Vol. XCII, No.

866

The American Naturalist

September-October,

958

SERUM ELECTROLYTE M.

LEVEI

IN HIBERNATING MAMMALS*

L.

RIEDESELT AND G. EDGAR FOLK, JR.

Department of Physiology, The State Universiry of lowa School of Medicine, Iowa City, Iowa
INTRODUCTION

of the hedgehog (Suomalainen, r)))); increased serum potassium with hibernation of the American woodchuck (McBirnie, er al., 1953), but decreased serum potassium (Suomalainen, t9j3), or no change (Biorch, et al., 19J6) with hibernation of the hedgehog. These contradictions may result from species characteristics or differences in the depth of hibernarion. Accordingly, the presenr study was designed to describe species differences in serum electrolytes as related to depth or type of hibernation. An earlier report by the senior author describes elevation of serum magnesium with the lowering of body temperarure in the little brown bat (Myotis lucifugus) (Riedesel, 1957). This presenrarion records serum calcium and potassium measurements on four species of hibernating animals, including two species of bats, hamsters, and thirreen-lined ground squirrels.
PROCEDURE AND TECHNIQUES

Reports on serum electrolyte changes with hibernation have been consistent in indicating an elevation of serum magnesium (Suomalainen, 1939; McBirnie, er al., l!Jl; Riedesel, 1957), but inconsistent with respecr ro other electrolytes. For example: hypo- and hyper-carcemia have been described with hibernation in the European marmor (Adler, 1926; Ferdmann and Feiaschmidt, 1932) but no change in the serum calcium with hibernation

sirnuitaneous serum calcium, potassium, specific gravity and blood hematocrit determinations were made on animals bled by decapitation. In the case of the bats, serum electrolyte and specific gravity determinations were made on samples obtained by pooling the blood of five animals. po-' tassium determinations were made by flame photometry. The spectrophotometric method of Natelson and Penniall (Igjj) was employed for the analysis of total serum calcium. Hematocrit values on the small bats were determined by the use of Van Allen hematocrit rubes. The Vinuobe method was employed for hematocrit determinations oo the other animals. In either

case the blood was centrifuged at 2800 r.p.m. for 45 minutes. The serum specific 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 were sacrificed. vhen initial srages of hibernation were studied, esophageal
rThis research was supported in part by a grant from the National science Foundation, Washington, D. C. tPtesent address: Department of occupational Health, Graduate School of public Health, University of Pittsburgh.
307

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THE AMERICAN NATURALIST

temperatures were measured at 30 minute intervals. The process of cooling to 18 and 1l oC. required from 30 minutes to L0 hours. During awakening frorn hibernation continuous measurements of esophageal temPeratures were made. The warming to 13 and 18oC. required from J to l0 minutes depending upon the initial depth of hibernation. The series were limited to males in the case of the rodents, but contained male and female with both species of bats. With them serum electrolyte and hematological data were analyzed separately, but sex differences were not found (p values were all greater than 10 per cenr). Therefore, the electrolyte and hematological data for bats are combined data from males and females. Each value presented in tables I and 2 represents the meao of at least four samples (20 animals). TABLE
1

SERUM ELECTROLYTE AND HEMATOLOGICAL DATA OF MYOTIS LUCIFUGUS IN COLD EXPOSURE

Calcium rl.Eq/L
Active
4 davs

Potassium .Eq/t

Specific
gravity

Hematocrit

*"rr

roorn Q4oC)

4.46

6.17
(0.93 )

t.0226
(0.0008)

(0.28)*
1.40
(0.1 1 )

46.0 (0.6)

1B-32 hrs cold room (60C)

7.24

r.0229
(o.o006) L.0228 (o.oo04)

(0.60)
7.05
(0.3 1)

Hibernating
12 hrs 18 hrs 32 hrs

J.28 (0.41) (0.92)

4.00

(0.0r)
(0.60)

6.81

t.0222
(0.0003 )

(1.6) 46.7 (2.6) 48.8 (4.1)

48.1

t0.r

6.t,
6.52

1.0221
(0.0003 )

2 days 4 days
10 days 9 wks +Standard deviation.

j.79

(0.t4)
4.28
(0.3 6)

(0.'')
6.e9 (0.60)
7.10

1.0239

(0.0014)
L.0243
(0.0003 )

(4.1)
t 1.8

2.40

(0.42) 3.r2
(0.40)

(0.44)

t.0247 (0.0009) L.0294 (0.0002)

(0.9)

The source and maintenance of these specimens wefe described earlier (Riedesel, 1957). Triplicate determinations were made on all samples obtained from rodents, Small sample statistical methods were used to ^nalyze the data and a five per cent or less level of confidence was accePted as a
significant difference.
RESI]LTS

The data describe primarily homeostasis of serum calcium and Potassium levels during hibernation, with some excePtions. Two control grouPs and

SERUM ELECTROLYTE LEVELS IN HIBERNATING

MAMMALS

309

TABLE

SERUM POTASSIUM AND HEMATOLOGICAL DATA OF MYOTIS LUCIFUGUS IN STAGES OF HIBERNATION

Conditions

Potassium

Specific

mEq/1
6.17

gravity
1,0226 (0.0008) r.027 t (0.0006)

Hematocrit 46.0 (0.6)

49.0
48.2

Active, 4 days
Cooled to E.T. of Cooled to E.T. of
warmed ro 13oC
Warmed

n-200c
0c 1 1-13

(0.94)* (0.36) (0.18)

7.25 7.20

(2.8) (2.8) (4.7)

(0.5r) (0.42) (0.29)

7.80 7.07

7.tt

(0.0007)
(o.oo06)

.02 81

t0.0
t5.2 5?t

to 18oC

1.028t

Active, I hour

r.0284
(0.0001 )

a.6)
(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 all cases mean calcium values for cold-exposed animals, hibernating and nonhibernating, were lower than control values (figure 1 and table 1). The differences betw.een the mean values of the group active four days and the groups which hibernated ten days and nine weeks resPectively are significant ar the 99 per cent level of confidence (table 1). The serum Potassium values did not change significantly; however, rhe mean values during hibernation were consistently higher (tables 1 and 2). The cell/plasma ratio and serum specific gravity usually did not change consistently (tables I and 2)'

/
J

././,

o td
I

? o
J

ACTIVE COOLED COOICO WARI'ED TO TO 4 DAYS TO

WARMED

ACTIVE

t7-20"c ll-13"c ls"c

l8'c

TO

I HR.

STAGES OF HIBERNATION
FIGURE 1. Serum calcium measurements of Myotis lucilugus in stages of hibernation. (Shaded alea reptesents ooe staodatd deviation above and below the
meao. )

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THE AMERICAN NATURALIST

The results for the first hour after arousal (table 2) showed unexpected trends. It is known that the first hour after arousal is characterized by drastic changes in homeostatic mechanisms. During this period the vascular 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 consistent with these changes (table 2, active one hour).
TABLE
3

SERUM ELECTROLYTE AND HEMATOLOGICAL DATA OF HIBERNATORS IN COLD EXPOSURE

Active in warm

Active in cold

Hibernating

Q40c)

t6ocl
Mean

2 days

Mean Std. dev.

Potassium

Std.

dev.

Mean

Std. dev.
0.1 8

Big Browa
Bat

(mEq/l)

6.16

0.19 0.0005 2.0 0.10 0.28 0.0002 2.7

Specific

5.88 44.7
4.89
8.16 0.96

n=4to30

Hematocrit
Calcium

gravity 1.0206 ,1,2

1.0240

0.0004 2.7 0.30 0.54 0.0007

(mEq/t) 4.66 (mEe/l) 5 .77 Squinel a=4to8 Specific gravity l.An, Hematocrit 46.8
Ground

Potassium

1.1 6.9
0.18 0.26

4.97 6.5 48.4

t.0245 r.0
5.78 6.12

0.0019 1.0232

2.r
0.10

Calcium
Hamster

Potassium Specific

a=2to4

.tt 0.02 (mEq/l) 8.61 o.0t

(mEs/i

1.59 9.46
,7

0.0*
1.4

Hematocrit

gravity L.0245 0.0010 I.0232 21.2 tr.9 14.!

4.0

0.0071 r.0234 0.004t

.4

*o=2. In contrast to the little brown bat, data on the big brown bat (Eptesicus fuscus) did not demonsffate consistent increase in the serum potassium (table 3). Ia the case of the ground squirrels, cold exposure. and hibernation did not alter the serum calcium, specific gravity or blood hematocrit, Control and hibernating ground squirrels also had similar serum porassium values. A 40 per cent increase in the mean porassium was observed with animals not hibernating during exposure to the cold. The two hamsters exposed to the cold had serum potassium levels much lower than their warm room conrols, but a larger sampling of this species is necessary to define the significance of this response. The increases in rhe serum potassium and specific gtavity with hibernation are similar to those observed *ith the little bfown bat and ground squirrel. No changes were observed in the serum calcium with hibernation of the hamsters.

SERUM ELECTROLYTE LEVELS IN HIBERNATING MAMMALS

DISCUSSION

3rt

The ranges of the serum electrolyte concentrations of the control.animals

in this study are similar to those reported for mammals which cannot hibernate (Albritton, lgjz). However, decreases in calcium and apparent increases in potassium concentrations w'ere observed with hibernation. It.is of particulat interest to oote the similarities between'the responses of the body temperature of the hibernators and the changes in these serum electrolyte concentrations. The little brown bat provided the first consistent evidence of low serum calcium with hibernation. The data in figure 1 suggest that the serum levels observed during hibernation may vary with depth of
hibernation. Observed similarities and differences in serum potassium levels may be related to differences in body temperature during cold exposure. Earlier studies have described lowered body temperatures for active bats and ground squirrels exposed to the cold whereas aitive hamsters do not extiSir lowering of body temperarures (Folk, 1957). Regarding changes "o"t in serum potassium, active hamsters were once again different in that the data indicate a decrease in the serum potassium concentration with cold exposure. The active bats and ground squirrels had an increase under the

same conditions. During hibernation

having higher serum potassium.

all three species are consistent in

The cause and effect relationship of the electrolyte changes observed during cold exposure and hibernation cannot be described completely until further studies have been made with emphasis on such pertinent factors as: renal excretion of cations, effect of cooling and pH changes oo the ion binding power of intracellular proteins, adequacy of circulation during various phases of hibernation, activity of the adrenal cortex, and activity of cell membrane electrolyte transfer systems.
SUMMARY

Earlier work has shown an increase in serum magnesium to be a characteristic of hibernation with the magnitude of the change by species in this order: ground squirrel ) bat ) hamster. The present study describes another prominent change in serum electrolytes: namely, a drop in serum calcium with active and hibernating little brown bats exposed to the cold. Active bats and ground squirrels both appear to have a rise in serum potassium during cold exposure with less definite rise in hibernation. The hamster, as usual, shows a different response, in this case a decrease in serum potassium when active during cold exposure. Consistent with the other species, the hamster shows a slight rise in this ion iq hibernation. On the whole, except for serum calcium in the little brown bat, and serum magnesium changes, the present experiments support the view that hibernation is characterized by the presence of serum electrolytes at control levels or slightly higher.

312

THE AMERICAN NATURALIST LITERATURE CITED

Adler, Leo,
1

192.6, Det Winterschlaf. Handbuch Normal. u. Pathol. Physiol. 17: 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:

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 mammals. 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 quantities of 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 diurnal sleep. 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'- Archibald, 1950, Measurernent of specific gravities of whole blood and plasma by standard coppet sulfate solution. J. Biol. Chem. 183: 30r-tt0.

28t-294.