Sodium and potassium in vertebrate cochlear endolymph as determined by flame microspectrophotometry

Comp. Biochem. Physiol., 1963, Vol. 9, pp. 335 to 341. Pergamon Press Ltd. Printed in Great Britain

SODIUM AND POTASSIUM IN VERTEBRATE COCHLEAR ENDOLYMPH AS DETERMINED BY F L A M E M I C R O SPECTROPHOTOMETRY* CLARE G. JOHNSTONE,~- ROBERT S. S C H M I D T $ and BRIAN M. J O H N S T O N E t § T h e Departments of Zoology and Surgery (Otolaryngology), T h e University of Chicago (Received 13 M a r c h 1963)

A b s t r a c t - - 1 . Concentrations of 1"8 mM/1 Na and 151 mM/1 K (K/Na quotient 84) were measured in cochlear endolymph of guinea pigs, using a simplified flame microspectrophotometer which is described. 2. This sodium concentration is low enough to admit of the possibility that the positive endocochlear potential is a sodium diffusion potential. 3. K / N a quotients were measured in other species, the highest values obtained for each being 42 (turtle), 30 (frog) and 20 (lizard).

INTRODUCTION

As SEENfrom Table 1, endolymph in several species has been shown to have high K and low Na concentrations; in this it resembles intracellular fluid. However, instead of being electrically negative with respect to extracellular fluids, the endolymph has been shown to be at a positive potential with respect to perilymph or blood (for review, see Davis, 1957). This positive potential is about 80 mV in the mammalian cochlea, but much less in the other parts of the vestibular system, although the ionic concentrations are similar throughout (Smith et al., 1954). Vertebrates other than mammals have smaller positive endocochlear potentials (Schmidt & Fernandez, 1962). Because the potential is profoundly affected by anoxia, and apparently unrelated to ionic concentrations, it was suggested that it is a direct result of oxidative metabolism (Davis, 1957). Recently this hypothesis has been questioned because the decline in potential during anoxia can be halted and reversed by perfusion with solutions having low oxygen tension (Honrubia et al., 1962). For ionic diffusion to give rise to a large electrical potential difference of the polarity observed between endolymph and perilymph, the interface would have * This work was aided by National Science Foundation Grants No. 12449 and G23837 and National Institute of Neurological Diseases and Blindness Grants BT-469(C2) and B-682(C8). t Present address: Department of Physiology, University of Western Australia, N e d lands, W.A., Australia. +*Address: Department of Surgery, The University of Chicago, Chicago 37, Ill., U.S.A. § Recipient of U.S.P.H.S. Training Grant 2 G - C I | . 335

3 36

CLARE G . JOHNSTONE,

ROBERT S. SCHMIDT AND BRIAN M . JOHNSTON~

to be selectively permeable to a positive ion at a relatively high concentration m perilymph, or a negative ion at a relatively high concentration in endolymph. C1 is unsuitable, being already at high concentration in perilymph. The pH of both fluids is similar (for comparison see Citron et al., 1956) making H, OH and HCO:~ unlikely. Na remains as the most likely possibility but the sodium concentration in the scala media would have to be lower than has so far been reported. 'l'he mean measured potential, 80 mV, would require the scala media sodium concentration to be less than 5 mM/1; the largest values reported, 110 mV (Konishi el a/., 1961), would require it to be less than 1.7 mM/1. "FABLE ] .--ENI)OLYMPH CATION CONCENTRATIONS IN MAMMALS AS MEASUtlED BY PREVI{HT~ INVESTIGATORS

Source Guinea pig; utricle

K (mM/1)

Na (mM/1)

K/Na

144

15"8 (lowest 5.6) (lowest 12) 26 66 13-16

9

Smith et al. (1954)

(26)

Smith et al. (1954)

(12) 5"5 2 12

Smith et al. (1954) Citron et al. (1956) Citron et al. (1956) Rauch & Kostlin ( 1958)

Guinea pig; cochlea Guinea pig; utricle Cat; cochlea Man ; cochlea

142 117 140-160

Authors

Because the likelihood of contamination of samples by high Na perilymph is very great, it must be assumed that the lower the Na value obtained, the less was the perilymph contamination. METHODS The procedures for obtaining access to the scala media were described previously (Schmidt & Fernandez, 1962). The endolymph was drawn up into a micropipette, pulled from 1 m m glass tubing, attached to a micrometer-driven syringe; syringe and pipette were initially filled with mineral (paraffin) oil. This endolymph sample was then expelled as a small droplet under oil. It was noticed that droplets larger than about 0.1/xl were usually heavily contaminated with perilymph. Samples containing red blood cells were rejected. Three to ten subsamples were then taken from the droplet with a similar micropipette in which a marker had been provided to indicate a constant volume. This marker was a piece of 44-gauge silver wire flattened to an oval section and wedged into the micropipette. The volume of the subsamples was about 0.01/xl; smaller volumes were difficult to manipulate. Using the same pipette, subsamples of known solutions were taken for calibration purposes, including estimation of the relative volume of each new pipette.

SODIUM AND POTASSIUM I N VERTEBRATE COCHLEAR E N D O L Y M P H

337

Each subsample was expelled onto a fine wire which had been cleaned by burning in the flame until no yellow glow was visible. Care was taken, in expelling the subsample, to avoid expelling oil with it, as oil prevented thorough drying and tended to burn explosively in the flame. The wire was dried in air, then for 5 min under an infra-red lamp. Endolymph of frogs and lizards was extremely viscous, and for such samples volumes could not be reliably measured. Therefore, concentrations could not be determined, but K/Na quotients were obtained. Turtle endolymph was less viscous and some volume measurements were attempted. The flame microspectrophotometer used was similar to those described by Ramsay et al. (1953), Mfiller (1958) and Bott (1960), in which a small sample carried on a wire is volatilized in a flame, the time integral of the intensity of the emitted light being measured. Details of the instrument, which is simple to construct and use, and low in cost, are presented in the appendix. RESULTS Guinea pigs: The quotient K/Na for samples of guinea pig cochlear endolymph varied from a little above 0.02 (its value in the perilymph) up to 100. Whereas twelve samples yielded values fairly evenly distributed between 0.07 and 20, seven samples (from five guinea pigs) gave values of K/Na grouped between 50 and 100. TABLE 2--MEAN

SODIUM AND POTASSIUM CONCENTRATIONS FROM EIGHTEEN SUBSAMPLES OF

COCHLEAR E N D O L Y M P H .

Tim

SAMPLES WERE OBTAINED FROM FIVE GUINEA PIGS.

T H E MEAN

VALUES FOR I N D I V I D U A L ANIMALS ARE ALSO LISTED

K/Na

Na

(Standard

K

(Standard

(mM/l)

deviation)

(raM/l)

deviation)

Number of

1.8 1.8 1.4

(0.5) (0.2) (0.6)

151 160 138

(16) (12) (5)

84 89 99

18 6 3

2.0 1.5 2.5

(0.2) (0.05) (0.2)

159 133 160

(8) (22) (8)

80 89 64

3 3 3

subsamples

These were regarded as the best samples, a high K/Na quotient being prima facie evidence of lack of perilymph contamination, and they are listed in Table 2. In the two instances where a second sample was obtained from the same cochlea, the second sample had a lower K/Na value than the first; both second samples have been omitted from the tabulation. As a eontrol, two perilymph samples were taken from the ears of another guinea pig; the mean values from a total of nine subsamples were Na 137 mM/l (S.D. 8), K 3.6 mM/l (S.D. 1"3), K/Na 0.026.

338

CLARE G . JOHNSTONE, ROBERT S. SCHMIDT AND BRIAN M .

JOHNSTONE

Other vertebrates: Some measurements (Table 3) were made on other vertebrates, which must be regarded as preliminary; higher K/Na quotients may be obtained as improvements are made in the dissection technique and sampling procedures so as to reduce contamination with perilymph. "FABLE3. ESTIMATES OF K/Na QUOTIENTS FOR REPRESENTATIVE VERTEBRATE SPECIES. FOR THE TURTLE SOME SAMPLE VOLUMES WERE MEASURED, THE MEANS OF FOUR SUBSAMPLES OF THE BEST SAMPLE GAVECONCENTRATIONS OF 2"7 mM/l Na, 114 mM/l K

Source

Number of samples

Number of samples with K/Na greater than unity

Highest K/Na value obtained

Turtle ; cochlea (Chrysemys picta) Frog; pars basilaris (Rana pipiens) Lizard; cochlea (Phrynosoma cornutum) Pigeon; cochlea (Columba livia)

10

8

42

7

3

30

14

8

20

2

2

3

DISCUSSION The cochlear sodium concentration determined for guinea pig is sufficiently low to admit of the possibility that the positive endocochlear potential is a sodium diffusion potential. The lowest value obtained (1.4 mM/1) would correspond to a sodium equilibrium potential of 60 log 137/1.4 = + 118 mV. Both a low sodium concentration and a high relative permeability to sodium are required to generate a large potential, so that a low concentration of sodium in the endolymph need not necessarily be accompanied by a large potential difference. For example, in the guinea pig vestibule, where sodium concentration is low (though not as low as in the cochlea), the potential is only 4 mV (Smith et al., 1958), presumably because of lack of differential permeability. Also in turtles and frogs, where the measured K/Na quotients of 42 and 30 suggest cochlear sodium concentrations of only 3-4 raM/l, the measured positive endocochlear potential is less than 6 mV (Schmidt & Fernandez, 1962), presumably for the same reason. APPENDIX The flame microspectrophotometer was simpler than those previously described (Ramsay et al., 1953; Miiller, 1958; Bott, 1960), in that no special gas regulatory mechanisms were used, and a hand-operated device advanced the wire into the flame. Sodium and potassium concentrations normally encountered in biological fluids could be handled in volumes up to 0.02/xl without dilution. When six subsamples of a sample were measured, the standard deviation of the recorded

SODIUM AND POTASSIUM IN VERTEBRATE COCHLEAR ENDOLYMPH

339

output was between 5 and 10 per cent of the mean, except at very low concentrations (3 mM/l), where it was 20 per cent. The variability could be reduced to some extent by making allowance for variations in the burning time.

"T"I

M <~(IOM for

l ~

uOqM F -

e ~ IntecJrate stand by

-

v' ,IM M

(

I I

I_~ i e" e Off _[On

,~100 F~ne balance

2K 182t< Coarse balance

VI

preamp ~182

931-A (7102 for K channel)

~]J _ 500V

V FIG. | . Photomultiplier and integrator circuit for the Na channel. T h e K channel was similar, differences are indicated. T h e K 2 W is a plug-in d.c. to 100 K C differential operational amplifier with a gain of 15,000 (made by George A. Philbrick Researches, Inc., of Boston, Mass.). It requires a regulated power supply of _+300 V d.c. and a constant voltage filament supply.

The flame housing was the arc housing of an old carbon arc projector mounted on a cast-iron base, and to it were attached two metal cylinders containing condensing lenses. The photomultiplier tubes and filters (589 F interference filter plus Corning type HR3-68 for Na and 768/z interference filter plus Corning type 7-69 for K) were on heat-insulating mounts attached to the cast-iron base, and were thermally insulated from the metal cylinders. It was found that this reduced heating of the photomultipliers and filters sufficiently to avoid trouble, even if the instrument was used all day. The lens systems used to focus the light onto the photomultipliers were adjusted so that the signals obtained with the largest amounts of sample used showed no evidence of overloading as checked at the photomultiplier anode with a cathode ray oscilloscope (i.e. pulses were sharp peaked, and less than 20 V). Also, diffusers were included in the optical pathways to avoid possible local overloading of the photomultiplier cathodes. Consequently, the instrument was not adjusted for maximum possible sensitivity. The photomultiplier and integrator circuits are shown in the figure. The integrated signals were recorded on a Grass dual channel polygraph, after passing

340

C L A R E G. JOHNSTONE, ROBERT S. SCHMIDT AND BRIAN ~1. JOHNSTONI~

through a voltage divider which enabled the Grass d.c. preamplifier to be used (with its calibration and sensitivity controls), and buffered the operational amplifier from chopper transients. The pulse height at the photomultiplier anode was in the range 0"1 to 5 V, the duration 0.1 to 5 sec. With the volume of solution used (~1.t 1 /,1), the signal obtained at the input to the preamplifier was about 20 mV for concentrations of 100 mM/1. Concentrations of 2 raM/1 gave signals of about 0.5 inV. Drift and flame noise amounted to about 0"05 inV. Thus the range of concentrations commonly encountered could be handled conveniently without the need for dilution, or adjustment of the instrument. To keep the flame noise low, other activities in the room had to be kept at a minimum; in particular, washing glassware with suds containing Na was found to increase flame noise markedly. The flame, obtained by burning a mixture of town (natural) gas and oxygen from a small welding nozzle, was about 7 cm high and 1 cm broad at its widest point. The proportion of oxygen was reduced until the flame was as coot as possible (which protected the wires) without yellow luminosity being visible at the center cone. This was found to be a reproducible condition. The burner nozzle was placed about 1 cm below the flame housing, leaving space to insert the wire carrying the sample into the lower part of the flame. Thus the wire, as it was heated by the flame, was not directly seen by the photomultipliers. The wire (100 /x diana 3(t~I,, iridium platinum), about 1 cm long, was mounted in a Luer hypodermic needle by crimping the needle onto it (Mfiller, 1958). A Luer-lok fitting xYas attached to a 2 in. rack and pinion. Both the rack and pinion and the burner nozzle were held in adjustable clamps fastened to the base of the instrument. It was found that a viewport could be open in the side of the flame housing without increasing the current of the photomultipliers; this proved an advantage, as readings could be roughly' verified by' eye. Mfiller (1958) describes sodium burning as having a fast (50 msec) and a slow (1-2 sec) phase. With the large amounts of Na used here, the light flash was often more complex. However, if the wire was introduced slowly into the flame, the very large and fast initial phase did not normally occur. Instead, a stead3: glow was obtained which commenced before the wire entered the visible part of the flame. The wire reached only a dull red heat until volatilization was complete and the recorded integral rose rapidly to a plateau. Thereafter the wire became hotter, and a slowly rising signal of constant gradient was recorded due to its luminosity. Variations in the position of the wire as it entered the flame were unimportant, provided it was kept clear of the center cone. The initial rise of the recorded integral was 0-1 to 5 sec in duration for steadvglow burning. It was found that the integrated value obtained for sodium decreased about 30 per cent as the burning time decreased over this range when the sample concentration was 100 mM/1. Although this nonlinearity was less pronounced at lower concentrations, no evidence of instrument overload was found. A similar nonlinearity of about 15 per cent occurred with high concentrations of potassium; at low concentrations the integrated values decreased with increased burning time if a high sodium concentration was also present. As only a small

SODIUM AND POTASSIUM IN VERTEBRATECOCHLEARENDOLYMPH

341

proportion of the atoms passing through the flame are excited to emit light (Margoshes, 1962), it m a y be that a change in this proportion occurred. I n the presence of oil, and perhaps if large crystals had formed, the sample burned explosively, appearing as a flash instead of a glow. I f this happened, little color was seen, even if the sample was high in sodium. U n d e r these circumstances, the burning time, as measured from the recorded integral, was m u c h less than 0.1 sec. T h e results obtained with this type of burning were very variable and they were discarded. It was observed that the calibration curves remained constant f r o m day to day provided that the burning times were similar. T h r e e curves (on log-log paper) were used to cover the range of burning times which resulted in some reduction of the variance in the results. Interference on the K channel due to high concentrations of N a was small (160 raM/1 N a read as 0.04 raM/1 on the K channel), but in the reverse direction it was larger (160 raM/1 K read as 1 mM/1 on the N a channel). T h i s was compensated for by the use of standard solutions reasonably close to the unknowns in concentration of both ions. At 2 m M / l Na, the increase in N a reading due to increasing K f r o m 2 m M / l to 160 mM/1 was 10 per cent.

Acknowledgements--The authors wish to thank Professor H. Burr Steinbach, of the Department of Zoology, University of Chicago, for his generous provision of support and facilities, and his unfailing encouragement during the course of this work. Thanks are also due to Professor C. A. G. Wiersma, Division of Biology, California Institute of Technology, for reading the manuscript and making valuable suggestions. REFERENCES BOTT PHYLLISA. (1960) The determination of sodium and potassium in biological fluids with the dual channel ultramicro flame photometer. Anal. Biochem. 1, 17-22. CITRON L., EXLEY D. & HALLPIKEC. S. (1956) Formation, circulation and chemical properties of the labyrinthine fluids. Brit. Med. Bull. 12, 101-104. DAvis H. (1957) Biophysics and physiology of the inner ear. Physiol. Rev. 37, 1-49. HONRUBIA V., JOHNSTONE B. M., BUTLER R. A. & FERNANDEZC. (1962) T h e maintenance

of cochlear potentials during anoxia. Fed. Proc. 21, 343. KONISHt T., BUTLERR. A. & FERNANDEZC. (1961) The effect of anoxia on cochlear potentials. J. Acoust. Soc. Amer. 33, 349-356. MARGOSHES M. (1962) Flame photometry. In Physical Techniques in Biological Research, Vol. 4 (Edited by NASTUKW. L.). Academic Press, New York. Mt~LLER P. (1958) Experiments on current flow and ionic movements in single myelinated nerve fibres. Exp. Cell Res. Suppl. 5, 118-152. RAMSAYJ. A., BROWNR. H. J. & FALLOONS. W. H. W. (1953) Simultaneous determination of sodium and potassium in small volumes of fluid by flame photometry. J. Expt. Biol. 30, 1-17. RAUCH S. & KOSTLINA. (1958) Aspects chimiques de l'endolymphe et de la perilymphe. Pract. Oto-rhino-laryng. 20, 287-291. SCHMIDTR. S. & FERNANDEZC. (1962) Labyrinthine DC potentials in representative vertebrates, ft. Cell. Comp. Physiol. 59, 311-322. SMITH C. A., LOWRY O. H. & Wu M. (1954) The electrolytes of the labyrinthine fluids. Laryngoscope, St. Louis 64, 141-153. SMITH C. A., DAVIS H., DEATrmRAOEB. H. & GESSERTC. F. (1958) DC potentials of the membranous labyrinth. Amer. dY. Physiol. 193, 203-206. 16