When hypxia is used as euthanasia in dgs the EEG becmes iselectric, with a nrmal r elevated bld pressure, then respiratin is stimulated befre being depressed and finally there is cardivascular cllapse. 21 A similar sequence f iselectric EEG, respiratry stimulatin, then depressin and final cardivascular cllapse was recently bserved in rats expsed t unilateral cartid ligatin and hypxia. 22 These patterns are cnsistent with the brain being the initial tissue cmprmised by hypxia fllwed by respiratry failure and ultimately cardivascular decmpensatin. Bld Glucse and BHB Analysis An additinal grup f mice was treated with bth an IV and IP injectin as described in the previus sectin. In rder t btian an assessment f the bld glucse and BHB levels at the nset f hypxia these animals were decapitated 30 minutes after the injectin instead f being expsed t hypxia. Because decapitatin was the methd used t btain the bld samples we were nly able t btain ne sample per animal at any time. The bld was cllected in a heparinized tube and immediately centrifuged t btain the plasma which was then deprteinized and neutralized befre analysis. Plasma glucse was measured enzymatically with the hexkinase methd 23 using reagents f Wrthingtn Bichemicals n a Gilfrd 3500 Cmputer-Directed Analyzer. BHB was detected enzymatically (beta-hydrxybutyrate dehydrgenase methd) 24 n a Farrand Rati Flurmeter 2. Data Analysis Statistical analysis was perfrmed with the aid f the Michigan Interactive Data Analysis System (MIDAS) n an Amdahl 470/v7 cmputer facility. Cmparisn f each experimental grup with the cntrl grup was dne with a tw-tailed Student's t-test using the Bnferrni crrectin fr multiple cmparisns where apprpriate. All results are expressed as mean ± ne standard errr f the mean (SEM); the sample size is designated (n). Brain metablites are all reprted in ^tmles/g wet weight and are nt crrected fr bld r cerebrspinal fluid metablite cntent. Entrapped bld in brain tissue represents apprximately 3% f the brain weight 25 and is f unknwn cmpsitin, thus making precise crrectin untenable. Results Brain Metablite Experiments General The average age fr 118 mice used in this prtcl was 53 ± 2 days and average brain weight used fr the analysis f metablites was 377 ± 5 mg and included bth fre- and hindbrain. Fd and water was available t all animals up t the time f the experiment. The average xygen cntent flushing the Dewar flask dur-

3 ing the hypxic perid was 4.72 ± 0.03%. With this lw an inspired xygen level the arterial xygen tensin althugh nt measured, culd nt exceed 20 mmhg at sea level regardless f respiratry rate and cardiac utput. Glyclytic Intermediates During nrmxia the brain tissue lactate was 2.5 /xmles/g and was nt changed significantly by pretreatment with BD r insulin. Each grup f animals subjected t hypxia shwed an increasing tissue lactate level with increasing duratin f hypxic expsure (fig. 1). The level f lactate accumulatin during hypxia was significantly (by Bnferrni weighting f Student t) lwer in bth BD (p = 0.02 at 90 sec andp = at 120 sec) and insulin [p = at 60 sec) pretreated animals as cmpared t saline pretreated cntrl animals. The difference between lactate in the saline and BD pretreated animals increases with the increase in duratin f hypxic expsure (fig. 1). During nrmxia brain pyruvate was 0.26 ± 0.02 jiimles/ g(n = 10) and was decreased significantly (P < 0.01) (34%) with BDpretreatment t 0.17 ± 0.02 (n = 10) and with insulin (P < 0.05) pretreatment t 0.17 ± 0.01 (n = 4). During hypxia the saline treated animals had a brain pyruvate level f 0.27 ± 0.02) (n = 14) which was nt statistically different frm levels in BD (0.23 ± 0.01) (n = 16) r insulin (0.24 ± 0.01) (n = 8) treated animals. In ne series f animals bth lactate and pyruvate were measured simultaneusly (table 1). In nrmxic animals the lactate-pyruvate rati was unaffected by BD pretreatment but increased by insulin pretreatment. With the exceptin f insulin pretreated animals, the lactate-pyruvate rati increased after expsure t hypxia. Whereas pretreatment with BD did nt significantly alter the hypxic lactate-pyruvate rati, insulin pretreated animals had a rati which was 37% lwer than cntrls (p < 0.05). Dwnladed frm by n September 27, 2018 Kreb Cycle Intermediates Neither brain malate r fumarate levels were significantly different frm saline cntrls in the nrmxic BD pretreated animals (fig. 2). With expsure t hypxia bth the saline and BD pretreated animals shwed marked increases in the accumulatin f these metablites. The hypxic accumulatin f malate hwever was slightly but significantly inhibited by BD when cmpared t the saline pretreated cntrl grup. HST Experiments The average age and bdy weight f mice used in these experiments was 39 ± 2 days and 27.3 ± 0.3 g respectively, n = 142. The average xygen cntent f the gas flushing the test chambers was 4.62 ± 0.01% during the hypxic perid. Animals given saline IV and IP had average HST, plasma glucse and plasma BHB levels (table 2) cmparable t thse we have published previusly. 15 As expected animals receiving IP injectins f BHB had HST and plasma BHB levels significantly higher than cntrl. In animals receiving nly IP BHB, plasma glucse levels were slightly lwer than cntrl. Elevatin f substrate availability by *>. O E UJ r- < O < pretreatment with dextrse IV prduced an increase in plasma glucse, a decrease in plasma BHB and n increase in HST. In cntrast animals pretreated with bth dextrse and BHB had HST values greater than wuld be expected fr a simple additive effect f the tw substrates. The plasma BHB level was significantly greater than the saline IV, saline IP cntrl and the plasma glucse was significantly (p = 0.05) greater than the saline IV, BHB IP grup. 12-* 3- (23) T 60 (6) I 120 HYPOXIC EXPOSURE (sec) FIGURE 1. On this graph brain lactate in pmlelg is pltted n the rdinate and the time f expsure t hypxia in secnds is pltted n the abscissa. This figure shws the average whle brain lactate cntents f mice that were fixed in liquid N after ne f the fllwing duratins f hypxia: 0 secnds, 30 secnds, 60 secnds, 90 secnds, r 120 secnds. The clsed circles represent saline cntrl mice, the pen circles represent animals treated with 1.4 mmles fbd, and the squares represent thse animals treated with 2 U f regular insulin. The numbers adjacent t the pints represent the sample size. The bars abve and belw the pints represent the standard errrs f the mean. Pints withut bars had standard errrs which were t small t graph. The star adjacent t several pints indicates that that pint is significantly different (p < 0.02 by Bnferrni) when it is cmpared t its hypxic saline cntrl clsed circle).

4 Dwnladed frm by n September 27, 2018 TABLE 1 Lactate-Pyruvate Rati Nrmxia Hypxia Saline 9.0 ±1.0= 30.1±2.6«.b (10) (14) BD = 29.1 ± 1.9».«> (10) (16) Insulin 14.7±1.8 a ' c 19.3±2.3».= (4) (8) In this table the lactate-pyruvate ratis are given fr each pretreatment grup sacrificed in a nrmxic envirnment r a hypxic envirnment. The numbers in parentheses are the sample size fr the grup, a = p < 0.05 when cmpared t saline nrmxic cntrls, b = p < 0.05 when cmpared t the same pretreatment, nrmxia vs hypxia, c = p < 0.05 when cmpared t saline pretreated hypxic animals. Discussin Methdlgical Cnsideratins Althugh many methds have been described fr rapid fixatin f brain metablites we will discuss nly thse reprted fr the unanesthetized muse. Three currently used fixatin methds are freeze blwing, 26 micrwave irradiatin (6 KW), 27 and immersin in liquid N 2. (28_30) With these three methds, values f brain metablites are generally cmparable. It was necessary that we use immersin in liquid N 2 because using either freeze blwing r micrwave irradiatin wuld require changes frm the test cnditins we had used previusly 1 ' 14,15 and hence wuld make the current data nt applicable as an explanatin fr the increased survival time bserved in these studies. Whle brain lactate measured in ur study is within the nrmal range reprted fr nrmxic mice using rr MALATE HFUMARATE BD (10) BD (10) NORMOXIA S (14) BD (16) HYPOXIA FIGURE 2. This figure represents average brain malate (pen bars) andfumarate (crss-hatched bars) cntents expressed as percent f saline-pretreated nrmxic cntrls. The number in parentheses is the sample size fr each bar. The line which extends abve each bar represents ne standard errr f the mean. The dashed lines represent literature values f malate and fumarate expressed as percent f cntrl fr animals treated with 225 mg/kg sdium phenbarbital Hi IP (line a) r 135 mg/ kg sdium amytal IP (line B) (Gldberg et al, 1966). = p< 0.01 as cmpared t saline pretreated hypxic animals. Bld Substrate and HST Values 30 Minutes after Pre TABLE 2 treatment Plasma HST glucse Plasma BHB IV IP (sec) (mg/dl) (mm) saline saline 118.3± ± (94) (14) (13) saline BHB >> 186.3±11.9 a b (17) (13) (9) dextrse saline ± " ±0.020" (9) (15) (15) dextrse BHB 188.7± 10.6" ±0.41<> (22) (14) (9) The first tw clumns indicate whether the animals received either saline r dextrse IV and whether they received saline r BHB IP. The ther clumns indicate the average HST, plasma glucse level, and plasma BHB level fr each f these grups when animals were sacrificed 30 minutes after their pretreatment. The numbers in parentheses are the sample size fr the grup, a = p = 0.05,b = p<0.05,c = p = 0.08 when the values are cmpared t saline, saline cntrls. liquid N 2 fixatin. 28 ' 29 Unaware f the lcal r glbal site f actin f BD in the brain, we included bth freand hindbrain fr tissue analysis. Duffy et al 30 have shwn marked reginal differences in brain lactate levels ranging frm 1.32 mmle/kg wet weight in the parietal crtex t 3.10 mmle/kg wet weight in the medulla f a muse. Therefre, cmpared t the literature values sme f the abslute differences in metablite levels presented in the current study may be attributed t the fact that thers restrict their tissue samples t brain frm the anterir cranial fssa. Since the cmparisns were made between cntrl and treated mice, that had been handled identically, such differences in abslute values shuld have little effect n the interpretatin f the data. BD was chsen fr the brain metablite experiments because we had previusly shwn 1 that 1.4 mmles BD given 30 minutes prir t hypxia increased bld BHB cncentratin 364% and HST 560%. BD is a dimer f ethanl which is cnverted t BHB by alchl dehydrgenase and aldehyde dehydrgenase, 31 bth f which are present in the liver and t a lesser extent in the brain. 32 Thus BD is reliable and effective fr elevating bld ketnes and cnsistently increasing HST. Because BD has ther systemic effects which may alter HST 1 direct administratin f BHB was chsen t fcus in n the substrate altering effects related t the increased HST. BHB rather than acetacetate was chsen fr measurement since preliminary studies indicated that the cntributin f acetacetate t the ttal ketne pl was small. BHB and acetacetate are in equilibrium and therefre an elevatin f ne f these substrates wuld tend t implicate an elevatin in the ther. Evaluatin f the Hyptheses We have prpsed tw pssible hyptheses t explain the increased tlerance t hypxia seen with the elevatin f bld ketnes by BD. The first is a decrease in lactate accumulatin, and the secnd invlves the maintenance f the cerebral energy state

5 BUTANEDIOL REDUCES BRAIN LACT ATE/Kirsch and D'Alecy 975 either directly r indirectly by mdificatin f substrate availability. Althugh neither f these explanatins exclude ne anther, r ther ttally different hyptheses, we will try t fcus n each thery as it is supprted r refuted by the current data. If it were assumed that death frm hypxia is linked t the accumulatin f lactic acid then ne wuld predict that BD-pretreated animals, with their augmented hypxic tlerance, wuld have less brain lactic acid accumulatin than cntrl animals. Cnversely in an animal with reduced hypxic tlerance an increase in lactic acid accumulatin wuld be predicted if tissue lactate were the sle determinant f hypxic tlerance. Mice pretreated with insulin have been shwn t have a decreased tlerance in hypxia. 8,14 This reduced hypxic tlerance culd f curse be explained by a clse assciatin between substrate availability and hypxic tlerance in which case either a reductin r n change in brain lactate wuld be predicted. On the ther hand, if insulin pretreatment prved t exacerbate lactate accumulatin this wuld supprt the hypthesis that lactate was the key cyttxic factr in this animal mdel. Recently, Rehncrna 11 has reaffirmed lactate as an imprtant factr which cntributes t the neurlysis assciated with ischemic hypxia. Furthermre, Barlet 28 has shwn that animals treated with 6-aminnictinamide, which is knwn t increase hypxic survival time, have substantially less lactate accumulatin during hypxia than saline cntrls. Our data are cnsistent with that f Rehncrna and Barlet in implicating tissue lactate as an imprtant factr in hypxic damage. In light f the insulin data, hwever, it is unlikely that lactate is the sle determinant f neurnal failure. We shwed that insulin pretreatment decreased HST 14 and in the current study the same insulin pretreatment was assciated with a reductin rather than an increase in brain tissue lactate as wuld be predicted if lactate was the sle determinant f neurnal damage. The decrease in brain lactate bserved in animals pretreated with BD culd be a direct r indirect effect. It is pssible fr example, that BD is acting directly t inhibit glyclysis r indirectly thrugh its ketne metablite, BHB, t inhibit glyclysis. Ketne metablism has been shwn t elicit ptent inhibitin f glyclysis via negative mdulatin f phsphfructkinase and hexkinase prbably because f elevated brain levels f citrate and ATP. 33 It is well dcumented in the literature 34 and cnfirmed here that the brain level f lactate increases during hypxia. The increase in the lactate-pyruvate rati during hypxia bserved in BD and saline pretreated animals indicates that the brain metablites are in a mre reduced state than during nrmxia. Since, hwever, pretreatment with BD did nt alter the lactate-pyruvate rati frm cntrl values during either nrmxia r hypxia it may be cncluded that the increase in HST assciated with BD is prbably nt due t a change in the brain's xidative state. These are static measurements and thus d nt exclude an effect due t dynamic changes which culd nt be detected with these methds. Regardless f hw Dwnladed frm by n September 27, 2018 BD reduces brain lactate, we are led t cnclude that a reductin in brain lactate cntributes t the prtective effects f BD. Barbiturates as well as induced ketsis affrd increased tlerance t hypxia and ischemia. The exact mechanism f barbiturate prtectin has yet t be identified, but barbiturates are knwn t alter brain metablism. 13 Specifically, phenbarbital and amytal cause a decrease r n change in brain citrate and iscitrate and a decrease in brain malate and fumarate. 35,36 A decrease in lactate, pyruvate and the lactate-pyruvate rati has als been reprted with general anesthetics. 37 Thus ne is led t examine patterns f change in brain tissue metablites that culd suggest a pssible mde f actin fr ptentially therapeutic agents like barbiturates and BD. Nevertheless, despite marked changes in cerebral metablites, it can nly be inferred that such changes have any bearing n hypxic tlerance. In ur study, pretreatment f nrmxic mice with BD prduced n statistically significant change in the brain levels f malate, fumarate, lactate r the lactate-pyruvate rati. Furthermre, Veech et al 26 shwed that rats given BD have increases in brain citrate and iscitrate rather than a decrease as anticipated with a general anesthetic effect. Steen and Michenfelder 13 have shwn that barbiturates' prtective effect is dependent n the anesthetic effect and distinct frm the anticnvulsant effect. They have als shwn that even slight mdificatins in barbiturate structure may greatly reduce its prtective effect. Neither BD r BHB have structural characteristics resembling thse f any f the barbiturates which have been shwn t increase HST. Mice treated with BD parenterally were nt anesthetized althugh they at times appeared intxicated by the alchl. Furthermre BD taken rally fr 12 days prtected frm hypxia withut any clinical signs f intxicatin r anesthesia. 1 Since the exact mechanisms by which barbiturates and BD affrd prtectin frm hypxia have nt been identified, ne can nt exclude the pssibility that they are the same. The clinical picture, the mlecular structure and the changes in tissue metablites all suggest a distinct mechanism f actin fr BD and barbiturates despite the fact that bth ffer prtectin frm hypxia. The data d nt exclude the pssibility that sme alchllike effect 38,39 cntributes t the verall prtectin affrded by BD. We, hwever, believe that the alchllike effects will prve t be additive rather than alternative t the dramatic changes assciated with altered substrate availability. The secnd hypthesis attributes the enhanced tlerance t hypxia bserved with BD pretreatment t an increased and altered substrate availability. The reduced tlerance fr hypxia bserved with the insulininduced hypglycemia may be explained by reduced substrate availability. Elevatin f bld ketnes whether by BD pretreatment r by BHB lading in the presence f hyperglycemia is assciated with increased hypxic tlerance. We have cncluded that the prtectin frm hypxia assciated with BD pretreatment may be attributed in part t a specific reductin in

6 Dwnladed frm by n September 27, 2018 brain lactate cntent. The decrease in brain lactate with a decrease in hypxic survival time in respnse t insulin, hwever, casts sme dubt n lactate accumulatin as the nly factr respnsible fr neurnal failure during hypxia in this mdel. This disassciatin between tissue lactate and HST des nt eliminate the pssibility that a reductin in brain lactate is the sle mechanism fr the prtectin frm hypxia seen with BD. The data, hwever, implicates sme additinal mechanism, pssibly elevated substrate availability, that allws fr maintenance f spntaneus ventilatin during hypxia and thus cntributes t the prtective effect assciated with BD administratin. Acknwledgments Dr. M. Shlafer's editrial and scientific assistance are greatly appreciated. Metablic substrate analyses were made pssible by assistance frm the Bichemistry Cre Labratry f the Michigan Diabetes Research and Training Center (USPHS AM 20572). The xygen analyzer and the temperature cntrl chamber were made available by Dr. M. J. Kluger. The secretarial assistance f Ms. Catherine Crsn and Ms. Kathy Yrk is appreciated. References 1. Kirsch JR, D'Alecy LG, Mngr PB: Butanedil induced ketsis increases tlerance t hypxia in the muse. Strke 11: , Myles WS: Survival f fasted rats expsed t altitude. Can J Pharmac 154: , Owen OE, Mrgan AP, Kemp HG, Sullivan JM, Herrera MG, Cahill GF: Brain metablism during fasting. J Clin Invest 46: , Ruderman NB, Rss PS, Berger M, Gdman MN: Regulatin f glucse and ketne-bdy metablism in brain f anaesthetized rats. Bichem J 138: 1-10, Perssn B, Settergsen G. Dahlquist G: Cerebral arteri-venus difference f acetacetate and D-/3-hydrxybutyrate in children. Acta Paediat Scand 61: , Hawkins RA, Williamsn DH, Krebs HA: Ketne-bdy utilizatin by adult and suckling rat brain in viv. Bichem J 122: 13-18, Avery RC, Jhlin JM: Relative susceptibility f adult and yung mice t asphyxiatin. Prc Sc Exp Bil Med 29: , Brittn SW, Kline RF: Age, sex, carbhydrate, adrenal crtex and ther factrs in anxia. Am J Physil 145: , Salfrd LG, Siesj BK: The influence f arterial hypxia and unilateral cartid artery cclusin upn reginal bld flw and metablism in the rat brain. Acta Physil Scand 92: , Friede RL, Van Huten WH: Relatins between pst mrtem alteratins and glyclytic metablism in the brain. Exp Neurl 4: , Rehncrna S, Rsen I, Siesj BK: Brain lactic acidsis and ischemic cell damage. I. Bichemistry and neurphysilgy. J CBF and Metab 1: , Wilheljelm BH, Arnfred I: Prtective actin f sme anesthetics against anxia. Acta Pharmacl Txicl 27: 93-98, Steen PA, Michenfelder JD: Cerebral prtectin with barbiturates relatin t anesthetic effect. Strke 9: Kirsch JR, D'Alecy LG: Effect f altered availability f energyyielding substrates upn survival frm hypxia in mice. Strke 10: , EigerSM, Kirsch JR, D'Alecy LG: Hypxic tlerance enhanced by /8-hydrxybutyrate-glucagn in the muse. Strke 11: , Artru AA, Michenfelder JD: A re-examinatin f physstigmineinduced cerebral prtectin in the hypxic muse. A critical assessment f the mdel. Strke 11: , Stupfel M, Vallern A, Demeestere M, Masse H: Hypxic survival variatins in male and female mice as functins f chrnlgical and envirnmental factrs. Aviat Space Envirn Med 49: , Gutmann I, Wahlefeld AW: L-( + )-lactate determinatin with lactate dehydrgenase and NAD. In: Methds f Enzymatic Analysis (Bergmeyer HV, ed). New Yrk, Academic Press, , Passneau JV, Lwry OH: Pyruvate flurimetric assay. In: Methds f Enzymatic Analysis (Bergmeyer HV, ed). New Yrk, Academic Press, , Gldberg ND, Passneau JV: L-malate and fumarate flurimetric determinatin. In: Methds f Enzymatic Analysis (Bergmeyer HV, ed). New Yrk, Academic Press, , Herin RA, Hall P, Fitch JW: Nitrgen Inhalatin as a Methd f Euthanasia in Dgs. JAVMA 39: , Lundy EF, Luyckx BA, Zelenck GB, D'Alecy LG: Butanedil induced cerebral prtectin in an imprved Levine rat. Submitted fr publicatin 23. Bergmeyer HU, Bemt D, Schmidt F, Stark H: D-Glucse. In: Methds f Enzymatic Analysis (Bergmeyer HU, ed) New Yrk, Academic Press, , Williamsn DH, Mellanby J: D-(-)-3-Hydrxybutyrate. In: Methds f Enzymatic Analysis (Bergmeyer HU, ed) New Yrk, Academic Press, , Kaliss N, Pressman D: Plasma and bld vlumes f muse rgans, as determined with radiactive idprteins. Prc Sc Expt Bil Med 75: 16-20, Veech RL, Harris RL, Vels D, Veech EH: Freeze-blwing: a new technique fr study f brain in viv. J Neurchem 20: , Medina MA, Jnes DJ, Stavianha WB, Rss DH: The levels f labile intermediary metablites in muse brain fllwing rapid tissue fixatin with micrwave irradiatin. J Neurchem 24: , Berlet HH: Cerebral metablic rates as determinants f hypxic survival f adult mice. In: Advances in Neursurgery 3 (Penhlz H, Brck M, Hamer J, Klinger M, Sperri O, eds), Berlin, Springer Verlag, 59-67, Pnten U, Ratchesn RA, Siesj BK: Metablic changes in the brains f mice frzen in liquid nitrgen. J Neurchem 21: , Duffy TE, Nelsn SR, Lwry OH: Cerebral carbhydrate metablism during acute hypxia and recvery. J Neurchem 19: , Tate RL, Mehlman MA, Tbin RB: Metablic fate f 1,3-butanedil in the rat: cnversin t beta-hydrxybutyrate. J Nutr 101: , Raskin NH, Sklff L: Enzymes catalysing ethanl metablism in neural and smatic tissues f the rat. J Neurchem 19: , DeViv DC, Leckie MP, Ferrendelli JS, McDugall DB, Jr: Chrnic ketsis and cerebral metablism. Ann Neurl 3: , Siesj BK, Nilssn L: The influence f arterial hypxemia upn labile phsphates and upn extracellular and intracellular lactate and pyruvate cncentratins in the rat brain, Scand J Clin Lab Invest 27: 83-96, Gldberg ND, Passnneau JV, Lwry OH: Effects f changes in brain metablism n the levels f citric acid cycle intermediates. J Bil Chem 241: , Chapman AG, Nrdstrm CH, Siesj BK: Influence f phenbarbital anaesthesia n carbhydrate and amin acid metablism in rat brain. Anesthesilgy 48: , Nilssn L, Siesj BK: The effect f anesthetics upn labile phsphates and upn extra- and intracellular lactate, pyruvate and bicarbnate cncentratins in the rat brain. Acta Physil Scand 80: , Emersn GA, Van Liere EJ, Mrrisn JL: Drug prphylaxis against lethal effects f severe anxia. II. Alchl amytal and pentbarbital. Prc Sc Bil Med 49: , Mursi MM, Luyckx BA, D'Alecy LG: The rle f ethanl in diluents f drugs that prtect the hypxic brain. Fed Prc 41: 1619, 1982