Arch. Oto-Rhino-Laryng.220, 259--264 (1978)
Archivesof Oto-Rhino-Laryngology 9 Springer-Vcrlag 1978
Chemical Composition in Various Compartments of Inner Ear Fluid* Kazuo Makimoto 1, Teiro Takeda 1, and Herbert Silverstein2 1 Department of Otolaryngology,School of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan, 2 Sarasota Memorial Hospital, 1901 Arlington Street, Sarasota, Florida 33579, USA
Summary. Sodium, potassium, chloride, glucose and total protein were determined in samples of scala vestibuli perilymph, scala tympani perilymph, CSF, cochlear endolymph and utricular endolymph from normal cats. Small but significant differences were evident in the concentrations of sodium and potassium between the scala vestibuli and scala tympani perilymph. It was also apparent that each compartment of endolymph has different values for sodium and potassium concentrations. Compared with the endolymph, the perilymph was found to contain a higher concentration of glucose and total protein. These findings are discussed from the view point of biological significance. Key words: Inner ear fluid biochemistry - Micro-chemical analysis - Osmotic pressure in inner ear fluids. There is a paucity in knowledge concerning chemical composition in inner ear fluids. In experiment series on inner ear fluid chemistry (Silverstein and Makimoto, 1973; Makimoto and Silverstein, 1974), we analysed sodium, potassium, chloride, glucose and total protein in samples of scala vestibuli perilymph, scala tympani perilymph, cochlear endolymph, utricular endolymph and CSF. 125 ears in 95 cats served as the materials. The present report is a summarization of the results heretofore obtained. Sodium and Potassium
Determination of sodium and potassium was done using a microflame photometer (Makimoto and Silverstein, 1973). The amount used in this analysis was 1.0 ~1 for perilymph, CSF and serum, and 0.5 p~l for endolymph. * Read before the 13th Workshop on Inner Ear Biologyin Dfisseldorfon August 23, 1976 This work was supported by Research Grant No. ROI-NS 1026801 from the National Institute of Neurological Diseases and Strokes, USA Offprint requests to: Kazuo Makimoto, M.D. (address see alcove)
0302-9530/78/0220/0259/$ 01.20
260
K. Makimoto et al.
These results are shown in Table 1. As demonstrated here, small but significant differences were evident between scala vestibuli and scala tympani perilymph both in sodium and potassium concentrations. It was also apparent that each compartment of endolymph has its own value for sodium and potassium concentrations. In the perilymph of scala vestibuli, a higher concentration.of potassium was noted. As to endolymph, a greater value was observed in the sodium concentration of utricular endolymph. This m a y be regarded as one of the biological features for utricular endolymph. In Figure 1, data on the concentrations of sodium and potassium in fluids o f extracellular type and serum are graphically illustrated. Regarding sodium concentration, perilymph of the scala vestibuli was found to be closest to serum. On the other hand, in potassium concentration, perilymph of the scala tympani showed the closest value to that of serum. When a comparison o f N a / K ratio between scala vestibuli perilymph and serum was made, it was obvious that a greater difference existed in this ratio. Accordingly, the perilymph of scala vestibuli m a y not be a simple blood filtrate.
Chloride Chloride was determined by the use of a Chloridemeter | With this instrument, the mEq/1 of chloride in a sample can b e measured by passing a current through the sample via two generating electrodes. The principle of this measurement is that silver ions are released from the anode and combine with chloride ions in the sample. The smallest amount required for this determination is 2.0 ~1. Table 1. Sodium and potassium concentrations in inner ear fluids, CSF, and serum
Sodium (mEq/1)
Potassium Difference
(mEq/1)
Perilymph of scala vestibuli (Pv) Perilymph of ~ scala tympani (Pt) Cochlear endolymph (Ee) Utricular endolymph (Eu)
147.76 (+ 0 . 9 4 ) ~ (N = 54) ~ . > 9.35' 157.11'(+ 1.06) j ~ ( P t - - P v ) (N = 5 8 ) ~ 6.07 (+ 0.40) ~ (N=41) " ~ 8 . 7 9 b 14.86 (+ 0.75) ~ (Eu-Ec) (N = 44)
CSF
152.91 (+ 1.00) (N = 32)
2.97 (+ 0.08) (N = 32)
Serum
140.94 (_+ 1.10) (N = 27)
3.93 (+ 0.16) (N = 27)
Difference
10.47 (+ 0.31) . (N = 54) ~ 6.68c 3.79 (+ 0.11) / / ~ (Pv--Pt) (N = 58) : 171.13 (+ 1.64) . (N = 41) ~ 1 5 . 7 0 a 155.43(+ 1.62) j (Ec--Eu) (N = 44) /
a t = 6.55 (significant, P < 0.001) b t = 10.15 (significant, P < 0.001) c t = 21.28 (significant, P < 0.001) a t = 6.81 (significant, P < 0.001) Each data is expressed as a mean (_+ standard'error); N = represents number of samples
Chemical Composition of Inner Ear Fluid
261 (rnEq/L) 160
150
140
130
6
d
1
I Sodium Concentration ~
Potassium Concentration
Fig. 1. Distribution pattern of sodium and potassium
in scala vestibuli perilymph, scala tympani perilymph, CSF and serum
Pt : Perilymph of Scala Tympani Pv : Perilymph of Scala Vestibuli
Table 2. Chloride concentrations in inner ear fluids, CSF, and Serum Chlorine (mEq/1) Perilymph of scala vestibuli (Pv)
138.51 (+_ 3.58)
Perilymph of scala tympani (Pt)
(N = 43) ~ 130.00 (_+ 2.31) (N = 46)
Cochlear endolymph (Ec)
169.82 (+ 5.83)
Utricular endolymph (Eu)
(N = 28) ~ 158.88 (+ 6.22) ( N = 24)
CSF
138.97 (_+ 1.97) (N = 39)
Serum
120.00 (+ 3.59) (N = 30)
Difference
8.51 a (Pv--Pt)
10.94b (Ec--Eu)
a t = 2.026 (significant, 0.02 < P < 0.05) b t = 1.283 (not significant, 0.2 < P < 0.3) Each data is expressed as a mean (_+ standard error); N =.represents number of samples
262
K. Makimoto et al.
As shown in Table 2, the concentrations of scala vestibuli perilymph, scala tympani perilymph and CSF showed values in a range from 130--140 mEq/1. Compared to these values, relatively high levels of chloride concentration were noted in both cochlear and utricular endolymph.
Glucose Glucose determination was carried out by adapting the enzymatic test kit of Boehringer to a micro-chemical technique (Silverstein, 1973; Makimoto and Silverstein, 1974). The principle of this enzymatic method is as follows: 1. Hexokinase catalyses the phosphorylation of glucose by ATP. Glucose + ATP -~ glucose-6-phosphate + ADP. 2. D-glucose-6-phosphate (G-6-P) is oxidized to 6-phosphogluconate (6-P-G) in the presence of TPN (NADP) by the enzyme G-6-P dehydrogenase. G-6-P + TPN -~ 6-PrG + T P N H + H +. The amount of T P N H arising in Equation (2) is equivalent to the amount of G-6-P or glucose, respectively. The amount of the samples used in this measurement was 0.5 ~1 for endolymph and 1.0 pxl for other fluids. The results are presented in Table 3. The glucose concentration of cochlear endolymph was appreciably low, being 11.2 mg/100 ml, whereas that of utricular endolymph was higher, being 41.9 mg/100 ml. As previously mentioned, utricular endolymph was found to contain a higher concentration of sodium as compared to Table 3. Glucose concentrations in inner ear fluids, CSF, and Serum
Glucose (rag/100 ml) Cochlear endolymph (Ec) Utricular endolymph (Eu) Perilymph of scala Tympani (P0 Perilymph of scala vestibuli (Pv) CSF Serum
Difference
11.17 (_+ 1.29) (N = 28) (+ 2,23) ~ 41.86 (N~ 30)/" 82.77 (+ 3.73) (N = 66) 79.65 (+ 3.46) (N = 59)
30.69a (Eu--Ec)
/
3.12b (Pt--Pv)
81.95 (+ 3.37) (N = 49) 122.48 (+ 7.12) (N = 45)
t = 11.71 (significant, P < 0.01) b t = 0.61 (not significant, 0.5 < P < 0.6) Each data is expressed as a mean (_+ standard error); N = represents number of samples
263
Chemical Composition of Inner Ear Fluid
cochlear endolymph. Such evidence indicates the difference in chemical composition between cochlear and utricular endolymph and may suggest that both compartments of endolymph are not freely communicable, and that each possesses its own independent nature in formation, absorption and circulation of endolymph. Perilymph of scala vestibuli, perilymph of scala tympani and CSF were almost identical in glucose concentration, i.e., approximately 80 mg/100 ml. This level is regarded as being about 40 mg/100 ml lower than that of serum.
Total Protein Total protein was determined by employing the Folin phenol reaction of Lowry et al. (1951). As the first step of this method, the sample is treated with copper sulfate in alkaline solution. Folin reagent is then added to the sample to develop the color reaction. According to the results shown in Table 4, the total protein contents of scala vestibuli perilymph and scala tympani perilymph exceeded those of cochlear and utricular endolymph. Also in CSF, the content of total protein was noted to be much lower than those in scala vestibuli perilymph and scala tympani perilymph. This pattern of concentration gradient in total protein did not coincide with that in glucose. The glucose study revealed that the glucose concentration of CSF was almost the same level as those of scala vestibuli perilymph and scala tympani perilymph. In Figure 2, the total amount of electrolytes (sodium plus potassium plus chloride) are illustrated on the left side of this figure. When this value is compared among various compartments of inner ear fluid, the values for endolymph are greater than those for perilymph.
Table 4. Amount of total Protein in inner ear fluids and CSF Total protein (rag-%) Perilymph of scala vestibuli (Pv) Perilymph of scala tympani (Pt) Cochlear endolymph (Ec) Utrieular endolymph (Eu) CSF
233.87 (+ 1 5 . 7 0 ) ~ (N = 23) ~ 212.52 (+ 1 0 . 6 6 ) ~ (N = 31) 61.50 (+ 7.49) (N = 18) 64.33 (+ 7.70) (N = 9) 37.89 (+ 6.24) (N = 9)
Difference a 21 35 (Pv--Pt) "
2.83b (Eu-Ec)
a t = 1.143 (not significant, 0.2 < P < 0.3) t = 0.229 (not significant, 0.8 < P < 0.9) Each data is expressed as a mean (_+ standard error); N = represents number of samples
264
K. Makimoto et al. I0 [ p
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I mE;q/L
Pt
::::::::::::::::::Na ......... ( m t ::' q /-L ): : . :.:.:.: .:. . . . .:.:.:-: .]. . ..
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Archivesof Oto-Rhino-Laryngology 9 Springer-Vcrlag 1978
Chemical Composition in Various Compartments of Inner Ear Fluid* Kazuo Makimoto 1, Teiro Takeda 1, and Herbert Silverstein2 1 Department of Otolaryngology,School of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan, 2 Sarasota Memorial Hospital, 1901 Arlington Street, Sarasota, Florida 33579, USA
Summary. Sodium, potassium, chloride, glucose and total protein were determined in samples of scala vestibuli perilymph, scala tympani perilymph, CSF, cochlear endolymph and utricular endolymph from normal cats. Small but significant differences were evident in the concentrations of sodium and potassium between the scala vestibuli and scala tympani perilymph. It was also apparent that each compartment of endolymph has different values for sodium and potassium concentrations. Compared with the endolymph, the perilymph was found to contain a higher concentration of glucose and total protein. These findings are discussed from the view point of biological significance. Key words: Inner ear fluid biochemistry - Micro-chemical analysis - Osmotic pressure in inner ear fluids. There is a paucity in knowledge concerning chemical composition in inner ear fluids. In experiment series on inner ear fluid chemistry (Silverstein and Makimoto, 1973; Makimoto and Silverstein, 1974), we analysed sodium, potassium, chloride, glucose and total protein in samples of scala vestibuli perilymph, scala tympani perilymph, cochlear endolymph, utricular endolymph and CSF. 125 ears in 95 cats served as the materials. The present report is a summarization of the results heretofore obtained. Sodium and Potassium
Determination of sodium and potassium was done using a microflame photometer (Makimoto and Silverstein, 1973). The amount used in this analysis was 1.0 ~1 for perilymph, CSF and serum, and 0.5 p~l for endolymph. * Read before the 13th Workshop on Inner Ear Biologyin Dfisseldorfon August 23, 1976 This work was supported by Research Grant No. ROI-NS 1026801 from the National Institute of Neurological Diseases and Strokes, USA Offprint requests to: Kazuo Makimoto, M.D. (address see alcove)
0302-9530/78/0220/0259/$ 01.20
260
K. Makimoto et al.
These results are shown in Table 1. As demonstrated here, small but significant differences were evident between scala vestibuli and scala tympani perilymph both in sodium and potassium concentrations. It was also apparent that each compartment of endolymph has its own value for sodium and potassium concentrations. In the perilymph of scala vestibuli, a higher concentration.of potassium was noted. As to endolymph, a greater value was observed in the sodium concentration of utricular endolymph. This m a y be regarded as one of the biological features for utricular endolymph. In Figure 1, data on the concentrations of sodium and potassium in fluids o f extracellular type and serum are graphically illustrated. Regarding sodium concentration, perilymph of the scala vestibuli was found to be closest to serum. On the other hand, in potassium concentration, perilymph of the scala tympani showed the closest value to that of serum. When a comparison o f N a / K ratio between scala vestibuli perilymph and serum was made, it was obvious that a greater difference existed in this ratio. Accordingly, the perilymph of scala vestibuli m a y not be a simple blood filtrate.
Chloride Chloride was determined by the use of a Chloridemeter | With this instrument, the mEq/1 of chloride in a sample can b e measured by passing a current through the sample via two generating electrodes. The principle of this measurement is that silver ions are released from the anode and combine with chloride ions in the sample. The smallest amount required for this determination is 2.0 ~1. Table 1. Sodium and potassium concentrations in inner ear fluids, CSF, and serum
Sodium (mEq/1)
Potassium Difference
(mEq/1)
Perilymph of scala vestibuli (Pv) Perilymph of ~ scala tympani (Pt) Cochlear endolymph (Ee) Utricular endolymph (Eu)
147.76 (+ 0 . 9 4 ) ~ (N = 54) ~ . > 9.35' 157.11'(+ 1.06) j ~ ( P t - - P v ) (N = 5 8 ) ~ 6.07 (+ 0.40) ~ (N=41) " ~ 8 . 7 9 b 14.86 (+ 0.75) ~ (Eu-Ec) (N = 44)
CSF
152.91 (+ 1.00) (N = 32)
2.97 (+ 0.08) (N = 32)
Serum
140.94 (_+ 1.10) (N = 27)
3.93 (+ 0.16) (N = 27)
Difference
10.47 (+ 0.31) . (N = 54) ~ 6.68c 3.79 (+ 0.11) / / ~ (Pv--Pt) (N = 58) : 171.13 (+ 1.64) . (N = 41) ~ 1 5 . 7 0 a 155.43(+ 1.62) j (Ec--Eu) (N = 44) /
a t = 6.55 (significant, P < 0.001) b t = 10.15 (significant, P < 0.001) c t = 21.28 (significant, P < 0.001) a t = 6.81 (significant, P < 0.001) Each data is expressed as a mean (_+ standard'error); N = represents number of samples
Chemical Composition of Inner Ear Fluid
261 (rnEq/L) 160
150
140
130
6
d
1
I Sodium Concentration ~
Potassium Concentration
Fig. 1. Distribution pattern of sodium and potassium
in scala vestibuli perilymph, scala tympani perilymph, CSF and serum
Pt : Perilymph of Scala Tympani Pv : Perilymph of Scala Vestibuli
Table 2. Chloride concentrations in inner ear fluids, CSF, and Serum Chlorine (mEq/1) Perilymph of scala vestibuli (Pv)
138.51 (+_ 3.58)
Perilymph of scala tympani (Pt)
(N = 43) ~ 130.00 (_+ 2.31) (N = 46)
Cochlear endolymph (Ec)
169.82 (+ 5.83)
Utricular endolymph (Eu)
(N = 28) ~ 158.88 (+ 6.22) ( N = 24)
CSF
138.97 (_+ 1.97) (N = 39)
Serum
120.00 (+ 3.59) (N = 30)
Difference
8.51 a (Pv--Pt)
10.94b (Ec--Eu)
a t = 2.026 (significant, 0.02 < P < 0.05) b t = 1.283 (not significant, 0.2 < P < 0.3) Each data is expressed as a mean (_+ standard error); N =.represents number of samples
262
K. Makimoto et al.
As shown in Table 2, the concentrations of scala vestibuli perilymph, scala tympani perilymph and CSF showed values in a range from 130--140 mEq/1. Compared to these values, relatively high levels of chloride concentration were noted in both cochlear and utricular endolymph.
Glucose Glucose determination was carried out by adapting the enzymatic test kit of Boehringer to a micro-chemical technique (Silverstein, 1973; Makimoto and Silverstein, 1974). The principle of this enzymatic method is as follows: 1. Hexokinase catalyses the phosphorylation of glucose by ATP. Glucose + ATP -~ glucose-6-phosphate + ADP. 2. D-glucose-6-phosphate (G-6-P) is oxidized to 6-phosphogluconate (6-P-G) in the presence of TPN (NADP) by the enzyme G-6-P dehydrogenase. G-6-P + TPN -~ 6-PrG + T P N H + H +. The amount of T P N H arising in Equation (2) is equivalent to the amount of G-6-P or glucose, respectively. The amount of the samples used in this measurement was 0.5 ~1 for endolymph and 1.0 pxl for other fluids. The results are presented in Table 3. The glucose concentration of cochlear endolymph was appreciably low, being 11.2 mg/100 ml, whereas that of utricular endolymph was higher, being 41.9 mg/100 ml. As previously mentioned, utricular endolymph was found to contain a higher concentration of sodium as compared to Table 3. Glucose concentrations in inner ear fluids, CSF, and Serum
Glucose (rag/100 ml) Cochlear endolymph (Ec) Utricular endolymph (Eu) Perilymph of scala Tympani (P0 Perilymph of scala vestibuli (Pv) CSF Serum
Difference
11.17 (_+ 1.29) (N = 28) (+ 2,23) ~ 41.86 (N~ 30)/" 82.77 (+ 3.73) (N = 66) 79.65 (+ 3.46) (N = 59)
30.69a (Eu--Ec)
/
3.12b (Pt--Pv)
81.95 (+ 3.37) (N = 49) 122.48 (+ 7.12) (N = 45)
t = 11.71 (significant, P < 0.01) b t = 0.61 (not significant, 0.5 < P < 0.6) Each data is expressed as a mean (_+ standard error); N = represents number of samples
263
Chemical Composition of Inner Ear Fluid
cochlear endolymph. Such evidence indicates the difference in chemical composition between cochlear and utricular endolymph and may suggest that both compartments of endolymph are not freely communicable, and that each possesses its own independent nature in formation, absorption and circulation of endolymph. Perilymph of scala vestibuli, perilymph of scala tympani and CSF were almost identical in glucose concentration, i.e., approximately 80 mg/100 ml. This level is regarded as being about 40 mg/100 ml lower than that of serum.
Total Protein Total protein was determined by employing the Folin phenol reaction of Lowry et al. (1951). As the first step of this method, the sample is treated with copper sulfate in alkaline solution. Folin reagent is then added to the sample to develop the color reaction. According to the results shown in Table 4, the total protein contents of scala vestibuli perilymph and scala tympani perilymph exceeded those of cochlear and utricular endolymph. Also in CSF, the content of total protein was noted to be much lower than those in scala vestibuli perilymph and scala tympani perilymph. This pattern of concentration gradient in total protein did not coincide with that in glucose. The glucose study revealed that the glucose concentration of CSF was almost the same level as those of scala vestibuli perilymph and scala tympani perilymph. In Figure 2, the total amount of electrolytes (sodium plus potassium plus chloride) are illustrated on the left side of this figure. When this value is compared among various compartments of inner ear fluid, the values for endolymph are greater than those for perilymph.
Table 4. Amount of total Protein in inner ear fluids and CSF Total protein (rag-%) Perilymph of scala vestibuli (Pv) Perilymph of scala tympani (Pt) Cochlear endolymph (Ec) Utrieular endolymph (Eu) CSF
233.87 (+ 1 5 . 7 0 ) ~ (N = 23) ~ 212.52 (+ 1 0 . 6 6 ) ~ (N = 31) 61.50 (+ 7.49) (N = 18) 64.33 (+ 7.70) (N = 9) 37.89 (+ 6.24) (N = 9)
Difference a 21 35 (Pv--Pt) "
2.83b (Eu-Ec)
a t = 1.143 (not significant, 0.2 < P < 0.3) t = 0.229 (not significant, 0.8 < P < 0.9) Each data is expressed as a mean (_+ standard error); N = represents number of samples
264
K. Makimoto et al. I0 [ p
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I mE;q/L
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