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Vol. XCII, No. 866 The American Naturalist September-October, 1 958SERUM ELECTROLYTE LEVEI S IN HIBERNATING MAMMALS*

M. L. RIEDESELT AND G. EDGAR FOLK, JR.Department of Physiology, The State Universiry of lowa School of Medicine,Iowa City, IowaINTRODUCTION

Reports on serum electrolyte changes with hibernation have been con-sistent in indicating an elevation of serum magnesium (Suomalainen, 1939;McBirnie, er al., l!Jl; Riedesel, 1957), but inconsistent with respecr roother electrolytes. For example: hypo- and hyper-carcemia have been de-scribed with hibernation in the European marmor (Adler, 1926; Ferdmannand Feiaschmidt, 1932) but no change in the serum calcium with hibernationof the hedgehog (Suomalainen, r)))); increased serum potassium with hiber-nation of the American woodchuck (McBirnie, er al., 1953), but decreasedserum potassium (Suomalainen, t9j3), or no change (Biorch, et al., 19J6)with hibernation of the hedgehog. These contradictions may result fromspecies characteristics or differences in the depth of hibernarion. Ac-cordingly, the presenr study was designed to describe species differencesin serum electrolytes as related to depth or type of hibernation. An earlierreport by the senior author describes elevation of serum magnesium withthe lowering of body temperarure in the little brown bat (Myotis lucifugus)(Riedesel, 1957). This presenrarion records serum calcium and potassiummeasurements on four species of hibernating animals, including two speciesof bats, hamsters, and thirreen-lined ground squirrels.

PROCEDURE AND TECHNIQUESsirnuitaneous serum calcium, potassium, specific gravity and blood hema-tocrit determinations were made on animals bled by decapitation. In thecase of the bats, serum electrolyte and specific gravity determinationswere made on samples obtained by pooling the blood of five animals. po-'tassium determinations were made by flame photometry. The spectrophoto-metric method of Natelson and Penniall (Igjj) was employed for the analy-sis of total serum calcium. Hematocrit values on the small bats were de-termined by the use of Van Allen hematocrit rubes. The Vinuobe methodwas employed for hematocrit determinations oo the other animals. In eithercase the blood was centrifuged at 2800 r.p.m. for 45 minutes. The serumspecific gravities were determined by the method of Van Slyke, et al. (1950).Esophageal temperat'res of the small bats were measured on a Rubicon

Potentiometer with a copper-coflsranran rhermocouple just before they weresacrificed. vhen initial srages of hibernation were studied, esophagealrThis research was supported in part by a grant from the National science Foun-dation, Washington, D. C.tPtesent address: Department of occupational Health, Graduate School of publicHealth, University of Pittsburgh.

307

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308 THE AMERICAN NATURALISTtemperatures were measured at 30 minute intervals. The process of coolingto 18 and 1l oC. required from 30 minutes to L0 hours. During awakeningfrorn hibernation continuous measurements of esophageal temPeratures weremade. The warming to 13 and 18oC. required from J to l0 minutes depend-ing upon the initial depth of hibernation. The series were limited to malesin the case of the rodents, but contained male and female with both speciesof bats. With them serum electrolyte and hematological data were analyzedseparately, but sex differences were not found (p values were all greaterthan 10 per cenr). Therefore, the electrolyte and hematological data forbats are combined data from males and females. Each value presented intables I and 2 represents the meao of at least four samples (20 animals).

TABLE 1SERUM ELECTROLYTE AND HEMATOLOGICAL DATA OFMYOTIS LUCIFUGUS IN COLD EXPOSURE

Calcium Potassium Specific Hematocritrl.Eq/L .Eq/t gravityActive4 davs*"rr roorn Q4oC)

1B-32 hrscold room (60C)Hibernating12 hrs

18 hrs32 hrs2 days4 days

10 days9 wks

4.46(0.28)*1.40(0.1 1 )

6.17(0.93 )7.24(0.60)

7.05(0.3 1)6.81(0.0r)6.t,(0.60)6.52(0.'')6.e9(0.60)7.10(0.44)

t.0226(0.0008)r.0229(o.o006)L.0228(o.oo04)t.0222(0.0003 )1.0221(0.0003 )1.0239(0.0014)L.0243(0.0003 )t.0247(0.0009)L.0294(0.0002)

46.0(0.6)

J.28(0.41)4.00(0.92)j.79(0.t4)4.28(0.3 6)2.40(0.42)3.r2(0.40)

t0.r(1.6)46.7(2.6)48.8(4.1)48.1(4.1)

t 1.8(0.9)+Standard deviation.

The source and maintenance of these specimens wefe described earlier(Riedesel, 1957). Triplicate determinations were made on all samples ob-tained from rodents, Small sample statistical methods were used to ^nalyzethe data and a five per cent or less level of confidence was accePted as asignificant difference.RESI]LTSThe data describe primarily homeostasis of serum calcium and Potassiumlevels during hibernation, with some excePtions. Two control grouPs and

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SERUM ELECTROLYTE LEVELS IN HIBERNATING MAMMALS 309TABLE 2

SERUM POTASSIUM AND HEMATOLOGICAL DATA OF MYOTIS LUCIFUGUSIN STAGES OF HIBERNATIONConditions PotassiummEq/1 Specificgravity Hematocrit

Active, 4 daysCooled to E.T. ofCooled to E.T. ofwarmed ro 13oCWarmed to 18oCActive, I hour

n-200c1 1-13 0c

6.17(0.94)*7.20(0.36)7.25(0.18)7.tt(0.5r)7.07(0.42)7.80(0.29)

1,0226(0.0008)r.027 t(0.0006)1 .02 81(0.0007)1.028t(o.oo06)r.0284(0.0001 )

46.0(0.6)49.0(2.8)48.2(2.8)t0.0(4.7)t5.2a.6)5?t(0.9)*Standatd deviation.E.T.-Body temperature measuted in the esophagus'

rwelve groups of little brown bats exposed to the cold were studied. In allcases mean calcium values for cold-exposed animals, hibernating and non-hibernating, were lower than control values (figure 1 and table 1). The dif-ferences betw.een the mean values of the group active four days and thegroups which hibernated ten days and nine weeks resPectively are significantar the 99 per cent level of confidence (table 1). The serum Potassium valuesdid not change significantly; however, rhe mean values during hibernationwere consistently higher (tables 1 and 2). The cell/plasma ratio and serumspecific gravity usually did not change consistently (tables I and 2)'

JotdIt?oJo

/ ././,

ACTIVE COOLED COOICO WARI'ED WARMED4 DAYS TO TO TO TOt7-20"c ll-13"c ls"c l8'cSTAGES OF HIBERNATIONFIGURE 1. Serum calcium measurements of Myotis lucilugus inhibernation. (Shaded alea reptesents ooe staodatd deviation above andmeao. )

ACTIVEI HR.stages ofbelow the

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310 THE AMERICAN NATURALISTThe results for the first hour after arousal (table 2) showed unexpectedtrends. It is known that the first hour after arousal is characterized bydrastic changes in homeostatic mechanisms. During this period the vascu-lar bed is opened, the wings are spread, the spleen contracts, the state of"emergency" results in hypercoagulability of the blood, rapid changes in

serum magnesium occur, and the animal probably becomes more dehydrated.The high values of potassiurn, specific gravity. and hematocrit are con-sistent with these changes (table 2, active one hour).TABLE 3

SERUM ELECTROLYTE AND HEMATOLOGICAL DATA OFHIBERNATORS IN COLD EXPOSUREActive in warmQ40c) Active in coldt6ocl Hibernating2 daysMean Std. dev. Mean Std. dev. Mean Std. dev.

Big BrowaBatn=4to30Potassium(mEq/l) 6.16Specific 0.19gravity 1.0206 0.0005Hematocrit ,1,2 2.0Calcium(mEq/t) 4.66 0.10Potassium(mEe/l) 5 .77 0.28Specificgravity l.An, 0.0002Hematocrit 46.8 2.7

0.96

5.88 0.1 81.0240 0.000444.7 2.74.97 0.30

1.1 6.5 0.540.0019 1.0232 0.00076.9 48.4 2.r

GroundSquinela=4to8

Hamstera=2to4

4.898.16t.0245

5 r.0Calcium(mEs/i ) 5 .tt 0.02 5.78Potassium(mEq/l) 8.61 o.0t 6.12Specificgravity L.0245 0.0010 I.0232Hematocrit 14.! 21.2 tr.9

0.180.26

1.59 0.109.46 0.0*

0.0071 r.0234 0.004t4.0 ,7 .4 1.4*o=2.

In contrast to the little brown bat, data on the big brown bat (Eptesicusfuscus) did not demonsffate consistent increase in the serum potassium(table 3). Ia the case of the ground squirrels, cold exposure. and hiberna-tion did not alter the serum calcium, specific gravity or blood hematocrit,Control and hibernating ground squirrels also had similar serum porassiumvalues. A 40 per cent increase in the mean porassium was observed withanimals not hibernating during exposure to the cold. The two hamsters ex-posed to the cold had serum potassium levels much lower than their warmroom conrols, but a larger sampling of this species is necessary to definethe significance of this response. The increases in rhe serum potassiumand specific gtavity with hibernation are similar to those observed *ith thelittle bfown bat and ground squirrel. No changes were observed in theserum calcium with hibernation of the hamsters.

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SERUM ELECTROLYTE LEVELS IN HIBERNATING MAMMALS 3rtDISCUSSION

The ranges of the serum electrolyte concentrations of the control.animalsin this study are similar to those reported for mammals which cannot hiber-nate (Albritton, lgjz). However, decreases in calcium and apparent in-creases in potassium concentrations w'ere observed with hibernation. It.isof particulat interest to oote the similarities between'the responses of thebody temperature of the hibernators and the changes in these serum electro-lyte concentrations. The little brown bat provided the first consistent evi-dence of low serum calcium with hibernation. The data in figure 1 suggestthat the serum levels observed during hibernation may vary with depth ofhibernation.

Observed similarities and differences in serum potassium levels may berelated to differences in body temperature during cold exposure. Earlierstudies have described lowered body temperatures for active bats andground squirrels exposed to the cold whereas aitive hamsters do not ex-tiSir "o"t lowering of body temperarures (Folk, 1957). Regarding changesin serum potassium, active hamsters were once again different in that thedata indicate a decrease in the serum potassium concentration with coldexposure. The active bats and ground squirrels had an increase under thesame conditions. During hibernation all three species are consistent inhaving higher serum potassium.The cause and effect relationship of the electrolyte changes observedduring cold exposure and hibernation cannot be described completely untilfurther studies have been made with emphasis on such pertinent factors as:renal excretion of cations, effect of cooling and pH changes oo the ionbinding power of intracellular proteins, adequacy of circulation during vari-ous phases of hibernation, activity of the adrenal cortex, and activity ofcell membrane electrolyte transfer systems.

SUMMARYEarlier work has shown an increase in serum magnesium to be a charac-teristic of hibernation with the magnitude of the change by species in thisorder: ground squirrel ) bat ) hamster. The present study describes anotherprominent change in serum electrolytes: namely, a drop in serum calciumwith active and hibernating little brown bats exposed to the cold. Activebats and ground squirrels both appear to have a rise in serum potassiumduring cold exposure with less definite rise in hibernation. The hamster,as usual, shows a different response, in this case a decrease in serum po-tassium when active during cold exposure. Consistent with the other spe-cies, the hamster shows a slight rise in this ion iq hibernation. On thewhole, except for serum calcium in the little brown bat, and serum magne-sium changes, the present experiments support the view that hibernation ischaracterized by the presence of serum electrolytes at control levels or

slightly higher.

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312 THE AMERICAN NATURALISTLITERATURE CITED

Adler, Leo, 192.6, Det Winterschlaf. Handbuch Normal. u. Pathol. Physiol. 17:1 0t-i33.Bicch, G., B. Johansson and S. Veige, 1956, Some laboratory data on hedgehogs,hibernating and non-hibernating. Acta Physiol. Scand., 37, Fasc. 4:28t-294.

Ferdarenr, D. and O. Feinschmidt, 1912, Der Vinterschlaf. Er8ebn. Biol. 8: 1-75.Folk, G. E., Jr,, 1957, Twenty-four hour rhythms of marrmals in a cold environment.Amer. Nat. 9Iz lJl-L66.Lytnan, Charles P. and Paul O. Chatfield, 1955, Physiology of hibernation in mam-mals. Physiol. Rev. 35:.403-421.McBirnie, J. E., F. G. Pearson, G, A. Trusler, H. H. Karachi aod V. G. Bigelow,19J3, Physiological studies of the groundhog (Marmota monax). Can' J.Med. Sci. 3L:421-430.Natelson, S. and R. Penniall, 1955, Colorimetric estimation of ultramicro quantitiesof calcium in human serum as the complex with alizarin. Anal. Chem. 27:434-437.Riedesel, Marvin L., 19t7, Serum magnesium levels in mammalian hibernation.Ttans. Kans. Acad. Sci. 6U 99-14L.Suomalainen, Paava, 1939, Hibernation of the hedgehog, VI. Setum Mg and Ca.Artificial hibernation also a contribu,tion to chenical physiology of diurnalsleep. Annal. Acad. Sci. Fennicae. Ser. A. Tom. !1, No. 7, l-71.1953, Proc. Finnish Acad. Sci & Letters: 131.Van Slyke, DonaldD., P. B. Hamilton, V. P, DoIe, K. Emetson, Jr. and R. M'- Archi-bald, 1950, Measurernent of specific gravities of whole blood and plasmaby standard coppet sulfate solution. J. Biol. Chem. 183: 30r-tt0.