Br. J. Pharmacol. (1989), 96, 9-16

Anaesthetic potencies of primary alkanols:implications for the molecular dimensions of theanaesthetic siteJames K. Alifimoff, Leonard L. Firestone & 'Keith W. Miller

Departments of Anaesthesia and Pharmacology, Harvard Medical School, Massachusetts General Hospital,Boston, MA 02114, U.S.A.

1 We have redetermined the anaesthetic potencies (EC50 s) for a series of primary alkanols, toresolve uncertainties about the molecular dimensions of the anaesthetic site resulting from the use

of data from different laboratories.2 For each alkanol, concentration-response relationships for loss of righting reflex (LRR) were

plotted for over one hundred tadpoles, and the median effective concentrations determined.Aqueous concentrations present during potency assays were determined independently, and foralkanols with chain length greater than nonanol, correction was made for depletion from theaqueous phase.3 The EC50 s were found to decrease logarithmically with increasing number of carbon atoms inthe hydrocarbon chain of the alkanol (CN), such that, on average, each additional methylene groupwas associated with an approximately four fold increase in potency.4 The relationship between log EC50 and CN was best described by the quadratic equation, logEC o = 0.022 (+0.0038) CN2 + 0.76 (±0.051) CN + 3.7 (±0.14) (r2 = 0.9951).5 A previously described correlation between the apparent changes in the free energy of binding ofan additional methylene group both to luciferase and to the sites for LRR in tadpoles was notconfirmed.6 A cut-off in potency beyond dodecanol was established in experiments where tadpoles were

maintained in supersaturated solutions of tridecanol for 20 h without demonstrable LRR.7 These findings indicate that the soluble enzyme firefly luciferase does not adequately model theanaesthetic site. Specifically, there are discrepancies in the position of cut-off, and the apparentchanges in the free energy of binding, per methylene group, of an alkanol to luciferase do notparallel that for tadpoles.

Introduction

It is still not known whether general anaestheticsproduce their effects by acting on central nervoussystem neurotransmitter receptor proteins directly,or indirectly through the lipids which surround thesereceptors. One approach to resolve these alternativesis to apply pharmacological criteria derived fromobservations of anaesthesia in animals to well-characterized model systems. For example, a particu-larly useful criterion is the 'cut-off' in anaestheticpotency, that is, the aburpt loss of potency amongthe higher molecular weight members of homologousfamilies of anaesthetics. The primary alkanols areone such series that can be used to probe structure-

1 Author for correspondence.

activity relationships in these model systems. Inanimals, cut-off was thought to occur beyond tri-decanol at tetradecanol (Meyer & Hemmi, 1935;Pringle et al., 1981), but it remains to be establishedwhether cut-off occurs due to depletion of theaqueous alkanol concentration or loss of intrinsicanaesthetic activity (Lee, 1976).

Proponents of the protein-based theories haveused data obtained with the lipid-free enzyme, fireflyluciferase, to argue that anaesthetics act directly atcentral nervous system receptors. Specifically, luci-ferase not only manifests a cut-off in inhibitorypotency for the primary alkanols and alkanes, butalso demonstrates changes in the free energy forbinding of alkanols which parallel those found in

© The Macmillan Press Ltd 1989

10 J.K. ALIFIMOFF et al.

animals (Franks & Lieb, 1985). However, the validityof this latter point has recently been questioned(Elliott & Haydon, 1986) because the potencies ofthe primary alkanols in vivo are not known with ahigh degree of precision. Problems with these valuesin the literature are that: (i) data have never beenderived in a single laboratory for the completehomologous series of n-alkanols (Vernon, 1913;Meyer & Hemmi, 1935; Pringle et al., 1981); (ii) thepotencies were roughly estimated by a 'bracketing'method rather than quantal concentration-responseanalysis, so that no standard errors could bederived; and (iii) measurements of the actual freeconcentration of alkanol in solution are lacking. Thisis especially important for the higher molecularweight compounds where adsorption might lead tosignificant depletion of the free concentration.Although such values in the literature may be usefulfor a first approximation, they are inadequate todefine the fine structure of their molecular targets.Therefore, we have systematically determined theloss of righting reflex (LRR) potencies in tadpoles forthe complete homologous series of primary aliphaticalkanols. With these values, we show that in vivopotency data from tadpoles do not parallel the inhi-bition of firefly luciferase by the primary alkanols,and we firmly establish the position of the cut-off inanaesthetic potency for the series of primary alka-nols.

Methods

Early pre-limb bud tadpoles, Rana pipiens, approx-imately 1.0-1.5cm in length (Carolina BiologicalSupply Co., Burlington, NC), were used for determi-nation of anaesthetic concentration-response curvesas previously described (Alifimoff et al., 1987).Briefly, groups of five tadpoles were placed incovered 100 ml beakers in neutral, oxygenated,aqueous solutions of alkanols prepared in twicedistilled water. No tadpole was used more than once.Anaesthesia was defined as the loss of righting reflex(LRR). After allowing 15 to 240 min for equilibrationwith the alkanol, the tadpoles were tipped manuallywith a flame polished glass pipette. Unrespon-siveness for greater than 5 s was scored as LRR, andthis test was repeated at 15min intervals. After eachexperiment, all tadpoles were placed in containers ofneutral distilled water until recovery of rightingreflex was confirmed.

['4C]-tetradecanol was assayed for its ability toinduce LRR in three ways. First, two groups of fivetadpoles each were incubated for 20h in separatebeakers containing 100ml of oxygenated, saturatedsolutions, and then assayed for LRR. Samples ofthese solutions were obtained for liquid scintillation

counting at 30 min intervals for the first 4 h, and thenagain at 20 h. Second, ten tadpoles were incubated ina beaker containing 500 ml of an oxygenated, super-saturated solution for 72 h; both LRR and aqueousconcentration were assayed at 24 h intervals. Thirdly,to determine whether tetradecanol shifted theconcentration-response curve of another anaesthetic,tadpoles were preincubated in an oxygenated, super-saturated solution of tetradecanol for 48 h (50ml ofsolution per animal), then assayed for their responsesto ["4C]-octanol. Tridecanol was also assayed for itsability to induce LRR. Two groups of ten animalswere incubated in 1000 ml of an oxygenated, satur-ated solution of tridecanol and in 1000 ml of a super-saturated solution prepared from an ethanolic stocksolution. Tadpoles were assayed for LRR at hourlyintervals for 6 h and then again at 20 h. Samples wereobtained for determination of free aqueous concen-trations by gas chromatography at the start of theexperiment and at 20 h.

Concentrations of alkanols present during assayswere verified either by gas chromatography or liquidscintillation spectroscopy. Concentrations of theshort chain alkanols (C1-C9; CN = number ofcarbon atoms in the hydrocarbon chain of thealkanol) were measured with a Beckman GC 72BGas Chromatograph (Porapak P column packing(Waters Co., Milford, MA) with column tem-peratures ranging from 130 to 215°C), while concen-trations of undecanol and tridecanol were measuredon a Varian 3700 Gas Chromatograph (Dexsil-300column packing (Supleco Inc., Bellfonte, PA) withcolumn temperatures from 140 to 200°C). Actualconcentrations of the short chain alkanols werewithin 5% of their nominal values during the timecourse of assays. For the long-chain alkanols(C1O-C14), the relationship between nominal andmeasured concentrations during concentration-response assays was more complex, and is describedin the Results section.For all agents, concentration-response assays were

performed using a minimum of 10 animals at each of5 different concentrations. Entire studies wererepeated at least once, and the data pooled. Therespective effective concentrations (EC5o) and slopeswere obtained using a logistic method for quantalresponses (Waud, 1972).With the more potent agents, it was convenient to

make aqueous solutions of alkanols from ethanolicstock solutions. To determine whether this made anydifference in potency, the EC50 of [14C]-octanol wasdetermined when alkanol solutions were prepared (i)by dilution from ethanolic stock solutions, (ii)directly in water or (iii) from a ["4C]-alkanol directlyin water. EC50 s obtained for octanol by eachmethod were compared. The estimated variance inthe respective EC50 values was calculated from the

MOLECULAR DIMENSIONS OF THE ANAESTHETIC SITE

Table 1 Time-course of ["C]-tetradecanol deple-tion during a concentration-response assay

Time (h) [ 4C]-tetradecanol (pmol I - 1 water) % LRR

0244872

48.5*43.122.212.7

See Methods for details. LRR = loss of rightingreflex.* Saturated solubility = 1.5 pM (Bell, 1973).

standard errors; the sum of these then yielded theestimated variance of the difference in the EC5obetween the octanol prepared from an ethanolicstock solution, and radiolabelled or unlabelledoctanol prepared directly in water. The ratio of thesedifferences to their respective standard errors wasthen referred to a standard normal distribution. Sta-tistical significance was assumed at the level ofP < 0.05.

Drugs and solutions

All alkanols were the highest quality available fromtheir respective manufacturers. Aqueous solutions ofmethanol (Mallinckrodt, Paris, KY; purity 99.8%),ethanol (Pharmco, Dayton, NJ; >99%), propanol

(Mallinckrodt, St. Louis, MO; 99.8%), butanol(Baker, Phillipsburg, NJ; 99.7%), pentanol (Aldrich,Milwaukee, WI; 99%), and hexanol (Sigma, St.Louis, MO; 99%) were prepared in gas-tight vials bystirring weighed aliquots of each compound intotwice distilled water for 1 h at room temperature.Solutions of heptanol (Eastman Kodak, Rochester,NY; 99%) and octanol (NuChek Prep, Elysian, MN;>99%) were prepared by stirring for longer (> 12 h)in gas-tight vials at 40'C. Solutions of undecanoland tridecanol (NuChek Prep; >99%) were pre-pared from ethanolic stock solutions. Gas chroma-tography revealed no impurities in saturated stocksolutions of these compounds.

[14C]-octanol (4.5 Ci mol '), [14C]-decanol(7.5 Ci mol- 1), ["4C]-dodecanol (56 Ci mol 1) (ICNBiomedicals, Irvine, CA) and ['4C]-tetradecanol(58 Ci mol 1) (NEN, Boston, MA) were used todetermine free aqueous concentrations of the longerchain alkanols during concentration-response assays.Radiochemical purities were >98.5% as determinedby thin layer chromatography, high performanceliquid chromatography and/or autoradiography bythe manufacturer. Aqueous solutions of each radiola-belled alkanol were prepared by diluting concen-trated stock solutions made in ethanol; final ethanolconcentrations were always <25 mm. Samples werecounted in Liquiscint (National Diagnostics, Man-ville, NJ), and efficiency was determined using

Table 2 Loss of righting reflex potency of primary alkanols in Rana pipiens tadpoles at 20+ 1°C

Alcohol

MethanolEthanolPropanolButanolPentanolHexanolHeptanolOctanolOctanol'OctanolbNonanolDecanola-bUndecanol'Dodecanola.bTridecanol'TetradecanolaRange:

EC50 + s.e. Slope ± s.e. n/T

590 41 mM190 + 16mM73 ± 2.4mM

10.8 + 0.77 mM2.9 +0.11 mM570 ± 37pM230 + 11,uM57 + 2.5 sM55 + 3.1 liM59 ± 3.1pM37 ± 2.4pm

12.6 ± 0.48 pM8.1 +0.81.uM4.7 + 0.33 pM

Not anaestheticNot anaesthetic

126,383 x

2.7 ± 0.442.3 + 0.317 ± 1.1

2.2 + 0.345.0± 1.23.9 ± 0.726+ 1.15 ± 1.0

3.6 ± 0.505 ± 1.1

2.3 ± 0.405.4 ± 0.812.5 ± 0.493.1 ± 0.47

3/1704/2703/1703/2303/1502/1202/1403/1504/2203/1704/2503/1702/1103/160

EC50 was determined after 15-30min for methanol through to nonanol; after 60min for decanol and undecanol,and after 120min for dodecanol. See Methods for curve-fitting procedure and estimation of standard errors. Allconcentrations were verified by gas chromatography unless otherwise noted.n = number of experiments, T = total number of animals in n experiments.a Aqueous alkanol solutions were prepared from concentrated ethanolic stock solutions; the final concentration ofethanol was < 25 mm. See Methods for details.b A ["4C]-alkanol was used to measure the aqueous concentrations.

11

12 J.K. ALIFIMOFF et al.

quenched samples of ['4C]-toluene as an externalstandard.

Results

The short-chain alkanols (Cl-C9) produced areversible LRR in tadpoles which plateaued in10min and remained unchanged when assayed at 15and 30min. For decanol and undecanol, a steadyLRR response was not observed until 60min; fordodecanol, 120min was required. Accordingly, theEC50 values presented for decanol and undecanolwere obtained at 60min, while the EC50 value fordodecanol was obtained at 120min. In contrast, tri-decanol did not produce LRR during 20h of expo-sure to both saturated and supersaturated solutions.Tetradecanol, on the other hand, did not produceLRR in tadpoles within 72h even when concentra-tions in excess of saturation were present (Table 1).At intervals greater than 72 h, tetradecanol was irre-versibly toxic. However, preincubation of tadpoles intetradecanol for 48 h, did not shift their response tooctanol (EC50: 52 + 5.2 with preincubation vs59 + 3.1 ym without preincubation; P > 0.19).For octanol, results monitored by gas chromatog-

raphy were in close agreement with those obtainedusing ["4C]-octanol (Table 2). However, the freeaqueous concentrations of [14C]-dodecanoldecreased by about 15% during 2h of incubationwith tadpoles (Figure 1). Depletion of ["4C]-dodeca-nol from aqueous solution continued between 2 and4h, but resulted in little further change in either theaqueous concentration or in EC50 values (4.7 + 0.33

(s.e.) vs 4.6 + 0.73ti1M, determined at 2 and 4h,respectively, P > 0.80). In comparison, free aqueousconcentrations of ['4C]-tetradecanol decreased byapproximately 18% and 30% at 2 and 4h, respec-tively. Depletion of free aqueous concentrations con-tinued so that there was approximately 13, 50, and68% depletion of decanol, dodecanol and tetra-decanol, respectively, at 20 h.

Final concentrations of ethanol present duringconcentration-response assays for the long-chainalkanols (C10-C14) were always <25mM; however,even as much as 40mm ethanol does not produceLRR in tadpoles. Moreover, when results fromassays performed in the presence and absence of25 mm ethanol were compared (Table 2), it was clearthat small concentrations of ethanol did not shift theconcentration-response curves of other alkanols.There was no significant difference in either the EC50or slope of concentration-response curves for octanolin the presence or absence of 25mm ethanol (EC50:55 + 3.1 vs 57 + 2.5 gM, respectively, P > 0.61;slope: 3.6 + 0.50 vs 5 + 1.0 respectively, P > 0.66).Likewise, the EC50 (59 + 3.1 yM, P > 0.62) and slope(5 + 1.1, P > 0.16) for [14C]-octanol were not differ-ent (Table 2).The EC50 s for the series of primary alkanols, stan-

dard errors, and slopes are presented in Table 2. TheECj0 s decreased in the series from methanolthrough to dodecanol, such that dodecanol wassome 126,000 times more potent than methanol. Thelog concentration-response curves were sigmoidalwith slopes ranging from 2.3 + 0.40 to 7 + 1.1(Figure 2), but no systematic trends (for example, asa function of CN) were observed. Similar degrees of

100

90

80

70

60

C0Dpp0p

50 [40 [30

0--

-1 1 2 3 4

Time (h)5 " 20

Figure 1 Time-course for depletion of long-chain alka-nols from aqueous solution; decanol (El), dodecanol(C), and tetradecanol (A). Each point represents themean of five separate determinations during a singleexperiment under the conditions of the concentration-response assay (see Methods for details). In all cases, thestandard deviations were <10% of the mean valuespresented.

1.0 - 4^^r °°00 03

Ct0.8-~ ~ 0

-0660.4 0q0 o o

+1

02 0

0 03 *

-30 -20 -1.0 0 10 2.0 3.0

log [Alkanol] (mM)Figwe 2 Log concentration-response relationships forthe primary alkanols with an even number of carbonatoms. Dodecanol is the left-most, while ethanol is thefarthest to the right. Curves were fitted according to themethod of Waud (1972). Each symbol represents themean response of ten animals, scored quantally asdescribed in Methods. In some instances, symbolsoverlap. The total number of animals tested appears inTable 2. LRR = loss of righting reflex.

c0

Q)a)

MOLECULAR DIMENSIONS OF THE ANAESTHETIC SITE

variation in the slopes of concentration-responsecurves for tadpoles have been previously found inour laboratory (Dodson et al., 1985; Alifimoff et al.,1987).The coefficients of variation in EC50 values

(s.e./EC50) x 100%) ranged from 3.3 to 9.9%, whichare comparable to those previously obtained by us(Pringle et al., 1981; Dodson et al., 1985; Alifimoff etal., 1987) and by others measuring either rightingresponse or minimum alveolar concentration (MAC)in mice (Deady et al., 1980), or MAC in man(Saidman et al., 1967). A similar degree of variationwas found between experiments. For example, themean of five individual ethanol concentration-response experiments which were fitted independent-ly was 190 + 22mM (s.e.).The relationship between EC50 and the alkanol

carbon chain length (CN) was logarithmic (Figure3a), and a least-squares line fitted to log EC50 versusCN had a slope of -0.49 + 0.024 (s.e.) (r2 = 0.9764).The deviations of the observed values for EC50 fromthe fitted line were assessed by calculating theresidual of each point (Figure 3b). This suggestedthat the data might be better fitted by a quadraticrather than a linear equation. Fitting the data to aquadratic equation (log EC5o = 0.022 (±0.0038) CN2+ 0.76 (±0.051) CN + 3.7 (±0.14) (r2 = 0.9951))resulted in a significantly better fit (P < 0.002; Ftest).

Discussion

This study was undertaken to resolve deficiencies inthe anaesthetic potency values for the primaryalkanol series available from the literature. Theproblems mentioned in the Introduction have beenaddressed in this study as follows: (i) the EC50 s pre-sented in Table 2 represent the only complete seriesof anaesthetic potencies for primary alkanols from asingle laboratory; (ii) the potencies were derived byanalysis of quantal concentration-response curves sothat estimates of standard errors around both EC50 sand slopes could be made; and (iii) free aqueous con-centrations of all alkanols were monitored either bygas chromatography or liquid scintillation spectros-copy. Finally, monitoring the free concentrationsallowed the exact position of the cut-off in anaes-thetic potency of primary alkanols to be established.Any model of the anaesthetic site should share the

pharmacological properties of general anaesthesiaitself, including pressure reversal (Lever et al., 1971);lack of stereoselectivity between enantiomeric pairsof secondary alkanols (Alifimoff et al., 1987); andcut-off (Janoff & Miller, 1982). Anaesthetic cut-offhas been observed in several series of homologous

2

iE0

LOLU

0

0

-2

2 4 6 8 10 12

Alkanol carbon chain length

)

050 e

(n

0

-0.50

0

0 00

03

[ ~~~~~~00

F 0 00

F 2 4 6 8 10L LI0 2 4 6 8 10 12

Alkanol carbon chain lengthFigure 3 (a) Relationship between log EC5o and thenumber of carbons in the hydrocarbon chain of theprimary alkanols (CN). Error bars are smaller than thesymbols. (b) Residual analysis of (a). The residuals (yaxis) are the difference between the observed value oflog EC50 and the calculated value from a linear fit ofthe data in (a).

compounds, including the primary alkanols, alkanes,and fluorocarbons (for a review see Miller, 1987).While luciferase has also been shown to manifest acut-off in inhibitory potency for the primary alka-nols, this occurs at a higher molecular weightmember of the series (beyond hexadecanol) than inanimals (beyond dodecanol). One conceivable expla-nation for this discrepancy involves the depletion ofanaesthetic from the aqueous phase, wherein thecut-off in vivo might actually occur at a longer chainlength than previously observed. Our data, however,clearly establish that cut-off in vivo is not an artifactof depletion and that it occurs at tridecanol. Thiswas demonstrated by the inability of both tridecanoland tetradecanol to produce LRR. Tridecanol did

13

L

14 J.K. ALIFIMOFF et al.

Table 3 Comparison of anaesthetic potencies of the primary alkanols obtained by different investigators

Vernon (1913)CN (mM)

Meyer & Hemmi (1935)(mM)

Pringle et al. (1981)(mM)

1 9902 410 3303 104 1104 22.3 305 7.06 0.97 0.35 0.388 0.127 0.139 - 0.02510 0.01011 0.00512 0.007513

Dashed lines indicate that values were not determined.

not induce LRR after exposure of tadpoles for 20 hto either saturated or supersaturated aqueous solu-tions of tridecanol. Even after 20 h of exposure tosupersaturated solutions, aqueous concentrationsstill remained above saturated solubility (Bell, 1973).Likewise, tetradecanol did not induce LRR despite(i) long-term exposure of tadpoles to solutions con-

taining in excess of 0.9 ym, and (ii) exposure for 72 hto supersaturated solutions of ["4C]-tetradecanol(Table 1). Although 74% of the total amount of[14C]-tetradecanol initially added was depletedduring the 72h, the aqueous phase still containedmore tetradecanol than the published saturated solu-bility of approximately 1.5 um (Bell, 1973).The observed percentage depletion increased with

the alkanol chain length. A priori, this might arisefrom increasing lipophilicity and surface activitycoupled with decreasing aqueous solubility. Simplecalculations show that uptake into the fatty tissuesof the tadpoles would account for much of the deple-tion within the range of error of the published lipid-buffer partition coefficients (Sallee, 1978; Franks &Lieb, 1986).When our results are compared to those pre-

viously obtained by Pringle et al. (1981), the majorityof their values are within the precision of our mea-surements. However, the values for both ethanol andpropanol observed by Pringle et al. (1981) were sig-nificantly lower than in the present study. In addi-tion, they demonstrated that tridecanol was a partialanaestheic, but in the present study, it did not induceLRR even after prolonged exposure to supersatu-rated solutions. Because the purities of the anaes-thetics used in that study as well as their freeaqueous concentrations were not monitored by gaschromatography, we can only explain the discrep-

ancies by possible contamination with potent highermolecular weight anaesthetic alkanols.That the relationship between log EC50 and CN is

best described by a quadratic equation rather than a

straight line can be interpreted to mean that theapproach to cut-off is gradual rather than abrupt,whereas the phenomenon of cut-off itself is evenmore abrupt than previously appreciated, in thatpotency disappears completely after dodecanol, i.e.,there are no partial anaesthetics. This gradualapproach to cut-off implies that the intrinsic anaes-thetic efficacy of dodecanol per methylene group isless than that of the other alkanols. Whether thissomewhat gradual loss of potency per methylenegroup occurs in other homologous series of anaes-thetics which display cut-off remains to be investi-gated.

While the approach to cut-off is gradual for theprimary alkanols in that the higher molecular weightmembers of the series significantly deviate from a

linear relationship, we found no such deviationsfrom linearity in the region of hexanol and heptanol.Indeed a fit of the EC50 s of propanol throughoctanol showed a strong linear correlation(r2 = 0.9912). This is in contrast to the results ofFranks & Lieb (1985) who compared a collection ofdata for tadpole anaesthesia with the inhibitorypotencies of primary alkanols on luciferase (ICio).They observed that the chain-length dependence ofthe IC50 s levelled off in the region of hexanol andheptanol (i.e., these two alkanols were approximatelyequal in potency) and that the change in the freeenergy of binding (AAG0) of alkanols to luciferaseappeared to parallel that found in animals. This was

interpreted to mean that the alkanol binding site onluciferase, and the in vivo anaesthetic site, share

Present study(mM)

1205412

0.7

0.06

0.013

0.00540.037

590190731130.570.230.0570.0370.0130.00810.0047Not

anaesthetic

MOLECULAR DIMENSIONS OF THE ANAESTHETIC SITE 15

similar molecular dimensions. However, recalcula-tion of their AAG' with our self-consistent set ofEC50s shows that the reported correlation betweenanaesthetic activity in tadpoles and luciferase doesnot exist (Figure 4). The disparity between ourEC50 s and those used by Franks & Lieb (1985)(Vernon, 1913; Meyer & Hemmi, 1935) are madeapparent in Table 3. It appears that Vernon's andMeyer's laboratories employed different anaestheticendpoints (suppression of spontaneous movement vssuppression of reflex response, respectively) as wellas different indices of relative potencies (EC1oo vsEC50, respectively). In addition, as pointed out byElliott & Haydon (1986), the value shown for penta-nol (Vernon, 1913) is actually an estimate based onthat of isopentanol. Thus, it would seem that com-bining these two sets of data for the purposes ofmolecular level arguments is inappropriate.

Linearity of log EC50 versus CN for the lowermolecular weight members of the series has beenobserved in mice (LRR for ethanol through hexanol,r = 0.993 (Lyons et al., 1981)) and the freshwater

cna)-0co

_.

0

C)

)-.

la -cm 0

*c Ea -2z-.- ~- 1

I

(u -2

C -3a)

CU0.0._

1/2 3/4 5/6 7/8 9/10 11/12

Number of carbon atoms (CN/CN+ 1

a)

_1 LU

a).._-2 0

-32

_a) OcC';

-6_0Icu

a)

Q)CLa

C)

Figure 4 The apparent changes in the free energy ofbinding (AAG') for a methylene group to luciferase(top) and for loss of righting reflex (LRR) in tadpoles(bottom) as a function of CN/CN 1. Note that the verti-cal axis scale for luciferase is shown on the right whilethat for tadpoles is shown on the left. Points were calcu-lated using the differences in thermodynamic freeenergy,

AAG' = -RT log[EC50(N)/EC5O(N + 1)]

for luciferase inhibition and LRR. The EC50 values forluciferase inhibition are from Franks & Lieb (1985 andpersonal communication). The EC50 values for LRR arefrom Table 2.

shrimp Gammarus (butanol through octanol,r2 = 0.998, Elliott et al., 1987). In the latter study,hexanol and heptanol were not equipotent, as theyare shown to be for luciferase. Thus whatever suchanomalies observed in luciferase may imply aboutthe molecular features of the alkanol site in vitro,inferences regarding the site in vivo must be madewith caution.One previous explanation for cut-off, derived from

lipid based theories of anaesthesia, was based on thereported decrease in the membrane/buffer partitioncoefficient for tetradecanol, such that the maximummembrane concentration obtained was less than thatnecessary for anaesthesia (Pringle et al., 1981).However, recent data obtained in cholesterol-containing bilayers (Franks & Lieb, 1986) revealedthat partition coefficients continue to increasebeyond dodecanol, making it unlikely that cut-off isa consequence of inability to achieve a sufficientmembrane concentration. Therefore, either bio-membranes behave differently in this respect fromlipid bilayers, which seems unlikely, or the alterna-tive explanation, that cut-off may arise from a loss inthe intrinsic pharmacological efficacy must be con-sidered (Pringle et al., 1981; Janoff & Miller, 1982).Indeed, observation of a gradual loss of lipid dis-ordering potency, or even an increase in order, bylong chain alkanols in spin labelled lipid bilayers(Richards et al., 1978) and postsynaptic membranes(L. Firestone, Miller, K.W., unpublishedobservations) support the latter alternative.

In summary, we present LRR potency values forthe complete series of anaesthetic primary alkanols.In contrast to the previously published values, theaqueous concentrations of all the alkanols weremonitored, and the EC50 values were obtained fromquantal concentration-response relationships. TheEC50 s show a relationship between potency andalkanol chain length from methanol through tododecanol which is best described by a quadraticequation, suggesting that the approach to cut-off isgradual. However, after dodecanol potency abruptlycuts off in that there are no partial anaesthetics.Depletion of longer chain alkanols, while significant,is unable to account for the potency cut-off.

This work was supported in part by grants from theNational Institute of General Medical Sciences to theHarvard Anaesthesia Research Center (GM-15904, GM-07592) and the Department of Anaesthesia, MassachusettsGeneral Hospital. J.K.A. was supported in part by an Anes-thesiology Young Investigator Award and is a Foundationfor Anesthesia Education and Research Investigator. L.L.F.was supported by a Starter Grant from the AmericanSociety of Anesthesiologists and a Fellowship from theCharles A. King Trust, Boston, MA. The authors thank DrDavid C. Hoaglin, Department of Statistics, Harvard Uni-versity, for assistance in statistical analysis.

16 J.K. ALIFIMOFF et al.

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ELLIOTT, J.R., HAYDON, D.A. & McELWEE, A.A. (1987). Acomparison of the potencies of n-alkanols and methylcarboxylic esters as general anesthetics in Gammarusand as local anaesthetics in axons of Loligo. J. Physiol.,384, 21P.

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(Received July 14, 1987Revised June 22, 1988

Accepted August 1, 1988)