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Brain amino acids decrease in chronic hyponatremia and rapid correction causes brain dehydration: Possible clinical significance
Life Sciences, Vol. 40, pp. 2539-2542 Printed in the U.S.A.
Pergamon Journal~
BRAIN AMINO ACIDS DECREASE IN CHRONIC HYPONATREMIA AND RAPID CORRECTION CAUSES BRAIN DEHYDRATION: POSSIBLE CLINICAL SIGNIFICANCE. Jean Holowach Thurston and Richard E. Hauhart Edward Mallinckrodt Department of Pediatrics, Washington University School of Medicine, Children's Hospital, St. Louis, MO 63110 (Received in final form April 16, 1987) Summary In animals, rapid correction of chronic hyponatremia produces brain lesions similar to those seen in central pontine myelinolysis. This is the first study of the effects of rapid correction (9 h) of chronic hyponatremia (3 d) on brain electrolyte, water, and amino acid contents in young mice. Despite profound hyponatremla, decreases in brain electrolytes and amino acids permitted an apparent osmotic balance between blood and brain with a normal brain water content. Rapid elevation of the depressed plasma sodium concentration to normonatremlc levels caused dehydration of the brain. Although brain Na + and K+ levels were returned to normal, the relatively brief interval of treatment was insufficient to allow complete recovery of brain amino acid levels. Findings support an osmotic disequilibrium - plasma osmolality higher than brain - in the pathogenesis of the brain lesions following rapid correction of chronic hyponatremia and suggest cautlon in the rate of elevatlon of the depressed plasma Na levels. •
.
.
+
The pathologic entity - central pontine myelinolysis - was first described by Adams, Victor and Mancall in 1959 (I). Reviewing their own cases and those reported in the literature, in 1977 Burcar, Norenberg and Yarnell (2) described a circumstantial relationship between hyponatremia and the development of central pontine myelinolysis. Subsequent research revealed that it was not the hyponatremia per se, but rather the rapid elevation of the depressed serum sodium level that may precipitate oligodendroglial necrosis and destruction of myelin (3-8). The mechanism of this action is unknown. To shed light on this unsolved problem, we studied the effects of chronic hyponatremia and its rapid correction on plasma and brain electrolytes and the brain water content in young mice. Since we have previously shown that brain amino acid levels fall in adaptation to acute hyponatremla (9), the effect of chronic hyponatremia on brain amino acids was also examined. The experimental protocol (below) was similar to those used in the neuropathologic studies of chronic hyponatremia and rapid correction in fasted and thirsted rats, dogs and rabbits (3-8). Materials and Methods Chronic hyponatremia was produced by three daily injections of vasopressin (5 U per kilogram subcutaneously) and 2.5% dextrose in water (50 milliliters per kilogram intraperitoneally) in 19 to 29-d-old fasted and thirsted Swiss Webster mice. During the 4-d experimental interval, fasted and 0024-32Q5/87 $3.Q0 + .QO Copyright (c) 1987 Pergamon Journals Ltd.
2540
Brain Amino Acids in Chronic Hyponatremia
Vol. 40, No. 26, 1987
thirsted control littermates received an equivalent number of injections and volumes of 0.9% NaCI. On the morning of the fourth day, some of the hyponatremic mice and the controls were killed. In others, the hyponatremia was corrected by intraperitoneal injection of molar NaCI (20 milliliters per kilogram at 8:00 a.m.) and two subcutaneous injections of 0.9% NaCI (30 milliliters per kilogram each dose, one at 11:00 a.m., the other at 3:00 p.m.). These animals were decapitated after nine h of therapy. After decapitation, blood was collected from the severed neck vessels in heparinized capillary tubes. The tubes were centrifuged immediately at 4°C and extracts of the plasma were prepared in perchloric acid (10). For water and electrolyte measurements, brain was dissected at room temperature. Methods for determination of brain water and brain and plasma electrolyte concentrations are described in Ref. 9. In other animals, the head was allowed to drop directly into liquid nitrogen with rapid stirring. Frozen brains were dissected free of blood and meninges in a cryostat at -35°C. Tissue extracts were prepared by the method of Lowry and Passonneau (10). Taurine was measured by the fluorescamine method of Orr et al. (11). Alanine, glutamate, and aspartate were measured by the methods of Lowry and Passonneau (10), glutamine by the method of Young and Lowry (12), and Y-aminobutyrate by a minor modification of the method of Hirsch and Robins (13). The method of glycine assay was that of Berger et al. (14). Data were analyzed by a one-way analysis of variance and Tukey's multiple comparison of the means (15). Results and Discussion Despite the profound, sustained hyponatremia, the brain water content was not increased (Table I)° Two mechanisms contributed prominently to this unexpected finding. It is well known that a decrease of the brain K + content is an important protective mechanism to limit the entry of water into the brain in hyponatremic states (16-23). Three d of chronic hyponatremia decreased the brain K + content 9 percent (Table I). Another factor contributing to a normal brain water content despite severe hyponatremia was the levels of brain amino acids. ~t is now well established that to prevent a loss of cell water and shrinkage of the brain in hypernatremic states, brain amino acids increase to maintain osmotic equilibrlum in amphibia and mammals (9,24-29). The present study reveals that a diametrically opposite effect is seen in hyponatremic states. Levels of each of the seven measured amino acids were greatly depressed (Table I). Five were decreased more than 50 percent, while taurine levels fell almost 70 percent. On a molar basis, the total decrease in the seven selected amino acids was greater than the drop in the brain K + content (wet weight). The normal water content in the brains of chronically hyponatremic mice is solid, albeit indirect, evidence for lack of a significant osmotic gradient between the brain and blood. In the chronically hyponatremic mice treated for nine h with saline, the plasma Na ÷ concentration was elevated some 35 mEq/l (3 to 4 mEq/1/h), to normonatremic levels (Table I). Although the brain Na + and K ÷ levels were also returned to normal, the brain water concentration decreased 1.7% on a wet weight basis and 6.3% on a dry weight basis - changes equivalent to a 6% shrinkage of the brain volume (30). With one exception (glutamine), treatment elevated the depressed brain amino acid levels (Table I). However, most of the values were still significantly lower than in control animals and the sum of the measured amino acids was only two-thirds of the control value. It is noteworthy that the brain aspartate content after treatment was one-third higher than in normonatremic controls.
Vol. 40, No. 26, 1987
Brain Amino Acids in Chronic Hyponatremia
2541
TABLE I Effect of Chronic Hyponatremia and Rapid Correction on Plasma and Brain Electrolyte and Brain Water and Amino Acid Contents
Measurement
Control (N=4 to 8)
Chronic Hyponatremia (N=6 to 10)
Rapid Correction (N=6 to 9)
Plasma Electrolytes Na +, mEq/1 K +, mEq/1
145 ± I 7.2 ± 0.2
104 ± 4a 6.6 ± 0.2
139 ± 3 6.6 ± 0.4
Brain Water and Electrolytes Water, g/100 g wet Water, g/100 g dry Na +, mEq/kg dry K +, mEq/kg dry
80.41 410.4 239 549
± ± ± _+
0.10 2.6 6 8
80.68 417.6 192 501
± ± ± -+
0.09 2.4 5a 6a
79.35 384.7 238 533
± 0.31 b'c ± 7.3 b'c ± 5c _+ 6 c
Brain Amino Acids Alanine Aspartate GABA Glutamate Glutamine Glycine Taurlne Total
All animals
0.442 + 0.022 2.46 --+ 0.09 2.22 T 0.07 9.00 ~ 0.29 8.63 ~ 0.17 1.42 ~ 0.08 12.7 T 0.6 36.9 ~ 0.6
0.212 + 0.017 a 1.07 --+ 0.08 a 1.44 T 0.07 a 5.08 ~ 0.30 a 3.87 ~ 0.20 a 0.634 ~ 0.048 4.18 ~ 0.59 a 16.1 T 0.7 a
0.358 + 0.021 b'c 3.36 --+ 0.21a'c 1.85 T 0.06 a,c 9.18 ~ 0.24 c 4.03 ~ 0.22 a 1.05 T 0.05 a,c 6.15 ~ 0.22 a'c 25.9 ~ 0.6 a'c
(including controls) were fasted and thirsted.
Amino acid levels are given as mmol/kg wet wt.; all values are mean ± SE. a-c p versus acontrol, <0.01; bcontrol, <0.05; Cchronic hyponatremia,
<0.01.
It is not known if a similar chain of water, electrolyte and biochemical events occurred in the brains of animals in which myelinolysis was observed after rapid correction to normonatremic levels (3-8). Similar biochemical changes in the brains of mice and other mammals in altered metabolic states, such as hypoxia-ischemia, hypoglycemia, anesthesia - to name a few - suggest that they do. Further, absence of lesions in the brains of chronically hyponatremic rabbits, rats and dogs (3-8) suggest that adaptive electrolyte and amino acid changes similar to those seen in mice had established a new osmotic balance between brain and blood in these species as well. Dehydration and shrinkage of mouse brain after rapid elevation of the chronically depressed plasma sodium concentration to normal levels support the suggestion of an osmotic disequilibrium between blood and brain (plasma osmolality higher than that in brain) in the possible pathogenesis of experimental central pontine myelinolysis (3-8). Whether the post-treatment elevation of the neuroexcitatory and neurotoxic brain amino acid, aspartic acid, to above normal, concomitant with below normal levels of the neuroinhibitory amino acids - GABA, glycine and taurine - plays a contributory role in the pathogenesis of brain lesions cannot be said. In any case, the
2542
Brain Amino Acids in Chronic Hyponatremia
Vol. 40, No. 26, 1987
findings support the recommendation of slow correction of chronically depressed plasma Na + levels in patients. Acknowledgements We thank David B. McDougal, Jr. for review of this manuscript and many helpful discussions, this work was supported in part by PHS grants NS 06163 and NS 15660, the Allen P. and Josephine B. Green Foundation, Mexico, MO, and Abbott Laboratories, Chicago, IL. References I. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
16. 17. 18.
R.D. ADAMS, M. VICTOR, and E.L. MANCALL, Arch. Neurol. Psychiatry 81 154-172 (1959). P.J. BURCAR, M.D. NORENBERG, and P.R. YARNELL, Neurology 27 223-226 (1977). B.K. KLEINSCHMIDT-DE MASTERS and M.D. NORENBERG, Science 211 1068-1070 (1981). B.K. KLEINSCHMIDT-DE MASTERS and M.D. NORENBERG, J. Neuropathol. Exp. Neurol. 41 67-80 (1982). M.D. NORENBERG, J. Neuropathol. Exp. Neurol. 40 319 (1981). R. LAURENO, Ann. Neurol. 1 3 232-242 (1983). B. ILLOWSKY and R. LAURENO, Kidney Int. 25 168 (1984). J.C. AYUS, R.K. KROTHAPALLI, and D.L. ARMSTRONG, Am. J. Physiol. 247 F711-F719 (1985). J.H. THURSTON, R.E. HAUHART, E.M. JONES and J.L. ATER, J. Neurochem. 24 953-957 (1975). O.H. LOWRY and J.V. PASSONNEAU, A Flexible System of Enzymatic Analysis (Academic Press, New York, 1972), pp. 120-128 and 146-186. H.T. ORR, A.I. COHEN and O.H. LOWRY, J. Neurochem. 26 609-611 (1976). R.L. YOUNG and O.H. LOWRY, J. Neurochem. 13 785-793 (1966). H.E. HIRSCH and E. ROBINS, J. Neurochem. 9 63-70 (1962). S,J, BERGER, J.A. CARTER and O.H. LOWRY, Anal. Biochem. 65 232-240 (1975). J.W. TUKEY, Ditto, Princeton University, Princeton, NJ (1953) cited by R.E. KIRK, Experimental Design: Procedures for the Behavioral Sciences ( Br co ks / Col e, B~a-on[~-,-CA]--~6-8[,--p-p.--6-9---9-8-................................. H. YANNETT, Am. J. Physiol. 128 683-689 (1940). D.M. WOODBURY, Am. J. Physiol. 185 281-286 (1956). P.R. DODGE, J.D. CRAWFORD and J.H. PROBST, Arch. Neurol. 3 513-529
(1960). 19.
20. 21. 22.
23. 24, 25. 26. 27. 28. 29.
30.
R. KATZMAN, Ann. Rev. Med. 17 197-212 (1966). M.A. HOLIDAY, M.N. KALAYCI and J. HARRAH, J. Clin. Invest. 47 1916-1928 ( 1 968). C.J. DILA and H.M. PAPPIUS, Arch. Neurol. 26 85-90 (1972). M.M, RYMER and R.A. FISHMAN, Arch. Neurol. 28 49-54 (1973). A.I. ARIEFF, F. LLACH and S.G. MASSRY, Medicine 55 121-129 (1976). J.H. THURSTON, R.E. HAUHART and J.A. DIRGO, Life Sol. 26 1561-1568 ( 198O ). J.H. THURSTON, R.E. HAUHART and D.W. SCHULZ, J. Neurochem. 40 240-245 (1983). A.H. LOCKWOOD, Arch. Neurol. 32 62-64 (1975). R.A. FISHMAN and P.H. CHAN, Trans. Am. Neurol. Assoc. 101 34-39 (1976). P.H. CHAN and R.A. FISHMAN, Brain Res. 161 293-301 (1979). A.I. ARIEFF, R. GUISADO and V.C. LAZAROWITZ, in Disturbances in Body Fluid Osmolality, T.E. ANDREOLI, J.J. GRANTHAM, F.C. RECTOR, Jr., Eds. (American Physiological Society, Bethesda, 1977), pp. 227-250. K.A.C. ELLIOTT and H. JASPER, Am. J. Physiol. 157 122-129 (1949).
Pergamon Journal~
BRAIN AMINO ACIDS DECREASE IN CHRONIC HYPONATREMIA AND RAPID CORRECTION CAUSES BRAIN DEHYDRATION: POSSIBLE CLINICAL SIGNIFICANCE. Jean Holowach Thurston and Richard E. Hauhart Edward Mallinckrodt Department of Pediatrics, Washington University School of Medicine, Children's Hospital, St. Louis, MO 63110 (Received in final form April 16, 1987) Summary In animals, rapid correction of chronic hyponatremia produces brain lesions similar to those seen in central pontine myelinolysis. This is the first study of the effects of rapid correction (9 h) of chronic hyponatremia (3 d) on brain electrolyte, water, and amino acid contents in young mice. Despite profound hyponatremla, decreases in brain electrolytes and amino acids permitted an apparent osmotic balance between blood and brain with a normal brain water content. Rapid elevation of the depressed plasma sodium concentration to normonatremlc levels caused dehydration of the brain. Although brain Na + and K+ levels were returned to normal, the relatively brief interval of treatment was insufficient to allow complete recovery of brain amino acid levels. Findings support an osmotic disequilibrium - plasma osmolality higher than brain - in the pathogenesis of the brain lesions following rapid correction of chronic hyponatremia and suggest cautlon in the rate of elevatlon of the depressed plasma Na levels. •
.
.
+
The pathologic entity - central pontine myelinolysis - was first described by Adams, Victor and Mancall in 1959 (I). Reviewing their own cases and those reported in the literature, in 1977 Burcar, Norenberg and Yarnell (2) described a circumstantial relationship between hyponatremia and the development of central pontine myelinolysis. Subsequent research revealed that it was not the hyponatremia per se, but rather the rapid elevation of the depressed serum sodium level that may precipitate oligodendroglial necrosis and destruction of myelin (3-8). The mechanism of this action is unknown. To shed light on this unsolved problem, we studied the effects of chronic hyponatremia and its rapid correction on plasma and brain electrolytes and the brain water content in young mice. Since we have previously shown that brain amino acid levels fall in adaptation to acute hyponatremla (9), the effect of chronic hyponatremia on brain amino acids was also examined. The experimental protocol (below) was similar to those used in the neuropathologic studies of chronic hyponatremia and rapid correction in fasted and thirsted rats, dogs and rabbits (3-8). Materials and Methods Chronic hyponatremia was produced by three daily injections of vasopressin (5 U per kilogram subcutaneously) and 2.5% dextrose in water (50 milliliters per kilogram intraperitoneally) in 19 to 29-d-old fasted and thirsted Swiss Webster mice. During the 4-d experimental interval, fasted and 0024-32Q5/87 $3.Q0 + .QO Copyright (c) 1987 Pergamon Journals Ltd.
2540
Brain Amino Acids in Chronic Hyponatremia
Vol. 40, No. 26, 1987
thirsted control littermates received an equivalent number of injections and volumes of 0.9% NaCI. On the morning of the fourth day, some of the hyponatremic mice and the controls were killed. In others, the hyponatremia was corrected by intraperitoneal injection of molar NaCI (20 milliliters per kilogram at 8:00 a.m.) and two subcutaneous injections of 0.9% NaCI (30 milliliters per kilogram each dose, one at 11:00 a.m., the other at 3:00 p.m.). These animals were decapitated after nine h of therapy. After decapitation, blood was collected from the severed neck vessels in heparinized capillary tubes. The tubes were centrifuged immediately at 4°C and extracts of the plasma were prepared in perchloric acid (10). For water and electrolyte measurements, brain was dissected at room temperature. Methods for determination of brain water and brain and plasma electrolyte concentrations are described in Ref. 9. In other animals, the head was allowed to drop directly into liquid nitrogen with rapid stirring. Frozen brains were dissected free of blood and meninges in a cryostat at -35°C. Tissue extracts were prepared by the method of Lowry and Passonneau (10). Taurine was measured by the fluorescamine method of Orr et al. (11). Alanine, glutamate, and aspartate were measured by the methods of Lowry and Passonneau (10), glutamine by the method of Young and Lowry (12), and Y-aminobutyrate by a minor modification of the method of Hirsch and Robins (13). The method of glycine assay was that of Berger et al. (14). Data were analyzed by a one-way analysis of variance and Tukey's multiple comparison of the means (15). Results and Discussion Despite the profound, sustained hyponatremia, the brain water content was not increased (Table I)° Two mechanisms contributed prominently to this unexpected finding. It is well known that a decrease of the brain K + content is an important protective mechanism to limit the entry of water into the brain in hyponatremic states (16-23). Three d of chronic hyponatremia decreased the brain K + content 9 percent (Table I). Another factor contributing to a normal brain water content despite severe hyponatremia was the levels of brain amino acids. ~t is now well established that to prevent a loss of cell water and shrinkage of the brain in hypernatremic states, brain amino acids increase to maintain osmotic equilibrlum in amphibia and mammals (9,24-29). The present study reveals that a diametrically opposite effect is seen in hyponatremic states. Levels of each of the seven measured amino acids were greatly depressed (Table I). Five were decreased more than 50 percent, while taurine levels fell almost 70 percent. On a molar basis, the total decrease in the seven selected amino acids was greater than the drop in the brain K + content (wet weight). The normal water content in the brains of chronically hyponatremic mice is solid, albeit indirect, evidence for lack of a significant osmotic gradient between the brain and blood. In the chronically hyponatremic mice treated for nine h with saline, the plasma Na ÷ concentration was elevated some 35 mEq/l (3 to 4 mEq/1/h), to normonatremic levels (Table I). Although the brain Na + and K ÷ levels were also returned to normal, the brain water concentration decreased 1.7% on a wet weight basis and 6.3% on a dry weight basis - changes equivalent to a 6% shrinkage of the brain volume (30). With one exception (glutamine), treatment elevated the depressed brain amino acid levels (Table I). However, most of the values were still significantly lower than in control animals and the sum of the measured amino acids was only two-thirds of the control value. It is noteworthy that the brain aspartate content after treatment was one-third higher than in normonatremic controls.
Vol. 40, No. 26, 1987
Brain Amino Acids in Chronic Hyponatremia
2541
TABLE I Effect of Chronic Hyponatremia and Rapid Correction on Plasma and Brain Electrolyte and Brain Water and Amino Acid Contents
Measurement
Control (N=4 to 8)
Chronic Hyponatremia (N=6 to 10)
Rapid Correction (N=6 to 9)
Plasma Electrolytes Na +, mEq/1 K +, mEq/1
145 ± I 7.2 ± 0.2
104 ± 4a 6.6 ± 0.2
139 ± 3 6.6 ± 0.4
Brain Water and Electrolytes Water, g/100 g wet Water, g/100 g dry Na +, mEq/kg dry K +, mEq/kg dry
80.41 410.4 239 549
± ± ± _+
0.10 2.6 6 8
80.68 417.6 192 501
± ± ± -+
0.09 2.4 5a 6a
79.35 384.7 238 533
± 0.31 b'c ± 7.3 b'c ± 5c _+ 6 c
Brain Amino Acids Alanine Aspartate GABA Glutamate Glutamine Glycine Taurlne Total
All animals
0.442 + 0.022 2.46 --+ 0.09 2.22 T 0.07 9.00 ~ 0.29 8.63 ~ 0.17 1.42 ~ 0.08 12.7 T 0.6 36.9 ~ 0.6
0.212 + 0.017 a 1.07 --+ 0.08 a 1.44 T 0.07 a 5.08 ~ 0.30 a 3.87 ~ 0.20 a 0.634 ~ 0.048 4.18 ~ 0.59 a 16.1 T 0.7 a
0.358 + 0.021 b'c 3.36 --+ 0.21a'c 1.85 T 0.06 a,c 9.18 ~ 0.24 c 4.03 ~ 0.22 a 1.05 T 0.05 a,c 6.15 ~ 0.22 a'c 25.9 ~ 0.6 a'c
(including controls) were fasted and thirsted.
Amino acid levels are given as mmol/kg wet wt.; all values are mean ± SE. a-c p versus acontrol, <0.01; bcontrol, <0.05; Cchronic hyponatremia,
<0.01.
It is not known if a similar chain of water, electrolyte and biochemical events occurred in the brains of animals in which myelinolysis was observed after rapid correction to normonatremic levels (3-8). Similar biochemical changes in the brains of mice and other mammals in altered metabolic states, such as hypoxia-ischemia, hypoglycemia, anesthesia - to name a few - suggest that they do. Further, absence of lesions in the brains of chronically hyponatremic rabbits, rats and dogs (3-8) suggest that adaptive electrolyte and amino acid changes similar to those seen in mice had established a new osmotic balance between brain and blood in these species as well. Dehydration and shrinkage of mouse brain after rapid elevation of the chronically depressed plasma sodium concentration to normal levels support the suggestion of an osmotic disequilibrium between blood and brain (plasma osmolality higher than that in brain) in the possible pathogenesis of experimental central pontine myelinolysis (3-8). Whether the post-treatment elevation of the neuroexcitatory and neurotoxic brain amino acid, aspartic acid, to above normal, concomitant with below normal levels of the neuroinhibitory amino acids - GABA, glycine and taurine - plays a contributory role in the pathogenesis of brain lesions cannot be said. In any case, the
2542
Brain Amino Acids in Chronic Hyponatremia
Vol. 40, No. 26, 1987
findings support the recommendation of slow correction of chronically depressed plasma Na + levels in patients. Acknowledgements We thank David B. McDougal, Jr. for review of this manuscript and many helpful discussions, this work was supported in part by PHS grants NS 06163 and NS 15660, the Allen P. and Josephine B. Green Foundation, Mexico, MO, and Abbott Laboratories, Chicago, IL. References I. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
16. 17. 18.
R.D. ADAMS, M. VICTOR, and E.L. MANCALL, Arch. Neurol. Psychiatry 81 154-172 (1959). P.J. BURCAR, M.D. NORENBERG, and P.R. YARNELL, Neurology 27 223-226 (1977). B.K. KLEINSCHMIDT-DE MASTERS and M.D. NORENBERG, Science 211 1068-1070 (1981). B.K. KLEINSCHMIDT-DE MASTERS and M.D. NORENBERG, J. Neuropathol. Exp. Neurol. 41 67-80 (1982). M.D. NORENBERG, J. Neuropathol. Exp. Neurol. 40 319 (1981). R. LAURENO, Ann. Neurol. 1 3 232-242 (1983). B. ILLOWSKY and R. LAURENO, Kidney Int. 25 168 (1984). J.C. AYUS, R.K. KROTHAPALLI, and D.L. ARMSTRONG, Am. J. Physiol. 247 F711-F719 (1985). J.H. THURSTON, R.E. HAUHART, E.M. JONES and J.L. ATER, J. Neurochem. 24 953-957 (1975). O.H. LOWRY and J.V. PASSONNEAU, A Flexible System of Enzymatic Analysis (Academic Press, New York, 1972), pp. 120-128 and 146-186. H.T. ORR, A.I. COHEN and O.H. LOWRY, J. Neurochem. 26 609-611 (1976). R.L. YOUNG and O.H. LOWRY, J. Neurochem. 13 785-793 (1966). H.E. HIRSCH and E. ROBINS, J. Neurochem. 9 63-70 (1962). S,J, BERGER, J.A. CARTER and O.H. LOWRY, Anal. Biochem. 65 232-240 (1975). J.W. TUKEY, Ditto, Princeton University, Princeton, NJ (1953) cited by R.E. KIRK, Experimental Design: Procedures for the Behavioral Sciences ( Br co ks / Col e, B~a-on[~-,-CA]--~6-8[,--p-p.--6-9---9-8-................................. H. YANNETT, Am. J. Physiol. 128 683-689 (1940). D.M. WOODBURY, Am. J. Physiol. 185 281-286 (1956). P.R. DODGE, J.D. CRAWFORD and J.H. PROBST, Arch. Neurol. 3 513-529
(1960). 19.
20. 21. 22.
23. 24, 25. 26. 27. 28. 29.
30.
R. KATZMAN, Ann. Rev. Med. 17 197-212 (1966). M.A. HOLIDAY, M.N. KALAYCI and J. HARRAH, J. Clin. Invest. 47 1916-1928 ( 1 968). C.J. DILA and H.M. PAPPIUS, Arch. Neurol. 26 85-90 (1972). M.M, RYMER and R.A. FISHMAN, Arch. Neurol. 28 49-54 (1973). A.I. ARIEFF, F. LLACH and S.G. MASSRY, Medicine 55 121-129 (1976). J.H. THURSTON, R.E. HAUHART and J.A. DIRGO, Life Sol. 26 1561-1568 ( 198O ). J.H. THURSTON, R.E. HAUHART and D.W. SCHULZ, J. Neurochem. 40 240-245 (1983). A.H. LOCKWOOD, Arch. Neurol. 32 62-64 (1975). R.A. FISHMAN and P.H. CHAN, Trans. Am. Neurol. Assoc. 101 34-39 (1976). P.H. CHAN and R.A. FISHMAN, Brain Res. 161 293-301 (1979). A.I. ARIEFF, R. GUISADO and V.C. LAZAROWITZ, in Disturbances in Body Fluid Osmolality, T.E. ANDREOLI, J.J. GRANTHAM, F.C. RECTOR, Jr., Eds. (American Physiological Society, Bethesda, 1977), pp. 227-250. K.A.C. ELLIOTT and H. JASPER, Am. J. Physiol. 157 122-129 (1949).