Treatment of osmotic disturbance with organic osmolytes

A method of treating an osmotic disturbance in an animal which comprises administering to an animal an effective amount of an organic osmolyte, wherein the organic osmolyte is a polyol. Specific polyols include myo-inositol and sorbitol. Also included are precursors of organic osmolytes including precursors of polyols. Other polyol precursors are selected from the group consisting of glucose, glucose polymers, and glycerol. Also inclu This invention was funded by a research grant from the National Institutes of Health, 1RO1 DK36031, which provides to the United States Government certain rights in the invention.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The invention relates to the use of organic osmolytes in oral or parenteral 
fluids to treat osmotic disturbances such as those substantially 
associated with acute and chronic hypernatremia and hyponatremia. 
2. Description of the Background Art 
A. Organic Osmolytes 
Study of osmoregulatory behavior in marine animals, plants, and bacteria 
demonstrated that organic solutes known as osmolytes accumulate 
intracellularly when the extracellular or environmental osmolality is 
significantly increased (Blundin, G. et al., Bot. Mar. 25:563-567 (1982); 
Pierce, S. K., Biol. Bull. Woods Hole 163:405-419 (1982); Somero, G. N., 
Am. J. Physiol. 251:R197-R213 (1986); Yancey, P. H. et al., Science 
217:1214-1222 (1982)). High levels of inorganic salts and urea are toxic 
to numerous enzymatic and other cellular processes. Certain organic 
solutes such as trimethylamines counteract the toxic effects of urea, 
enabling cells to tolerate high concentrations of urea (Somero, G. N., 
supra: Yancey, P. H. et al., supra). 
In the renal medulla, where osmolality and urea concentration can be very 
high during antidiuresis, the relative significance of the individual 
osmolytes has not been clearly delineated. During antidiuresis an 
intracellular osmotic gap exists in the renal inner medulla (IM) such that 
the concentration of electrolytes plus urea is lower in the cell than in 
the surrounding extracellular fluid (ECF) (Beck, F. et al., Kidney Int. 
25:397-403 (1984); Bulger, R. E., Kidney Int. 31:556-561 (1987)). The 
magnitude of this gap is controversial, ranging from 100 to 650 mosmol/kg 
H.sub.2 O. Several investigations have identified organic solutes 
(osmolytes) as significant constituents of this gap. Over 30 years ago 
Ullrich (Ullrich, K. J., Pfluegers Arch. 262:551-561 (1956)) observed a 
large pool of glycerophosphorylcholine (GPC) in the dog IM, and others 
later discovered high myo-inositol levels in the dog IM (Cohen, M. A. H. 
et al., Proc. Soc. Exp. Bio. Med. 169.380-385 (1982)). More recently, with 
the use of nuclear magnetic resonance (NMR) spectroscopy, gas 
chromatography, and enzymatic analysis, trimethylamines and polyols were 
identified in the rabbit and rat renal IM (Bagnasco, S. et al., J. Biol. 
Chem. 261:5872-5877 (1986); Balaban, R. S, et al., Kidney Int. 31:562-564 
(1987); Balaban, R. S. et al., Am. J. Physiol. 245:C439-C444 (1983); 
Corder, C. N. et al., J. Histochem. Cytochem. 25:1-8 (1977); Yancey, P. H. 
et al., supra). In particular, significant levels of GPC and glycine 
betaine (betaine) as well as myo-inositol and sorbitol were observed. 
Trimethylamines have also been implicated in human renal function since 
both glycine betaine and proline betaine were identified in urine 
(Chambers, S. T. et al., J. Clin. Invest. 79:731-737 human urine 
(Chambers, S. T. et al., J. Clin. Invest. 79:731-737 (1987)). Finally, 
several reports suggest that significant levels of amino acids are 
accumulated in the mammalian renal IM (Balaban, R. S. et al., 1983, supra: 
Law, R. O. et al., J. Physiol. Lond. 386:45-61 (1987); Robinson, R. R. et 
al., Comp. Biochem. Physiol. 19:187-195 (1966)). 
Bagnasco and co-workers (supra) compared diuretic with antidiuretic rabbits 
and found that a 105% increase in urine osmolality was accompanied by an 
increase in IM urea (73%) and betaine (101%) content; however, sorbitol 
and GPC were not significantly elevated. Cohen et al., (supra) showed that 
acute (2-3 hour) water diuresis produced a 36% decrease in IM myo-inositol 
content. Amino acids, in comparison, either did not change (Robinson, R. 
R. et al., supra) or changed by only 28% (Law, R. O. et al., supra ) 
during in vivo antidiuresis. This observation is in striking contrast to 
in vitro tissue slice studies which indicated that acute increases in ECF 
osmolality caused a dramatic (148%) and rapid (15-25 min) rise in cellular 
amino acids in IM tissue (Law, R. O. et al., supra). Interestingly, this 
increase was blocked by trimethylamine N-oxide, a prominent osmolyte in 
elasmobranchs. 
B. Clinical Disorders of Osmoregulation 
1. Hypoosmolar States 
Hypoosmolality or hyponatremia is the most common disorder of body fluid 
and electrolyte balance encountered in the clinical practice of medicine 
(Anderson, R. J. et al., Ann Intern. Med. 102:164-168 (1985)), with 
incidence ranging from 15-22% in both acutely (Flear, C. T. G. et al., 
Lancet 2:26-81 (1981) and chronically (Kleinfeld, M. et al., J. Am. 
Geriat. Soc. 27:156-161 (1979) hospitalized patients. Hyponatremia is a 
major cause of morbidity, though its contribution to mortality is not yet 
settled. 
In its severe form, hyponatremia is the most frequent cause of metabolic 
coma with or without seizures or other neurologic manifestations. In 
chronic hyponatremia, the patient or animal can be remarkably free of 
central nervous system (CNS) manifestations, with serum sodium 
concentrations between 110-115 mEq/liter. This observation is a sign of 
volume regulation in response to brain cell overhydration and interstitial 
fluid volume expansion. 
The causes of hyponatremia are well known. In all cases, the hypoosmolar 
state results from either an absolute or relative excess of water in the 
body. It can occur with or without edema, and with or without salt 
depletion or reduced ECF volume. In all cases, water intake is excessive 
relative to the kidney's ability to excrete it. The morbidity, clinical 
presentation, and mortality rate of patients with hyponatremia are related 
to the age of the patient (the oldest and youngest are most affected), the 
acuteness of the decline in serum sodium, the severity of the 
hyponatremia, and the concomitant presence of certain medical conditions. 
A. I. Arieff (N.Eng. J. Med. 314:1529-1534 (1986)) studied 15 women in whom 
hyponatremia occurred after elective surgery, leading to death in 4 and 
recovery of 8 in a persistent vegetative state. It was not certain whether 
hyponatremia itself was the direct cause of neurological damage. (See, 
also: Arieff, A. I. et al., Medicine 55:121-129 (1976); Clin Endocrinol. 
Metab. 13:269-294 (1984); Sterns, Ann Int. Med. 107:656-664 (1987). 
The cellular transport processes responsible for volume regulation during 
hypotonicity in the brain are poorly understood. Volume regulation has 
been demonstrated in many cell types in response to hypotonicity of the 
ECF, however, and ion loss is similar to that of brain. Such losses could 
include a marked increase in potassium conductance and/or K.sup.+ 
/Cl.sup.- cotransport, or an inhibition of the Na.sup.+ -K.sup.+ 
/2Cl.sup.- cotransporter in response to volume expansion. 
The correction of hyponatremia performed in too rapid a manner is thought 
to lead to neurological deficits due to brain myelinolysis in man, similar 
to the production of demyelinating lesions in animals by rapid correction 
of hyponatremia (Wright, D. G. et al., Brain 102:361-385 (1979); Sterns, 
R. H. et al., N. Eng. J. Med. 314:1535-1541 (1986); 
Kleinschmidt-Demasters, B. K. et al., Science 211:1068-1070 (1981); 
Laureno, R., Ann Neurol. 13:232-242 (1983); Ayus, J. C. et al., Am. J. 
Physiol. 248:F711-F719 (1985); and Illowsky, B. P. et al., Brain 
110:855-867 (1987). However, it is not yet settled whether, and under what 
circumstances, hyponatremia and subsequent changes in plasma osmolality 
cause cellular and tissue damage and death (Ayus et al., Am J. Med. 
78:897-902 (1985); Narins, R. G., N. Eng. J. Med. 314:1573-575 (1986)). 
Much is known about cellular adaptation to hypoosmolar conditions in vitro 
(Grantham, J. J., in Disturbances in Body Fluid Osmolality (Andreoli, T. 
E. et al., Eds.), Amer. Physiol. Soc., Bethesda, 1977, pp 217-225; 
Hoffmann, E. K., in Transport of Ions and Water in Animals. (Gupta, BL et 
al., Eds.), Academic Press, London, 1977, pp 285-332). However, much 
remains to be learned about such adaptive changes and their consequences 
for mammals in vivo (Grantham, J. J. et al., Circ. Res. 54:483-491 (1984). 
Useful animal models have been developed in which chronic severe 
hyponatremia in rats can be maintained for prolonged periods by 
subcutaneous (SC) infusions of the antidiuretic vasopressin analogue, 
1-deamino-(3-D-arg) vasopressin (DDAVP) (Verbalis J. G. et al., Kidney. 
Int. 34:351-360 (1988). This allows the animals to be maintained for 
prolonged periods without a need for excess fluid administration, and in 
the absence of tissue catabolism, morbidity and mortality. Analysis of 
brain water and electrolyte contents has revealed normalization of brain 
water content, demonstrating the ability of brain tissue in vivo to volume 
regulate completely in response to hyponatremia of sufficient duration. 
In a similar model involving treatment of rats with dextrose in water and 
vasopressin, Ayus, J. C. et al. (Am. J. Physiol. 257:F18-F22 (1989)) found 
that (1) spontaneous correction of severe symptomatic hyponatremia (serum 
sodium &lt;120 mEq/l) resulted in 68% mortality as compared with 15% 
mortality in rats with asymptomatic mild hyponatremia (serum sodium 
between 120 and 130 mEq/l); (2) rapid correction of severe hyponatremia by 
hypertonic saline at a rate of change of absolute serum levels of &lt;25 
mEq/l in the first 24 hr. improved survival to 100%; and (3) rapid 
correction of severe hyponatremia of &gt;25 mEq/l in the first 24 hr. 
resulted in histological brain damage and 88% mortality. It was concluded 
that correction of hyponatremia can be safe if the rate of change of serum 
sodium is kept between 14 and 25 mEq/l during the first 24 hr. 
Sterns, R. H. et al. (Kidney Int. 35:69-75 (1989) demonstrated that rats 
adapted quickly to hyponatremia and survived with extremely low plasma 
sodium levels for prolonged periods. Slow correction (0.3 mEq/l/hr) 
permitted 100% survival. Rapid correction was well tolerated when 
hyponatremia was of brief duration. However, in animals that had already 
adapted to the osmotic disturbance, more rapid correction by treating with 
IM NaCl, or by withdrawal of DDAVP, caused brain dehydration leading to 
demyelinating brain lesions and over 40% mortality. 
2. Hyperosmolar States 
Hypernatremia results from water loss in excess of isotonic proportions of 
sodium chloride or sodium bicarbonate, or from an increase of one of these 
salts without a proportionate gain in the amount of water. Thus, 
total-body water can be normal, reduced, or elevated, but in all cases 
water is lost (to varying degrees) from every cell in the body. Cellular 
dehydration, of course, is secondary to the movement of water along its 
osmotic gradient because the permeability of body cells to sodium from the 
basolateral or circulatory side of the cell is quite low. When the rise in 
extracellular osmolality is acute, the fractional loss of water from cells 
examined is quite uniform. Later, however, various cells or organs deviate 
from the water loss predicted by the assumption that the cells or organs 
behave as perfect osmometers. 
Arieff and colleagues infused hypertonic glucose rapidly into rabbits so as 
to elevate their plasma glucose to 60 mM (1100 mg/dl) in 1 hour (Arieff, 
A. I. et al., J. Clin. Invest. 52:571-583 (1973)). This level was then 
maintained for 4 to 6 hours. At 2 hours, the fractional losses of water 
from brain and skeletal muscle were equal, slightly more than 10%. By 4 
hours, however, brain water content had returned to normal, whereas that 
of muscle remained depressed. This remarkable recovery of the water 
content of the brain to normal despite sustained and marked 
hyperosmolality of the plasma is an example of complete "volume regulatory 
increase." Such regulation of cell volume is observed to varying degrees 
in many areas of the body, but nowhere is it more complete than in the 
brain. In the Arieff et al. study (supra), skeletal muscle cells at 4 
hours showed no volume regulatory increase. 
In contrast to systemic tissues, the unique characteristics of the 
blood-brain barrier (BBB) and blood-cerebrospinal fluid (CSF) barrier 
allow for maximal maintenance of brain volume despite the hyperosmolality 
of the blood. The "tight" epithelium-like properties of the capillary 
endothelium of the brain, together with the intimate relationship on both 
a hydrostatic and compositional basis between the brain ECF and the CSF 
(Cserr, H. F., Ann. N.Y. Acad. Sci. 529:9-20 (1988)), allow for both the 
intracellular and the ECF volume to minimize deviations from normal. The 
brain cells regain normal water content with an increased solute content 
after the acute hyperosmolar stress has caused the loss of ECF and 
cellular water, despite the sustained ECF hyperosmolality. 
All animals studies of acute hypernatremic states have demonstrated varying 
degrees of volume regulatory increase in the brain. The more acute and 
severe the hypernatremic or hyperosmolar state (induced by hypertonic 
saline, urea, or glucose), the more severe is the initial dehydration and 
contraction of the brain, and the resulting neurologic damage. If the 
initial hypernatremia and hyperosmolality are less severe, the majority of 
animals survive despite a hyperosmolar state sustained for many hours or 
even for days. After this period, the volume regulatory increase is 
maximal. The cells have gained sodium, chloride, and potassium as well as 
nonelectrolyte solutes (including certain amino acids), and brain volume 
has returned to normal (Katzman, R. et al., Brain Electrolytes and Fluid 
Metabolism. Baltimore, Williams & Wilkins (1973); Arieff, A. I., in Fluid, 
Electrolyte and Acid-Base Disorders. (Arieff, A. I. et al., Eds.), New 
York, Churchill-Livingstone, p. 969 (1987); Kleeman, C. R., Hosp. Pract., 
pp. 59-73 (May 1979); Arieff, A. I. et al., in Disturbances in Body Fluid 
Osmolality. (Andreoli, T. E. et al., Eds.), Bethesda, Am. Physiol. Soc., 
pp. 227- 250 (1977); Lockwood, A. H., Arch. Neurol 32:62-64 (1975); 
Holiday, M. A. et al., J. Clin. Invest. 47.1916-1928 (1968); Culpepper, R. 
M. et al., in Clinical Disorders of Membrane Transoort Processes, 
(Andreoli, T. E. et al., eds.), New York, Plenum, p. 173 (1986); Sotos, J. 
F. et al., Pediatrics 26:925-937 (1960)). 
Studies of "volume regulatory increase" and "volume regulatory decrease" in 
other cells and tissues suggest that dehydration of the cells in response 
to hyperosmolar stress leads to activation of the coupled Na.sup.+ 
/K.sup.+ /2Cl.sup.- co-transport system and/or the chloride/bicarbonate 
exchanger and the sodium/hydrogen antiporter in the plasma membrane 
(Sotos, J. F. et al., supra). The parallel activation leads to a gain in 
cellular sodium and chloride in exchange for hydrogen and bicarbonate 
respectively, and a probable decrease in potassium conductance out of the 
cell. The net effect is a cellular gain in sodium, potassium, and 
chloride, a rise in intracellular osmolality, and a return of cell volume 
toward normal. It is probable that these cellular mechanisms are 
responsible for part of the "volume regulatory increase" occurring in 
brain cells. However, in numerous experiments carried out by Arieff and 
associates, the gain in osmolality in brain cells after administration of 
hypertonic sodium chloride and hypertonic glucose could not be explained 
by the osmotic equivalent of the gained electrolytes (see Arieff, A. I., 
1987, supra). 
The term "idiogenic osmoles," was adopted to define the undetermined 
solutes (Arieff, A. I. et al., 1973, supra), and was first used with 
respect to systemic tissues (McDowell, M. E. et al., Am. J. Physiol. 
180:545-558 (1955)) or brain (Sotos, J. F. et al., supra). The relative 
contribution of electrolyte uptake or nonelectrolyte "idiogenic" osmole 
accumulation to the total increment in cell osmolality differs depending 
on the nature of the solute causing the hyperosmolar state (Culpepper, R. 
M. et al., supra). The acute achievement of osmotic equilibrium is almost 
solely due to cellular water loss. Subsequently, electrolyte gain and 
idiogenic osmole accumulation account for the solute gain and return of 
brain water to normal. About 50% to 60% of the osmoles responsible for 
volume regulation during chronic hypernatremia were found to comprise 
amino acids (Arieff, A. I. et al., 1977, supra: Lockwood, A. H., Arch. 
Neurol. 32:62-64 (1975)). During hyperglycemia, about 50% of the volume 
regulatory increase was found to consist of electrolytes, a small amount 
of glucose, and idiogenic osmoles (Arieff, A. I. et al., 1973, 1977, 
supra). The exact nature of all these osmoles was not determined, though 
they were not likely to be amino acids. The increment of idiogenic osmoles 
appeared to be the consequence of hyperglycemia and not hyperosmolality, 
because a comparable increase in osmolality with glycerol, sucrose, or 
mannitol did not generate idiogenic osmoles and did not cause a volume 
regulatory increase. The lack of volume regulation in response to 
glycerol, sucrose, and mannitol accounts for their usefulness in reducing 
brain volume in patients with cerebral edema (Culpepper, R. M. et al., 
supra). 
In contrast to the amino acids that accumulate during hypernatremic states, 
the "idiogenic" osmoles of hyperglycemia disappeared rapidly as the plasma 
glucose fell (Arieff, A. I. et al., 1973, 1977, supra). Thus, rapid 
reduction in plasma glucose in hyperglycemic hyperosmolar coma leads to 
rapid and progressive improvement in the comatose state whereas comparably 
rapid reduction in serum osmolality to normal in hypernatremic states can 
precipitate convulsions. 
More recently, it was shown that chronic hypernatremia resulted in 
generation of idiogenic osmoles which, in addition to electrolytes, 
accounted for cellular osmolality. In rats made hypernatremic by NaCl 
injection and water restriction, myo-inositol increased in brain and 
kidney and sorbitol increased in the kidney. Water content was unchanged. 
The authors concluded that polyols play a significant role in brain and 
kidney cellular osmoregulation (Lohr, J. W. et al., Life Sci. 43:271-276 
(1988). Nakanishi, T. et al. (Am. J. Physiol. 255:C181-C191 (1988)) 
surveyed several renal-derived cell lines for their ability to survive in 
vitro under high concentrations of NaCl and urea, and for the accumulation 
of organic osmolytes. The same osmolytes which are found in renal IM were 
accumulated by several of the cell lines growing in vitro. For example, 
cells of the MDCK cell line, which proliferated in hyperosmotic medium, 
contained higher levels of myo-inositol, GPC, and betaine than they did in 
iso-osmotic medium. MDCK cells accumulated myo-inositol in response to 
hyperosmolality via a high affinity myo-inositol transporter, the level of 
which increased in response to high salt (Nakanishi, T. et al. (Proc. 
Natl. Acad. Sci. USA 86:6002-6 (1989)). 
If the initial acute hypernatremia insult is severe, a subject can die 
without evidence of volume regulation. A partial explanation for this 
comes from the demonstration that the tight junctions of the endothelial 
cells that restrict intercellular diffusion of ions, proteins, and 
water-soluble nonelectrolytes, can be opened by osmotically induced 
shrinkage of the endothelial cells of the BBB (Rapaport, S. I. et al., 
Ann. N.Y. Acad. Sci. 481:250-267 (1986)). The more water-soluble the 
solute and the higher its reflection coefficient, the more it is able to 
"open" the BBB when in contact with the endothelium at hypertonic 
concentrations (for example, molar). This "opening" of the BBB would leave 
the brain tissue unprotected. 
Examination of the brains of humans and animals following severe acute 
hyperosmolar salt loads revealed normal or shrunken brains, severely 
engorged vessels, capillary rupture and petechial hemorrhages, and larger 
parenchymal and subarachnoid bleeding. This damage usually was associated 
with plasma osmolalities of 350 to 450 mosm/liter (Fineberg, L., 
Pediatrics 23:40-48 (1958); Fineberg, L. et al., Pediatrics 23:46-53 
(1959)). The mortality rate in patients with hyperosmolality of this 
severity can exceed 50% (Arieff, A. I. (1987), supra). In humans and 
animals, the more chronic the development of hypernatremia and the less 
extreme the hypertonicity (serum sodium less than 160 mEq/liter), the less 
symptomatic the subject and the lower the mortality rate. 
In the treatment of human hypernatremia, the rate of return of serum sodium 
to normal should be a function of the severity and the rapidity of its 
development. In a patient who developed hypernatremia over 4 hours, for 
example, it is probable that little volume regulation took place. Thus, 
the serum sodium probably could be returned to normal over, at most, a 12 
to 24 hour period without fear of adverse CNS reactions. On the other 
hand, if the duration of the hypernatremic state is unknown, especially 
when serum sodium is above 160 mEq/liter, it is suggested that correction 
should be extended over 2 to 4 days. Arieff (Arieff, A. I. (1987), supra) 
suggested that correction take place over at least 48 hours, or at a rate 
of decrease of serum osmolality of 2 mosm/kg/hour. 
The longer the duration of the hypernatremic state, the more likely that 
volume regulation will be complete; that is, the brain cell content of 
solutes is increased and brain volume is normal. Too rapid a correction of 
hypernatremia by free water administration at this time will result in 
overexpansion of the cell volume and possibly cerebral edema. Time is 
required for the volume regulatory effects on the brain to dissipate. Slow 
correction should be the password, e.g., a rate of reduction of serum 
osmolality and serum sodium of about 1 mosm/kg and 0.5 mEq/liter per hour, 
respectively. 
Current treatment of osmotic disturbances, such as those discussed above is 
confined to the infusion of salt solutions, such as sodium chloride, in an 
attempt to correct serum sodium levels or serum osmolality. The solutes 
present in these solutions have the disadvantage of adversely affecting 
cells. These salt solutions do not contain organic osmolytes, which are 
known to be non-perturbing and/or "counteracting" solutes, and which, in 
contrast to electrolytes such as NaCl, are generally considered non-toxic 
(Yancey, P. H. et al., supra). 
Parenteral nutrition solutions, on the other hand, have been designed to 
meet nutritional needs. Although these fluids do contain some osmolytes, 
especially amino acids, they are not formulated to treat osmotic 
disturbances. The concentrations of organic osmolytes in these solutions 
is not sufficient for their function in the correction of osmotic 
disturbances. Thus, the use of solutions containing organic osmolytes to 
treat osmotic disturbances is not known in the art. 
BRIEF SUMMARY OF THE INVENTION 
The novelty of this invention lies in the use of organic osmolyte compounds 
to treat osmotic disturbances. Currently, NaCl solution is used in such 
situations. In general, exogenous fluids given intravenously and dialysis 
fluids are associated with many untoward side effects. There have been few 
major advances in the improvement of such fluids in recent years. The 
inventors, in their investigation of the concentration of organic 
osmolytes in various tissues, especially kidney and brain, under hyper- or 
hypoosmotic conditions, have made the discovery that many of these 
osmolytes are useful alone, in combination, or as additives to existing 
solutions, in the treatment of osmotic disturbances. 
This invention is directed to a method to treat an osmotic disturbance in 
an animal comprising providing to the animal an effective concentration of 
an organic osmolyte compound. 
The invention is also directed to a method to treat an osmotic disturbance 
in an animal comprising providing to the animal an effective concentration 
of a precursor of an organic osmolyte compound. 
The organic osmolyte compounds useful in this invention include, but are 
not limited to, three major classes of compounds: polyols (polyhydric 
alcohols), methylamines, and amino acids. The polyols considered useful in 
the practice of this invention include, but are not limited to, 
myo-inositol, and sorbitol. The methylamines of the invention include, but 
are not limited to, choline, betaine, phosphorylcholine, 
glycerophosphorylcholine, creatine, and creatine phosphate. The amino 
acids of the invention include, but are not limited to, glycine, alanine, 
glutamine, glutamate, aspartate, proline and taurine. 
The osmolyte precursors of this invention include, but are not limited to, 
glucose, glycerol, choline, phosphatidylcholine, and inorganic phosphates, 
which are direct precursors of polyols and methylamines, and to proteins, 
peptides, and polyamino acids which are precursors of amino acid 
osmolytes. 
This invention comprises selected organic osmolytes or their precursors in 
combinations and concentrations calculated to provide cells with the 
appropriate milieu for intact osmoregulation. The osmolytes of this 
invention are added as supplements to fluids administered enterally or 
parenterally. Since these compounds have been shown by the inventors and 
by others to have intracellular osmoprotective effects, especially in 
kidneys and brain, their administration protects subjects from cellular 
dehydration which is especially important in the treatment of hyponatremia 
and acute hypernatremia. 
Osmolyte concentrations in the fluids of the invention are in the range of 
about 0.01 to 4000 mM when used to supplement saline or in other standard 
solutions. Preferably, the osmolyte concentration is between about 0.1 and 
1500 mM. A solution comprised entirely of one or more organic osmolytes 
may contain concentrations as high as 4 M. It is understood that the 
concentration of one or more osmolytes in a solution of the invention will 
vary depending upon the other constituents of the solution, and the 
particular purpose for which the solution is formulated. 
The invention is directed to the treatment of osmotic disturbances 
substantially associated with acute hyponatremia, chronic hyponatremia, 
central pontine myelinolysis associated with hyponatremia, diabetic 
ketoacidosis, acute hypernatremia, hyperglycemic hyperosmolar coma, 
chronic uremia, chronic hypernatremia, including accidental salt loading 
in high sodium dialysis or baby formula, alcoholism-related dehydration, 
diabetes insipidus, diabetes mellitus, AIDS, or dehydration from other 
causes. 
This invention is also directed to treatment of osmotic disturbances 
associated with renal dialysis comprising addition to a dialysis fluid of 
an effective concentration of an organic osmolyte.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The term "osmolyte" as used herein, refers to a compound which is a solute 
in body fluids, can circulate in an animal, can enter cells in response to 
changes in the osmotic milieu, and can protect the cell from damage due to 
excessive loss or uptake of water. An organic osmolyte is an osmolyte 
which is an organic compound. 
The organic osmolyte compounds useful in this invention include, but are 
not limited to, three major classes of compounds: polyols (polyhydric 
alcohols), methylamines, and amino acids. The polyols considered useful in 
the practice of this invention include, but are not limited to, 
myo-inositol, and sorbitol. The methylamines of the invention include, but 
are not limited to, choline, betaine, phosphorylcholine, 
glycerophosphorylcholine, lyso-glycerophosphorylcholine, creatine, and 
creatine phosphate. The amino acids of the invention include, but are not 
limited to, glycine, alanine, glutamine, glutamate, aspartate, proline and 
taurine. 
The term "osmolyte precursor" as used herein refers to a compound which is 
converted into an osmolyte by a metabolic step, either catabolic or 
anabolic. The osmolyte precursors of this invention include, but are not 
limited to, glucose, glucose polymers, glycerol, choline, 
phosphatidylcholine, lyso-phosphatidylcholine and inorganic phosphates, 
which are precursors of polyols and methylamines. Precursors of amino acid 
osmolytes within the scope of this invention include proteins, peptides, 
and polyamino acids, which are hydrolyzed to yield osmolyte amino acids, 
and metabolic precursors which can be converted into osmolyte amino acids 
by a metabolic step such as transamination. For example, a preferred 
precursor of the amino acid glutamine is poly-L-glutamine, and a preferred 
precursor of glutamate is poly-L-glutamic acid. 
Also intended within the scope of this invention are chemically modified 
osmolytes or osmolyte precursors. Such chemical modifications involve 
linking to the osmolyte (or precursor) an additional chemical group which 
facilitates transport across the blood brain barrier or gastrointestinal 
tract, or inhibits degradation of the osmolyte molecule. Such chemical 
modifications have been utilized with drugs or prodrugs and are known in 
the art. (See, for example U.S. Pat. Nos. 4,479,932 and 4,540,564; Shek, 
E. et al., J. Med. Chem. 19:113-117 (1976); Bodor, N. et al., J. Pharm. 
Sci. 67:1045-1050 (1978); Bodor, N. et al., J. Med. Chem. 26:313-318 
(1983); Bodor, N. et al., J. Pharm. Sci. 75:29-35 (1986); 
Osmolytes can be used in a number of disease states which involve an 
osmotic disturbance. The term "osmotic disturbance" as used herein refers 
to a condition wherein plasma osmolality is outside the range of about 
280-290 mosm/kg H.sub.2 O, or wherein plasma osmolality is not outside 
this range, but the plasma sodium concentration is outside the range of 
about 135-145 mEq/liter. In the case of an osmotic disturbance as used 
herein, osmotic constituents of the extracellular fluids cause swelling or 
shrinkage of cells. 
Disease states which involve osmotic disturbances include, but are not 
limited to, acute hyponatremia, chronic hyponatremia, central pontine 
myelinolysis associated with hyponatremia, diabetic ketoacidosis, acute 
hypernatremia, hyperglycemic hyperosmolar coma, chronic hypernatremia, 
such as that associated with accidental salt loading in high sodium 
dialysis or feeding with high sodium baby formula. Osmotic disturbances 
are also associated with alcoholism (wherein alcoholic individuals are at 
risk for demyelination), diabetes mellitus, diabetes insipidus, and 
Acquired Immunodeficiency Syndrome (AIDS). 
The invention is also directed to uremia, such as chronic uremia, wherein 
intracellular osmolytes may be depleted. Restoration of intracellular 
osmolytes to desired levels is achieved through the administration of an 
osmolyte (or osmolyte precursor) of this invention, either enterally or 
parenterally. In a preferred embodiment, a patient with chronic uremia 
undergoing dialysis is provided with an effective concentration of the 
osmolyte or osmolyte precursor in the dialysis fluid. 
Osmotic disturbances included within the scope of this invention also 
include those substantially associated with particular medical or surgical 
treatments. Acute hyperosmolar conditions occur with the use of 
dehydrating agents, such as, for example, mannitol (in association with 
neurosurgery) or glycerol (as a treatment for cerebral edema). 
Hyponatremia can also occur with the use of hypoosmolar glycine solution 
to flush the bladder, as in transurethral prostate surgery. Dialysis 
disequilibrium syndrome occurs when uremic patients are dialyzed too 
rapidly leading to a rapid decrease in plasma urea, and resultant brain 
swelling. 
Also intended within the scope of the invention is acute hyponatremia which 
occurs in association with physical activity or exercise which is 
accompanied by loss of fluids and salts through, for example, 
perspiration. Therefore, in one embodiment of this invention, a fluid 
supplemented with an osmolyte or osmolyte precursor of this invention is 
administered enterally to a subject prior to, or during the course of, the 
physical activity or exercise, e.g., as in a marathon race. Such 
"osmo-loading" protects cells, especially in the brain, from damage due to 
transient acute hypernatremia, and is intended to reduce fatigue and other 
symptoms known to be associated with prolonged physical activity or 
exercise. 
The term "substantially associated with," as applied to the osmotic 
disturbances or symptoms for which the methods of the invention are 
effective, means those disturbances wherein the metabolic or osmotic 
demand for regulation of serum sodium or of intra- or extracellular 
osmolytes occurs during or after the event or disease precipitating the 
osmotic disturbance and is related thereto. 
By the term "treating" is intended preventing, ameliorating, or curing a 
symptom or set of symptoms constituting, or substantially associated with, 
an osmotic disturbance. 
The term "enteral" is intended to indicate a method of administration of 
osmolytes to that portion of the alimentary canal from the stomach to the 
anus. 
The term "parenteral" denotes method of administration of osmolytes to that 
region outside of the digestive tract. 
Examples of parenteral routes of administration include, but are not 
limited to, subcutaneous (SC), intramuscular (IM), intravenous (IV) or 
intraperitoneal (IP) injection or infusion, and nasopharyngeal, mucosal or 
transdermal absorption. In most cases, the osmolyte or precursor is 
administered IV. In IV administration, the therapeutically effective 
amount of the osmolyte or osmolytes, in liquid form, is directly 
administered from a reservoir from which tubing connects to a needle which 
is placed into a large vein of the recipient. 
These IV fluids are sterile solutions composed of simple chemicals such as, 
for example, sugars, amino acids, and electrolytes, which can be easily 
assimilated. 
Regardless of which route of administration is utilized, the osmolyte can 
be administered either singly or as a supplement. When used as a 
supplement to known solutions, the osmolyte can be mixed with an existing 
enteral or parenteral solution (or diet) prior to administration to the 
recipient. It is also possible to administer the osmolyte without mixing 
it directly with the other components of a diet as, for example, in IV 
infusion wherein the osmolyte is not directly added to the main IV bottle, 
but instead is added to a common reservoir using a "piggy-back" bottle. 
This invention is also directed to osmolyte supplementation of formulas 
used in "total parenteral nutrition" (TPN) wherein patients derive their 
entire dietary requirements from the formula administered IV. TPN formulas 
do not normally contain the organic osmolytes of this invention or contain 
them or their precursors in concentrations too low for them to be 
effective in osmoregulation as is intended in this invention. Amino acids, 
some of which can also function as osmolytes, are added present in current 
TPN formulas for their nutritional value. There was no recognition in the 
art prior to the present inventors' discovery that such compounds in 
parenteral solutions could serve an osmoregulatory function. For this 
reason, the concentrations of glutamine, glycine or myo-inositol present 
in current parenteral formulas is too low for these compounds to exert an 
osmoprotective role via their action as organic osmolytes. 
The therapeutically effective dose ranges for the administration of 
osmolytes are those large enough to prevent clinically significant brain 
shrinkage or swelling capable of altering neurologic function (by criteria 
which are well known in the art) or increasing the risk of hemorrhage or 
structural damage. For example, the dose should be capable of preventing 
demyelination. 
It will be readily apparent to one of skill that the dosage of osmolyte or 
osmolyte precursor administered will be dependent upon the age, health, 
and weight of the recipient, the nature of any concurrent treatment, the 
frequency of treatment, and the nature of the effect desired. 
The rate of administration for an osmolyte when administered IV is greater 
than or equal to about 1 .mu.mole/kg body weight/day. Such administration 
rates could be 1 .mu.mole/kg/day to 3 moles/kg/day preferably 2 
.mu.moles/kg/day to 240 mmoles/kg/day, and more preferably 200 
.mu.moles/kg/day to 120 mmoles/kg/day. 
For enteral administration, the osmolyte is administered at a rate greater 
than or equal to about 5 .mu.moles per kilogram of body weight per day. 
Such administration rates could be 25 .mu.mole/kg/day to 15 mole/kg/day, 
preferably 10 .mu.moles/kg/day to 1 mole/kg/day, and more preferably 1 
mmole/kg/day to 600 mmole/kg/day. 
According to the method of the invention, an osmolyte may be administered 
by simply modifying existing enteral or parenteral dietary formulas or 
infusion solutions to contain the proper concentration of the osmolyte. 
The Na.sup.+ concentration in the osmolytetration supplemented fluid also 
comprises the invention, and is about 75-154 mEq/L, and the osmolarity of 
the fluid is about 150-300 mosm/L. In one embodiment directed to the 
treatment of acute hyponatremia as an emergency measure, such as during 
surgery, a high concentration of Na.sup.+, such as about 513 mEq/L 
(osmolality of about 1 osm/L) is used. 
In one embodiment, the osmolyte would remain in a dry form such as, for 
example, a sterile lyophilized powder which is aseptically hydrated at the 
time of administration and mixed at the proper concentration with the 
other components of the dietary composition. 
Alternatively, the osmolyte could be premixed with the other components of 
a dry formula which is aseptically rehydrated at time of administration, 
or stored as a frozen concentrate which is thawed and mixed at the proper 
concentration at time of use. 
The use of the osmolyte by the method according to the invention is ideally 
suited for the preparation of compositions. These compositions may 
comprise the osmolyte or combination of osmolytes, either alone or in 
combination with other chemicals. These other chemicals can be 
pharmaceutically acceptable carriers, as well as other active substances 
present in a dietary composition, such as, for example, free amino acids, 
protein hydrolysates, or oils. 
Preparations for parenteral administration include sterile aqueous or 
non-aqueous solutions, suspensions, and emulsions. Carriers or occlusive 
dressings can be used to increase skin permeability and enhance cutaneous 
absorption. 
Other pharmaceutically acceptable carriers comprise excipients and 
auxiliaries which facilitate processing of the active compounds into 
preparations which can be used pharmaceutically. Examples of excipients 
are water, saline, Ringer's solution, dextrose solution and Hank's 
balanced salt solution. The formulation may also contain minor amounts of 
additives such as substances that maintain isotonicity, physiological pH, 
and stability. Other formulations, known in the art, can be found in 
Remington's Pharmaceutical Sciences (latest edition), Mack Publishing 
Company, Easton, PA, which is hereby incorporated by reference. 
Preparations which can be administered orally such as tablets, and 
capsules, and also preparations which can be administered rectally, such 
as suppositories, as well as suitable solutions for administration by 
injection or orally, contain from about 0.01 to 99 percent, preferably 
from about 20 to 75 percent of active compound(s), together with the 
excipient. 
Other suitable excipients are, in particular, fillers such as saccharides 
and/or calcium phosphates, as well as binders such as starches, methyl 
cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, 
and/or polyvinyl pyrrolidone. If desired, disintegrating agents may be 
added such as the above-mentioned starches, carboxymethyl-starch, 
cross-linked polyvinyl pyrrolidone, agar, alginic acid or a salt thereof, 
such as sodium alginate. Auxiliaries are, above all, flow-regulating 
agents and lubricants, for example, silica, talc, stearic acid or salts 
thereof, and/or polyethylene glycol. In order to produce coatings 
resistant to gastric juices, solutions of suitable cellulose preparations 
such as acetylcellulose phthalate or hydroxypropymethyl-cellulose 
phthalate are used. Dye stuffs or pigments may be added for identification 
or in order to characterize combinations of active compound doses. 
Other pharmaceutical preparations which can be used orally include push-fit 
capsules made of gelatin, as well as soft, sealed capsules made of gelatin 
and a plasticizer. The push-fit capsules can contain the osmolyte in the 
form of granules which are mixed with fillers, and, optionally, 
stabilizers. In soft capsules, the active compounds are preferably 
dissolved or suspended in suitable liquids, such as fatty oils or liquid 
paraffin. In addition, stabilizers may be added. 
Possible pharmaceutical preparations which can be used rectally include, 
for example, suppositories which consist of a combination of the active 
compounds with a suppository base. Suitable suppository bases are, for 
example, natural or synthetic triglycerides, or paraffin hydrocarbons. In 
addition, it is also possible to use gelatin rectal capsules which consist 
of a combination of the active compounds with a base. Possible base 
materials include, for example, liquid triglycerides, polyethylene 
glycols, or paraffin hydrocarbons. 
Suitable formulations for parenteral administration include aqueous 
solutions of the osmolytes in water-soluble form, for example, 
water-soluble salts. Aqueous injection suspensions may contain substances 
which increase the viscosity of the suspension, including, for example, 
sodium carboxymethyl cellulose. Optionally, the suspension may also 
contain stabilizers. 
The invention also relates to a medicament or pharmaceutical composition 
comprising the components of the invention, the medicament being used for 
treating osmotic disturbances. 
The preferred animal subject of the present invention is a mammal. By the 
term "mammal" is meant an individual belonging to the class Mammalia. The 
invention is particularly useful in the treatment of human subjects. 
EXAMPLE I 
Accumulation of Major Organic Osmolytes In Rat Renal Inner Medulla (IM) in 
Dehydration 
The purpose of this study was to evaluate the significance of organic 
osmolytes in the renal IM and cortex of normal and dehydrated rats. In 
addition to considering whether there are significant changes in the 
osmolytes during antidiuresis, we examined the stoichiometric 
interrelationships that exist between the various osmolytes. The results 
indicate that dehydration for 3 days is accompanied by a significant rise 
in the osmolyte content of the IM. However, the individual osmolytes 
increased to different extents from rat to rat, suggesting complex 
regulation. 
A. METHODS 
Wistar-Kyoto rats (Charles River, Wilmington, MA), weighing 110-300 g, were 
fed ad libitum (Purina rodent laboratory chow 5001). Rats were housed 
individually in metabolic cages for at least 1 wk before the experiment. 
Control animals were given free access to water, whereas dehydrated rats 
were deprived of water for 72 h. On the day of the experiment, the morning 
urine was collected under mineral oil for 2-4 h for a measurement of urine 
osmolality. Blood was collected into heparinized (300 U) 15-ml centrifuge 
tubes, centrifuged at 1,000 .times.g, and the plasma was frozen for later 
analyses. 
The kidneys were rapidly excised after sacrifice, and the inner medulla 
(25-50 mg/kidney) and superficial cortex (-600 mg/kidney) were dissected 
and minced in 1 and 3 ml, respectively, of ice cold 6% perchloric acid 
(PCA). The tissue from either 2 or 4 kidneys were pooled for each sample. 
After determining wet weight, the tissue was finely minced with scissors 
and homogenized by hand using a Dounce glass homogenized (Wheaton). The 
acid homogenate was kept ice-cold for -2 h and then centrifuged at 1,000 
.times.g for 10 min. The pellet was saved for analysis of protein, and the 
supernatant was neutralized (pH 7.0-7.4) with 2M KOH. The samples were 
centrifuged once more (1,000 .times.g for 10 min to remove the KCIO.sub.4 
precipitate, and the supernantant was frozen (-40.C). Each sample was run 
through an ion-exchange column (Chelex 100, Bio Rad) to remove 
paramagnetic ions such asCr.sup.2+ and Mn.sup.2+ that would diminish 
resolution. The column was prepared by hydrating the resin in distilled 
water, packing 5 ml of slurry in a 10-ml syringe containing a glass-wool 
plug, and washing with 5 ml of water. The sample was loaded on the column, 
chased with 10 ml of water, and the eluant was titrated with HCI to pH 
7.0-7.4. Each column was used for only three samples and then was either 
discarded or regenerated using 5 ml HCl (IN), 5 ml NaOH (IN), and 10 ml of 
water. The samples were subsequently frozen, lyophilized (Labconco Freeze 
Dryer 8) for 24-48 h, and reconstituted in 3 ml of D.sub.2 O. Each sample 
was then centrifuged (1,000 .times.g for 10 min) and filtered through a 
0.45-.mu.m filter (Millipore) that was rinsed with an additional 0.5 ml of 
D.sub.2 O. The sample was lyophilized again and then frozen pending 
analysis. 
To consider whether significant metabolite degradation occurred during 
dissection, extracts of minced whole kidneys were prepared as above and 
compared with extracts of whole kidneys that were rapidly frozen in liquid 
nitrogen. Each sample contained both kidneys from a single rat. The frozen 
kidneys were fragmented on a bed of dry ice using a hammer and a steel 
plate. The tissue fragments were then placed in ice-cold PCA (6%). These 
acid extracts were then centrifuged, neutralized, filtered, and 
lyophilized as described above. 
NMR soectroscopy--Lyophilized samples were reconstituted in 3 ml of D.sub.2 
O containing 5 mM sodium 3-trimethylsilylpropionate-2,2,3,3-d.sub.4 (TSP), 
a chemical shift and content standard, and placed in a 12 mm NMR tube 
(Wilmad, NJ). The D.sub.2 O was used to attenuate the large portion signal 
from water and thereby allow better resolution and quantitation of the 
osmolyte peaks. NMR spectra were obtained using a Nicolet NT360 WB NMR 
spectrometer tuned to 360.09 MHz for protons. The D.sub.2 O signal was 
used for shimming. Either 64 or 128 transients were collected into 4K or 
8K data blocks using a 90.degree. tip angle, a spectral width of .+-.3,000 
Hz, and a 12 s delay time. Because all protons of interest had 
spin-lattice relaxation times (TI), of .ltoreq.2.2 s, fully relaxed 
spectra were obtained. The free induction decay data were filtered for 
optimum resolution by apodization with a double exponential function, zero 
filled, and Fourier transformed. The integral of each peak was analyzed 
relative to the TSP peak to quantitate the amount of each metabolite in 
the sample. 
In general, the predominant osmolytes were quantitated from the integral of 
a specific peak for each. GPC was quantitated by integrating the peak at 
4.32 ppm. Because choline (3.20 and 4.07 ppm) was not significant in any 
sample, myo-inositol was quantitated by integrating the peak at 4.06 ppm. 
Betaine was quantitated by integrating the prominent trimethylamine peak 
at 3.27 ppm. However, to correct for a small contribution of myo-inositol 
to this signal (.about.15%), the myo-inositol measured at 4.06 ppm was 
subtracted from the integrated peak of 3.27 to yield the actual betaine 
content. When evident, sorbitol was most apparent from a peak at 3.85 ppm. 
To confirm the identity of the osmolyte species, solutions of known 
composition were prepared and peak assignments were made for each 
compound. Consequently, each osmolyte could be identified in a sample by 
observing peak positions, relative intensifies of companion peaks, or by 
spiking samples with known compounds. In addition, to assess whether 
osmolyte degradation, transformation, or dissipation occurred during the 
extraction and reconstitution procedures, solutions of known composition 
were analyzed before and after the extraction and reconstitution 
procedure. The results indicated that &gt;95% of the osmolyte contents were 
recovered. 
Biochemical assays and other measurements. IM urea content was measured 
fluorometrically in the PCA extracts according to Roman, R. J. et al., 
Anal. Biochem. 98:136-141 (1979)). The urease (US Biochemical, type III) 
was dissolved in a sodium phosphate buffer (0.2 M, pH 7.4) and dialyzed 
(Spectrapor, 3,500 MW cutoff) for 24 h in 11 of the same phosphate buffer. 
IM amino acids content was estimated spectrophotometrically by assaying 
NPS according to Lee, V. P. et al., Anal. Biochem. 14:71-77 (1966)). The 
assay was performed on the PCA extracts with glycine as standard. The NPS 
assay did not detect betaine, GPC, myo-inositol, or sorbitol. Very high 
concentrations of urea were detectable as NPS but the cross-reactivity 
(.about.0.1%) was not sufficient to significantly affect the results. In 
addition, the NPS assay is known to detect the 20 principal amino acids as 
well as a variety of other primary amines including ethanolamine, 
phosphorylethanolamine and taurine. IM sorbitol content was measured in 
the PCA extracts using sorbitol dehydrogenase and measuring the resultant 
production of NADH (Bergmeyer, H. U. et al., Methods of Enzymatic 
Analysis. Academic Press, New York, 1974, p. 1323-1330). This assay can 
detect other polyols such as xylitol; however, it does not detect 
myo-inositol, betaine, or GPC. Urine and plasma osmolality were determined 
by either freezing-point depression (Advanced Instruments, Needham, MA) or 
vapor pressure (Wescor 5100C, Logan, UT). Arginine vasopressin (AVP) 
levels were assayed in plasma extracts (Glick, S. M. et al., Methods in 
Hormone Radioimmunoassay, New York: Academic, 1979, p. 341-351) using a 
commercial polyclonal antibody (Arnel Pharmaceuticals) at a final dilution 
of 1:75,000, a cold vasopressin standard (Bachem), and 8-arginine .sup.125 
I-labeled vasopressin (New England Nuclear) (Majzoub, J. A. et al., Am. J. 
Physiol. 252:E637-E642 (1987)). 
Protein content of IM tissue was measured with the Lowry assay by first 
dissolving the PCA precipitate in 0.1 N NaOH-5% deoxycholate and using 
bovine serum albumin as the standard (Lowry, 0.H. et al., J. Biol. Chem. 
193:265-275 (1951)). Percent tissue water was determined in renal cortical 
tissue according to [(wet wt-dry wt)/wet wt].times.100. Wet weight was 
measured using pretared glass vials and fresh tissue. Dry weight was 
measured after the tissue had been dried for at least 24 h in an oven at 
100.degree. C. 
Materials. D.sub.2 O (99.8%) was obtained from either Sigma or Aldrich. 
Betaine, GPC, myo-inositol, sorbitol, choline, chloride, sorbitol 
dehydrogenase, and NAD were obtained from Sigma. 
Statistics. Each datum represents a pooled sample of either two or four 
kidneys. The data were calculated per wet weight and per protein content. 
Comparisons between control and dehydrated groups were made using the 
unpaired Student's t test. Values are expressed as means .+-.SE for n 
samples. Linear correlations were determined by least-squares regression 
analysis of individual data points. 
B. RESULTS 
Water deprivation for 3 days produced dramatic changes in body weight, 
urine, and plasma. The dehydrated animals lost 20.3.+-.0.8% (n=25) of 
their body weight and experienced a 2.5-fold increase in urine osmolality 
from a control value of 1,503.+-.68 (n=19) to 3,748.+-.142 (n=18) 
mosmol/kg (p&lt;0.001). Plasma osmolality rose from a control value of 
289.+-.2 (n=20) to 311.+-.2 (n=25) mosmol/kg (p&lt;0.001), and this was 
associated with a 10-fold rise in plasma AVP levels from 0.57.+-.0.08 
(n=16) to 6.7.+-.0.9 (n=16) pg/ml plasma (p&lt;0.001). 
Whole kidney - FIG. 1 is a typical .sup.1 H-NMR spectrum of a whole kidney 
extract from a control rat. The most prominent peaks have been identified 
as GPC, betaine, and creatine, Although many peaks are evident, only the 
characteristic osmolyte peaks have been labeled. When extracts of rapidly 
frozen kidneys (n=4) were compared with nonfrozen kidneys (n=4) the 
spectra were identical. Furthermore, quantitation of several peaks (in 
.mu.mol/g wet wt) showed that there was no significant difference between 
these preparation methods in the amounts of betaine (2.8.+-.0.6 vs. 
3.1.+-.0.5), GPC (6.4.+-.0.4 vs. 6.3.+-.0.6), creatine (1.4.+-.0.1 vs. 
1.4.+-.0.1), or myo-inositol (3.3.+-.0.3 vs. 3.2.+-.0.3). Therefore the 
time required to dissect the IM and the cortex did not appear to alter 
significantly the osmolyte composition of the extracts. 
Inner medulla--FIG. 2A is a typical NMR spectrum of a renal IM that shows 
characteristic osmolyte peaks was well as several smaller peaks to the 
right (upfield) of the osmolytes. The smaller peaks in the range 2.0-2.5 
ppm represent primarily amino acids. In addition, lactate (1.3 ppm) and 
creatine (3.04 ppm) are evident but they were present in smaller 
quantities than the trimethylamines and polyols. To focus on the principal 
osmolytes of the IM, FIG. 2B shows an expanded view of FIG. 2A from 2.9 to 
4.4 ppm. The two most prominent peaks (.about.3.2 ppm) represent 
trimethylamine peaks of betaine (3.27 ppm) and GPC (3.22 ppm). Companion 
methyl and methylene protons of betaine (3.90 ppm) and GPC (4.32, 3.91, 
3.67, and 3.63 ppm) are also evident. A unique myo-inositol peak is 
visible at 4.06 ppm with several other myo-inositol peaks also apparent 
(3.61 and 3.65 ppm). Although sorbitol was evident in some samples, there 
is none clearly apparent in this spectrum (e.g., 3.85 ppm). Because NH3 
protons can freely exchange with the deuterium in D.sub.2 O there is no 
signal from urea, although it was present in the sample. 
To evaluate whether we had indeed accounted for the major proton-containing 
solutes that exist in the IM, we prepared a solution containing only GPC 
(27 .mu.mol), betaine (16 .mu.mol), myo-inositol (14 .mu.mol, and TSP (15 
.mu.mol) in D.sub.2 O. As shown in FIG. 2C, a spectrum of this known 
solution looks virtually identical to the IM spectrum (FIG. 2B). This 
apparent identity, coupled with previous gas chromatography measurements 
of renal IM (Bagnasco, S. et al., supra) suggests that these compounds are 
the major organic osmolytes of the rat renal IM. 
Quantitation of the IM organic osmolytes in the NMR spectrum indicated that 
there was a general increase in solute content in dehydrated rats. Because 
dehydration is associated with a 20% loss of body weight, these organic 
solutes were quantitated per protein content to assess content changes. IM 
urea content increased from 2,036.+-.230 (n=9) to 4,405.+-.501 (n=13) 
nmol/mg protein (p&lt;0.001) with dehydration. In addition, as shown in FIG. 
3A, dehydration significantly increased GPC (in nmol/mg protein) from 
265.+-.32 (n=9) to 517.+-.34 (n=13) (p&lt;0.001 and betaine from 110.+-.15 
(n=9) to 214.+-.40 (n=13) (p&lt;0.05), whereas myo-inositol was not 
significantly elevated [103.+-.16 (n=9) to 178.+-.32 (n=13) (p&lt;0.10)]. 
When normalized per gram wet weight (FIG. 3B), GPC increased by 137% from 
17.1 .+-.2.2 (n=12) to 40.5.+-.2.8 (n=16) .mu.mol/g (p&lt;0.001), and 
myo-inositol significantly increased by 87% from 6.8.+-.1.0 (n=12) to 
40.5.+-.2.8 (n=16) .mu.mol/g (p&lt;0.02) but betaine was not significantly 
changed from 8.1.+-.1.4 (n=9) to 14.0.+-.2.4 (n=16) .mu.mol/g. IM urea 
also increased significantly from 123 17 (n=12) to 363 35 (n= 16) 
.mu.mol/g (p&lt;0.001). These data indicate that a net increase in the IM 
contents (i.e., per protein) of betaine and GPC occurred during hydration, 
whereas the apparent concentrations (i.e., per wet wt) of GPC and 
myoinositol increased with dehydration. 
Several reports indicate that significant levels of sorbitol exists in the 
rabbit and rat IM (Bagnasco, S. et al., supra: Corder, C. N. et al., 
supra). However, NMR analysis of our samples generally detected little or 
no sorbitol. Because NMR analysis was unable to quantitatively resolve 
sorbitol levels of .ltoreq.0.5 .mu.moles, a spectrophotometric assay was 
used that confirmed that each IM contained between 0.1 and 0.7 .mu.moles 
of sorbitol. In fact, the spectrophotometric measurements correlated with 
out NMR analysis; sorbitol was evident only in spectra of samples that 
contained the higher quantities. Dehydration caused a 105% increased in IM 
sorbitol (FIG. 3A) from a control value of 64.+-.9 (n=9) to 119 t 18 
(n=13) nmol/mg protein (p&lt;0.05). On a tissue weight basis (FIG. 3B), 
sorbitol content increased by 131% from 4.2.+-.0.5 (n=12) to 9.7.+-.1.3 
(n=16) (p&lt;0.002). 
Analysis of NPS indicated that the renal IM contained significant levels of 
amino acids; however, IM NPS did not significantly change during 
dehydration (FIG. 3A and 3B). The control rats contained 344.+-.44 (n=9) 
nmol/mg protein or 22.0.+-.4.6 (n=12) .mu.mol/g wet wt, and the dehydrated 
animals contained 337.+-.54 (n=13) nmol/mg protein or 23.2.+-.2.2 (n=16) 
.mu.mol/g wet wt. 
FIG. 4 provides a comparison of the total nonurea organic osmolyte pools in 
the IM of control and dehydrated rats. The data show that there was a 54% 
elevation of total osmolytes in dehydration from 885.+-.77 (n=9) to 
1,365.+-.127 (n=13) nmol/mg (p&lt;0.01). If one evaluates only the non-NPS 
osmolytes, however, there was a 90% increase from 541.+-.52 to 1,027.+-.92 
nmol/mg protein (p&lt;0.0001). Based on wet weight, the total osmolytes 
increased significantly from 58.5.+-.8.5 (n=12) to 100.1.+-.7.0 (n=16) 
.mu.mol/g (p&lt;0.002) and non-NPS osmolytes increased by 113% from 
36.1.+-.4.4 (n=12) to 76.9.+-.6.0 (n=16) .mu.mol/g (p&lt;0.001). FIG. 4 also 
compares relative osmolyte contents. Although NPS did not change, it 
represented a substantial fraction of the total osmolyte pool in both the 
control (39%) and dehydrated (25%) states. Furthermore, in both groups the 
trimethylamines were more abundant than the polyols, and these two classes 
of compounds were increased in dehydration. Total trimethylamines 
(GPC+betaine) significantly increased in dehydration from 375.+-.34 (n=9) 
to 731.+-.64 (n=13) nmol/mg protein (p&lt;0.001) or, per weight, from 
25.1.+-.3.2 (n=12) to 54.5 4.1 (n=16) .mu.mol/g (p&lt;0.001). Total polyols 
(inositol+sorbitol) increased in dehydration from 167.+-.18 (n=9) to 
297.+-.33 (n=13) nmol/mg protein (p&lt;0.01, or per wet weight, from 11.0 N 
1.2 (n=12) to 22.4 (n=16) .mu.mol/g (p&lt;0.001). 
Renal cortex - To address whether the increase in osmolytes was localized 
to the renal IM, we evaluated the osmolyte content of the renal cortex. 
FIG. 5 is a typical spectrum of a cortex, which is strikingly more complex 
than the IM spectrum. Similar to the IM however, there are two strong 
trimethylamine peaks that represent betaine and GPC. In addition, 
myo-inositol (e.g., 4.06 ppm) is also visible. In comparison to the IM, 
the osmolyte contents of the cortex were relatively low. In control rats, 
cortical betaine and GPC averaged 2.1.+-.0.4 (n=8) and 2.7.+-.0.2 (n=8) 
.mu.mol/g wet wt, respectively, and dehydration did not significantly 
alter betaine [2.0.+-.0.2 (n=9)] or GPC [3.3 0.3 (n=9)]. Furthermore, 
because cortical tissue water in control [79.6.+-.0.8% (n=5)]and 
dehydrated [78.7.+-. 0.4% (n=7)] rats was no different, these data 
indicate that betaine and GPC content clearly did not increase in 
dehydration. In contrast, dehydration was associated with a significant 
rise in myo-inositol from 1.2.+-.0.2 (n=8) to 2.1.+-.0.2 (n=9) .mu.mol/g 
wet wt (p&lt;0.002). Urea, sorbitol, and NPS were not measured in the cortex. 
C. DISCUSSION 
The role of organic osmolytes in the mammalian kidney has been poorly 
understood. Using NMR spectroscopy and biochemical assays, we confirmed 
the presence of high levels of trimethylamines (GPC and betaine), polyols 
(inositol and sorbitol), and amino acids (NPS) in the rat renal IM. To 
investigate the role of these osmolytes in antidiuresis, their quantities 
were assayed in the IM and cortex of kidneys from control and dehydrated 
animals. Three days of dehydration caused a 10-fold increase in plasma AVP 
and approximately doubled urine osmolality and IM urea content. 
Concurrently, the IM experienced a 54% increase in nonurea organic 
osmolyte content and a 90% increase in the nonurea, non-NPS (i.e., 
trimethylamines and polyols) osmolyte contents. The individual pools of 
total trimethylamine and total polyols increased by 95 and 78%, 
respectively, in dehydration. When individual osmolytes were quantitated 
per tissue protein content, dehydration caused significant increases in 
GPC (106%), betaine (95%), and sorbitol (130%) but not myo-inositol (73%) 
or NPS (-2%). When evaluated per tissue weight, dehydration significantly 
elevated all the osmolytes except betaine and NPS. Overall, the magnitude 
of the trimethylamine and polyol changes were comparable to the changes in 
urine osmolality and IM urea; all were increased about two-fold. Therefore 
these osmolytes were accumulated in antidiuresis as one would predict if 
they serve an osmoprotective function in the adaptation of the IM to a 
hypertonic environment. 
IM osmolyte contents reported in this study are consistent with those 
observed by other investigators. In the present study, GPC was 553 nmol/mg 
protein or 40.5 .mu.mol/g wet wt with 3 days of dehydration. Wirthensohn 
and co-workers (Wirthensohn, G. et al., Pfluegers Arch. 409:411-415 
(987)) used a biochemical assay and measured rat IM GPC levels of 451.8 
nmol/mg protein or 33.4 .mu.mol/g wet wt in rats deprived of water for 16 
h. Compared with our control GPC level of 17 .mu.mol/g wet wt in rabbit 
IM, whereas Ullrich (supra) measured 125 .mu.mol/g wet wt of GPC in the 
dog IM. In rat IM, sorbitol was measured as 0.34 (Corder, C. N. et al., 
supra) compared with 4.2 .mu.mol/g wet wt in the present study, whereas 
rabbit contained 10 .mu.mol/g wet wt (Bagnasco, S. et al., supra). 
Myo-inositol was detected at comparable levels in rabbit (10 .mu.mol/g wet 
wt) ((Bagnasco, S. et al., supra) and dog (15 .mu.mol/g wet wt (Cohen, M. 
A. H. et al., supra) as that observed by us in rat (6.8 .mu.mol/g wet wt). 
At least 25 amino acids have been identified in the renal IM, and the 
total amino acid content ranges from 25 to 39 .mu. mol/g wet wt in several 
species, including rat (Law, R. O. et al., supra); Robinson, R. R. et al., 
supra). Our NPS levels were comparable (22 .mu.mol/g wet wt) with no 
particular amino acid appearing dominant in the NMR spectrum. Because we 
did not quantitate specific amino acids, it is conceivable that there were 
selected changes in the relative abundance of particular amino acids but 
this was not expressed as a significant increment in the total pool 
content. 
Yancey and co-workers (Somero, G. N., supra); Yancey, P. H. et al., supra) 
showed that trimethylamines counteract the toxic effects of urea on 
numerous enzymatic processes. They established that a 2:1 concentration 
ratio of urea to trimethylamines was the optimal stoichiometric 
relationship for maintaining normal activity of many cellular enzymes. 
Therefore we calculated the urea-to-trimethylamine ratio in the IM. Under 
control conditions the ratio was 5.0.+-.0.4 (n=13), and it was 
significantly elevated to 6.8.+-.0.6 (n=16) (p&lt;0.05) in the dehydrated 
animals. Urea, however, is presumed to be equally distributed throughout 
the intracellular and extracellular compartments of the IM, whereas GPC 
(Wirthensohn, G. et al., supra) and the other osmolytes are thought to be 
localized intracellularly. Assuming that the intracellular volume is 40% 
of the total tissue volume (Pfaller, W., In: Advances in Anatomy. 
Embryology, and Cell Biology. Hild, W. et al., Eds. Springer-Verlag, 
Berlin, vol. 70, chap. 5, p. 21), then these urea-to-trimethylamine ratios 
would reflect intracellular ratios of 2.0 and 2.7, values in the range of 
2.0 described by Yancey and coworkers. The reason for the increase in the 
ratio with dehydration is unknown but could reflect a real change or a 
change in the relative volumes of the intracellular and extracellular 
compartments. 
Renal cortex is known to contain both GPC-hydrolyzing enzyme activity 
(Wirthensohn, G. et al., supra) as well as choline dehydrogenase for 
conversion of choline to betaine (Wirthensohn, G. et. al., In: 
Biochemistry of Kidney Function. F. Morel, ed., Elsevier, New York, 1981). 
Our data show that the renal cortex also contained significant quantities 
of betaine, GPC, and myo-inositol (FIG. 5); however, only myo-inositol was 
significantly elevated by dehydration. 
NMR spectroscopy proved to be a highly sensitive method for detecting 
several classes of organic osmolytes (&gt;0.5 .mu.mol). In addition, 
traditional biochemical assays were used to reliably quantitate the levels 
of sorbitol, amino acids, and urea for the following reasons. Sorbitol was 
present in amounts not generally detected in our NMR spectra. Amino acids 
were evident in our NMR spectra; however, the quantities of individual 
amino acids could not be reliably quantitated using NMR so they were 
measured collectively as NPS. Urea was undetectable by .sup.1 H-NMR 
spectroscopy because its protons are exchanged completely with deuterium 
(i.e., --NH.sub.2 converted to --ND.sub.2). Several other small peaks were 
present in the NMR spectrum, including lactate and 
phosphocreatine/creatine. However, the quantity of these organic 
osmolytes, was considerably less than that of the triethylamines, polyols, 
and NPS and thus did not appear to constitute a significant osmolyte pool. 
In addition to developing procedures for quantitating the major organic 
osmolytes, we demonstrated that each of these compounds is stable during 
the tissue harvesting and extraction procedures (see METHODS). Although it 
is possible that alternative extraction and/or detection methods might 
uncover additional osmolytes, our results, together with the data reported 
by others, offer no evidence that the rat IM contains significant 
quantities of other organic solutes. 
To assess whether the IM osmolytes were uniformly increased in response to 
dehydration, the individual osmolytes were compared in each sample. FIG. 
6A is a plot of betaine vs. GPC showing that these parameters are not 
directly correlated. With dehydration there was a generalized increase in 
GPC, whereas betaine showed significant scatter with some rats exhibiting 
control levels of betaine despite elevated GPC. Similar results were 
observed when myo-inositol and sorbitol were compared with GPC. Because 
urine osmolality, plasma osmolality, plasma antidiuretic hormone, and IM 
urea content were always elevated to similar levels in the dehydrated 
animals, these data suggest that additional modulators must exist to 
account for the animal-to-animal variations observed in the responses of 
betaine, myoinositol, and sorbitol. FIG. 6B provides some additional 
insight, indicating that there was a strong linear correlation (r=0.87) 
between betaine and myo-inositol in the IM. The regression line indicates 
that the IM accumulated 1.1 betaine per myo-inositol with an intercept not 
different from zero. The highest values were observed only in dehydrated 
individuals, although there was significant overlap between the control 
and dehydrated groups. This linear correlation suggests that these 
osmolytes are either regulated by the same effector(s) or that one of 
these osmolytes modulates the other. Finally, FIG. 6C is a composite 
comparing both betaine and myoinositol with sorbitol, indicating that here 
were no direct correlations. Interestingly, the control values are all 
tightly clustered, whereas the dehydrated samples tended to show an 
increase either in betaine and myo-inositol or in sorbitol but not 
concomitantly in all three. This mutually exclusive modulation of 
myo-inositol and betaine vs. sorbitol suggests multifactorial regulation 
of the osmolytes. 
E. CONCLUSION 
The rat renal IM contains high concentrations of trimethylamines and 
polyols that increase during antidiuresis (dehydration). The relative 
increases in total trimethylamines and total polyols were comparable to 
the changes in urine osmolality, suggesting that they were accumulated in 
response to the hypertonic environment. 
EXAMPLE II 
Amino Acids, Polyols, and Methylamines in Rat Brain: Response to Induced 
Hypernatremia 
A. INTRODUCTION 
Most studies have focused on the role of amino acids as a component of the 
idiogenic osmoles in brain. Specific amino acids shown to accumulate with 
hypernatremia in salt loading include glutamate, glutamine, aspartate, 
.gamma.-aminobutyric acid (GABA), alanine, glycine, serine, ornithine, and 
taurine. However, the relative importance of any particular amino acid has 
varied somewhat in the different models of hypernatremia. In the brain 
there have been no studies of the methylamines. The polyols, myo-inositol 
and sorbitol, were shown to accumulate within 4 hours of hyperglycemia 
(Prockop, L. D., Arch. Neurol 25:126-140 (1971)), and work in 
hypernatremic rats showed an elevated myo-inositol concentration (Lohr et 
al., supra). 
The purpose of the present study was to identify and quantify the organic 
solutes which accumulate in the brain in response to chronic salt loading 
or water deprivation, two different models known to cause hypernatremia. 
These two models were chosen because 5 days of salt loading is a 
well-known stimulus for accumulation of idiogenic osmoles in the brain, 
and 3 days of water deprivation is known to promote accumulation of 
methylamines and polyols in renal IM. Since previous studies have not 
attempted to identify all classes of organic compounds which could 
constitute idiogenic osmoles in a single model, we employed .sup.1 H NMR 
spectroscopy as an established technique for identification of multiple 
classes of organic compounds in brain and kidney. This approach was used 
successfully to identify organic osmolytes in the renal IM (see Example 
I). Our results indicated that three classes of compounds, amino acids, 
methylamines and polyols accumulate in the brain of salt loaded rats. 
Furthermore, the major compounds which accumulated were myo-inositol, 
glutamine, glutamate, phosphocreatine+creatine (PCr+Cr), 
glycerophosphorylcholine (GPC) and choline, in decreasing order of 
abundance. In contrast, no organic solutes were found to accumulate in 
water deprived rats. 
B. METHODS 
Animals Male Sprague-Dawley rats (250-350 g) were obtained from Charles 
River Breeding Co. and assigned to control (n=8), salt-loaded (n=9), and 
water-deprived (n=4) groups. All rats, housed individually in metabolic 
cages, were allowed free access to tap water and standard rodent chow 
(Purina Chow #5001) during one week of equilibration prior to the 
experimental regimens. The experimental periods were 4 days for control 
rats, 5 days for salt-loaded rats, and 3 days for water-deprived rats. 
During the experiment, all rats were allowed free access to food. In 
addition, control rats were allowed free access to tap water, salt-loaded 
rats were allowed free access to NaCl drinking water, and water-deprived 
rats had their water bottles removed. Chronic salt-loading was achieved by 
a combination of NaCl (320 mM) in the drinking water and daily gavage with 
6 ml of 10% NaCl. This salt-loading protocol was found in our pilot 
studies to produce a sustained elevation of P.sub.Na (&gt;155 meq/l). Body 
weights, water intake, and food intake were monitored daily. At the end of 
each protocol, rats were sacrificed by decapitation under light ether 
anesthesia. 
Plasma sodium concentration was measured in plasma collected from the tail 
at the very beginning of each protocol and in plasma collected in trunk 
blood at the time of sacrifice. Sodium was measured in plasma by flame 
photometry (Instrumentation Laboratories Instruments). 
Brain extracts: Following decapitation, each brain was rapidly removed from 
the cranium with a spatula and immediately freeze-clamped in liquid 
nitrogen. This entire procedure was performed in 5-10 seconds. The samples 
were pulverized in liquid nitrogen, and transferred to vials containing 3 
ml of ice-cold 6% PCA. Each sample was left in the ice-cold PCA for 2 
hours, centrifuged (1000.times.g) for 10 minutes, and the supernatant was 
decanted and neutralized (pH 7.0-7.4) with 1.0 M KOH. The PCA precipitates 
were saved for analysis of protein content. The neutralized extracts were 
then passed through a 5 ml volume chelex column (Chelex 100-200, Bio-Rad) 
to remove paramagnetic ions which are known to diminish NMR signal 
resolution. The filtrates were frozen at -40.degree. C. and subsequently 
lyophilized to dryness (48 hours). The lyophilisates were then 
reconstituted in 4 ml of D.sub.2 O (99.8%, Sigma Chemical Co.) and the 
residual potassium perchlorate precipitates were removed by centrifugation 
(1000.times.g for 10 minutes) and subsequent filtration using a 0.45 .mu.m 
syringe (Millipore). The syringe and filter were rinsed with two 0.5 ml 
washes of D.sub.2 O. These extracts were frozen again, lyophilized (24 
hrs), and then stored in the freezer (-40.degree. C.) until they were 
analyzed. This extraction procedure preserves organic solutes in studies 
of the kidney (Example I). 
.sup.1 H NMR Spectroscopy: The extracts were prepared as described in 
Example I. .sup.1 H NMR spectra were recorded as described in Example I. 
Individual organic components were identified in the spectra using three 
criteria: (i) comparison of peak positions and relative peak intensities 
to those observed following addition of known compounds to the samples 
(i.e., "doping"); (ii) comparison of the relative peak intensities of 
companion peaks associated with each compound; and (iii) observation of 
peaks at characteristic resonance frequencies as determined with pure 
compounds. Many of the peak assignments were also available from 
previously reported assignments in the brain and our assignments generally 
verified those assignments. In addition, the contents of organic compounds 
were comparable to previously published results in the literature. Once a 
compound was identified, one or two peaks which exhibited no significant 
overlap with other resonances in the spectrum were integrated to measure 
the content of that compound in the sample. Contents of organic compounds 
were verified by adding known quantities of a compound to a sample and 
observing the change in signal intensity and/or by comparison of the NMR 
measurement to a biochemical assay (e.g., myo-inositol and sorbitol). The 
individual peaks integrated to quantitate the methylamines, polyols, and 
amino acids are listed in Tables 1, 2, and 3. The lower limit of the NMR 
measurements was approximately 10 nmoles per sample or about 0.1 nmol/mg 
protein (each sample contained approximately 100 mg protein) as determined 
from quantification of the betaine peak at 3.27 ppm. Some other organic 
compounds which may have been more abundant than 0.1 nmol/mg (e.g., 
glycine) were not measurable in the spectra because they did not produce a 
high intensity peak such as that seen with the trimethylamines with 9 
resonating protons of three adjacent methyl groups. This large peak of 
trimethylamines allows for lower contents of these compounds to be 
detected and quantitated compared to some more abundant compounds which 
lack the trimethyl moiety and the associated high intensity peak. As will 
be discussed below, some compounds were detected (e.g., taurine) in the 
spectra which could not be quantitated. 
Biochemical Assays: myo-Inositol was measured spectrophotometrically by 
measuring the reduction of NAD.sup.+ in the presence of myoinositol 
dehydrogenase as described previously (Weissbach, A., In: Methods of 
Enzymatic Analysis 3:1333-1336 (1974)). Sorbitol was measured 
spectrophotometrically as measuring the reduction of NAD.sup.+ in the 
presence of sorbitol dehydrogenase. The protein content was measured as in 
Example I. 
Chemicals: All chemicals were analytical grade and were obtained from 
standard commercial sources. D.sub.2 O (99.8%) was obtained from either 
Sigma or Aldrich Chemical Co. TSP was obtained from Aldrich. Methylamines, 
polyols, and amino acids used to prepare standards were obtained from 
Sigma Chemical Co. myo-Inositol dehydrogenase, sorbitol dehydrogenase, and 
NAD.sup.+ were also purchased form Sigma Chemical Co. 
Statistics: All values represent the mean.+-.SEM for n animals in each 
group. Comparison of contents of individual compounds to those in the 
control group were made by one-way analysis of variance (ANOVA, STAT PAK) 
followed by the unpaired Student's t test when the F statistic was found 
to reach significance (p&lt;0.05). The paired t test was used to determine 
significant differences on consecutive days for body weight, water intake, 
and food intake. 
C. RESULTS 
Animals: FIG. 7 indicates the daily water intake for each group of rats 
throughout the experiment. In the 24 hrs. preceding the start of the 
experimental regimens (Day 0), the three groups of rats drank comparable 
amounts of water, about 38 ml/day, and the control rats maintained this 
water intake level throughout the protocol. In contrast, salt-loaded rats 
rapidly increased their fluid intake and achieved a stable intake of 80 to 
100 ml/day on days 2 to 5. On day 4 the water intake of salt-loaded rats 
(101.+-.3 ml/day) was 146% higher, and on day 5 (103.+-.2 ml/day) 151% 
higher than controls on day 4 (p&lt;0.001). Fluid intake of water-deprived 
rats was maintained at 0 ml/day on days 1 through 3 of the protocol. 
FIG. 8 indicates the daily food intake of the 3 groups of rats throughout 
their respective protocols. Prior to initiating the experimental regimens 
(Day 0), all 3 groups consumed approximately 26 gm of food per day. As 
seen with water intake, the food intake of the control group was 
relatively constant throughout the experiment. In contrast, both the 
salt-loaded and water-deprived groups of rats displayed significant and 
parallel decreases in their food intakes such that on day 2 and thereafter 
both groups consumed comparable amounts ranging from 5 to 10 g/day. On the 
final day of the respective protocols, the food intakes of the salt-loaded 
(4.7 g/day) and water-deprived (5.0 g/day) groups were significantly less 
than the control group (26 g/day) (p&lt;0.001). Food intake of salt-loaded 
rats on day 4 (9.6 g) was also significantly less than controls on day 4 
(p&lt;0.001). 
A plot of the cumulative percent changes in daily body weights for the 
three groups is shown in FIG. 9. Over the course of the experiment, the 
control group demonstrated a 6% gain in body weight (p&lt;0.05). In contrast, 
both the salt-loaded and water-deprived groups exhibited significant and 
parallel decreases in body weight. The salt-loaded animals lost 22% of 
their body weight in 5 days (p&lt;0.001). The water-deprived rats lost 18% of 
their body weight in 3 days (p&lt;0.001). 
Plasma On day 0, all three groups had similar P.sub.Na values with control 
rats at 143.+-.1 meq/l, salt-loaded rats at 144.+-.3 meq/l, and 
water-deprived rats at 140.+-.2 meq/l. At the end of the study the control 
rats exhibited no significant change in P.sub.Na at 141.+-.3 meq/l. In 
comparison, the P.sub.Na of both the salt-loaded (165.+-.5 meq/l) and the 
water-deprived (151.+-.2 meq/l) groups were significantly greater than the 
control group (p&lt;0.005). In addition, the P.sub.Na of salt-loaded rats was 
significantly higher than that of water-deprived rats (p&lt;0.05). 
Consequently, compared to the control group, three days of water 
deprivation produced a P.sub.Na 7% higher, and five days of hypertonic 
salt loading produced a P.sub.Na 17% higher. 
Organic Compounds: A typical .sup.1 H NMR spectrum of a brain extract from 
a salt-loaded rat is shown in FIG. 10. Only a portion of the entire 
spectrum is shown (from -1.5 ppm to -4.5 ppm) since this is where the 
methylamines, polyols, and amino acids are located. This spectrum is 
qualitatively similar to previously published spectra of brain and shows 
characteristic large resonances for total phosphocreatine and creatine 
(PCr+Cr) and N-acetyl aspartate (NAA), two compounds known to be 
relatively abundant in brain. Also evident in this spectrum are regions 
which contain peaks representing methylamines including GPC, amino acids, 
and myo-inositol resonances. 
Amino acids: Characteristic proton resonances for NNA, glutamine, 
glutamate, GABA, aspartate, alanine, glycine, taurine, and serine were 
identified in the extracts; however, the relative contributions of 
individual amino acids to the total pool of amino acids differed markedly. 
The most abundant amino acids were identified in the region of 2.0 to 2.8 
ppm, and these included NAA, glutamate, glutamine, and GABA. FIG. 11 
compares scaled spectra of the "amino acid region" of .sup.1 H NMR spectra 
(i.e., 2.2 to 2.8 ppm) obtained from a control (dashed line) and a 
salt-loaded (solid line) rat. These spectra contain characteristic peaks 
for NAA (peaks 1-8), glutamine (peaks 9-11), glutamate (peaks 12-14) and 
GABA (peak 15). In addition, these spectra indicate that compared to 
control, the brain of the salt-loaded animal contained more glutamate and 
glutamine, but equivalent amounts of NAA and GABA. 
Table 1 summarizes the brain contents of these four amino acids in the 
three groups of rats. Glutamate, the most abundant amino acid, was 27% 
higher in the salt-loaded animals (101 nmol/mg) than in the controls (79.6 
nmol/mg). The water-deprived animals (68.1 nmol/mg) showed no significant 
change in glutamate content compared to controls. Glutamine content in 
salt-loaded rats (58.1 nmol/mg) was also significantly greater than in 
controls (35.3 nmol/mg), but was unchanged in water-deprived rats (35.9 
nmol/mg). Unlike glutamate and glutamine, neither NAA nor GABA was 
significantly changed in salt-loaded or water-deprived groups compared to 
the control group. 
TABLE 1 
______________________________________ 
Brain Contents of Major Amino Acids in Control, 
Salt-Loaded, and Water-Deprived (-H.sub.2 O) Rats 
Brain Contents (nmol/mg protein) 
Amino Acid Control Salt-Loaded -H.sub.2 O 
______________________________________ 
Glutamate 79.6 .+-. 6.1 
101.0 .+-. 6.9.sup.1 
68.1 .+-. 4.3 
Glutamine 35.3 .+-. 3.2 
58.1 .+-. 4.7.sup.2 
35.9 .+-. 7.6 
NAA 50.9 .+-. 3.7 
60.0 .+-. 5.5 
48.0 .+-. 2.4 
GABA 13.5 .+-. 1.9 
15.2 .+-. 2.2 
16.8 .+-. 1.0 
Total 180 .+-. 15 
235 .+-. 13.sup.1 
167 .+-. 11 
Amino Acids 
______________________________________ 
Contents of individual amino acids in perchloric acid extracts of brain 
were measured with .sup.1 H NMR spectroscopy. Abbreviations: NAA for 
Nacetyl aspartate, and GABA for .gamma.-aminobutyric acid. .sup.1 p &lt; 
0.02, .sup.2 p &lt; 0.002. 
Several other less abundant amino acids were identified in other portions 
of the spectrum including alanine (FIG. 10, e.g., 1.46/1.48 ppm), 
aspartate (FIG. 10, e.g., 2.78 ppm), glycine (FIG. 10, e.g., 3.35 ppm), 
taurine (FIG. 10, e.g., triplet at 3.41, 3.43 and 3.45 ppm), and serine 
(3.99 ppm). The contents of these amino acids appeared to be low when 
compared to other peaks in the spectrum and they frequently overlapped 
with neighboring peaks; therefore, they could not be quantified in many of 
the samples. The apparent low contents of these amino acids in all three 
groups of rats suggested they were unlikely to play a significant role in 
brain osmoregulation in this study. This analysis of the amino acids 
indicated, in agreement with previous observations, that glutamate, 
glutamine, NAA, and GABA are among the most abundant amino acids in the 
brain. Calculation of the total brain content of these four amino acids 
indicated that salt-loaded rats contained 31% more of these amino acids 
than control rats. However, this elevation reflected selective increases 
in glutamate and glutamine only. 
Methylamines: The inset in FIG. 12 shows that PCr+Cr (peak 9) and GPC (peak 
5) are the two most abundant methylamines in the brain. The expanded view 
of that portion of the NMR spectrum (3.14 ppm to 3.34 ppm) indicates 
several additional methylamines were present. For comparison, 
representative brain spectra from both a control (dashed line) and a 
salt-loaded (solid line) rat are shown; the spectra from water-deprived 
rats were comparable to the control rat spectrum. Identifiable in these 
spectra are betaine (peak 3), glycerophosphorylcholine or GPC (peak 5), 
phosphorylcholine or PCholine (peak 6), and choline (peak 7). The only 
form of betaine detected was glycine betaine (betaine). Although proline 
betaine has been found in human urine, this form of betaine has not been 
detected in our studies of rat brain and renal IM. Three methylamines, 
PCr+Cr, GPC and choline, were significantly elevated in salt-loaded rats 
compared to controls. 
A quantitative comparison of the methylamine contents in the brain of 
experimental and control rats is given in Table 2. In all three groups, 
PCr+Cr and GPC were the most abundant methylamines, constituting 
approximately 72-84% of the total pool. In addition, the brain content of 
PCr+Cr in salt-loaded rats (26.1.+-.1.4 nmol/mg) was 32% higher than in 
control rats (19.8.+-.1.5 nmol/mg). GPC was also significantly higher in 
salt-loaded rats (13.1.+-.1.0 nmol/mg) as compared to control rats 
(7.5.+-.0.7 nmol/mg). Neither PCr+Cr nor GPC was significantly changed 
with 3 days of water deprivation. Though less abundant, choline was 
elevated 114% in the salt-loaded group (4.7 nmol/mg) Brain choline content 
of water-deprived rats (1.5.+-.0.3 nmol/mg) was similar to controls (2.2 
nmol/mg). PCholine content was 4 nmol/mg in controls, and was unchanged in 
the two experimental groups. Betaine was relatively scarce (2.4 nmol/mg in 
controls) and failed to accumulate in either salt-loaded or water-deprived 
rats. Total methylamines (i.e., PCr+Cr+PCholine+choline+betaine) in the 
salt-loaded animals (52.1.+-.2.3 nmol/mg) were 45% higher than in control 
rats (36.0.+-.3.3 nmol/mg protein). Total brain methylamine contents of 
the control and water-deprived rats were not significantly different. 
Therefore, in salt loading, there was a significant increase in the total 
methylamine content of the brain, and PCr+Cr, GPC and choline were largely 
responsible for this change. Three days of water deprivation produced no 
significant change in methylamine contents. 
TABLE 2 
______________________________________ 
Brain Contents of Major Methylamines in Control, 
Salt-Loaded, and Water-Deprived (--H.sub.2 O) Rats 
Brain Contents (nmol/mg protein 
Methylamine 
Control Salt-Loaded --H.sub.2 O 
______________________________________ 
PCr + Cr 19.7 .+-. 1.5 
26.1 .+-. 1.4.sup.1 
18.6 .+-. 1.0 
GPC 7.5 .+-. 0.7 
13.1 .+-. 1.0.sup.3 
8.0 .+-. 0.1 
Choline 2.2 .+-. 0.3 
4.7 .+-. 0.6.sup.2 
1.5 .+-. 0.3 
PCholine 4.0 .+-. 1.0 
5.0 .+-. 1.0 
3.0 .+-. 1.0 
Betaine 2.4 .+-. 0.5 
3.4 .+-. 0.8 
0.7 .+-. 0.2 
Total 36.0 .+-. 3.3 
52.1 .+-. 2.3.sup.1 
31.7 .+-. 1.4 
Methylamines 
______________________________________ 
Contents of individual methylamines were measured with .sup.1 H NMR 
spectroscopy. Abbreviations: PCr + Cr for phosphocreatine plus creatine. 
GPC for glycerophosphorylcholine, PCholine for phosphorylcholine. .sup.1 
&lt;0.005, .sup.2 p &lt; 0.002, .sup.3 p &lt; 0.001. 
Polyols: Two polyols, myo-inositol and sorbitol, are known to exist in the 
brain and are also known to accumulate in the renal IM. .sup.1 H NMR 
spectra indicated the presence of significant quantities of myo-inositol 
but not sorbitol in the three groups of rats. FIG. 12 contains three peaks 
(1, 2 and 4) attributable to myo-inositol which appear larger in the 
salt-loaded rat compared to the control. Other proton resonances from 
myo-inositol were also clearly identified including seven peaks clustered 
in the region 3.5 to 3.7 ppm (FIG. 10) as well as a triplet at 4.06 ppm. 
Sorbitol was now visible in .sup.1 H NMR spectra from any of the brain 
extracts (e.g., 3.85 ppm, 3.67 ppm, or 3.65 ppm). 
In addition to .sup.1 H NMR spectroscopy, biochemical assays were used to 
quantify myo-inositol and sorbitol contents and these assays confirmed the 
.sup.1 H NMR analysis. The mean content of myo-inositol assayed in the 
brain extracts from the three groups was 101.+-.4% of that measured by NMR 
spectroscopy. Furthermore, though present, brain sorbitol content (about 
0.4 nmol/mg protein) was below the level detectable by NMR spectroscopy. 
The polyol contents of the three groups are listed in Table 3. 
Myo-inositol was greater than 100-fold more abundant than sorbitol in the 
brain of control animals (65.7 vs. 0.40 nmol/mg protein). Moreover, 
myo-inositol was 36% higher in salt-loaded rats (89.5 nmol/mg). Brain 
myo-inositol with 3 days of water deprivation (57.8 nmol/mg) was not 
significantly different from controls. In contrast to myo-inositol, 
sorbitol failed to change in either experimental group. Total polyols 
(i.e., myo-inositol+sorbitol) were significantly elevated in the 
salt-loaded rats (90.0.+-.8.3 nmol/mg). Total polyols in the brain 
extracts of water-deprived rats (58.2t2.0) were similar to controls 
(66.It5.1 nmol/mg). 
TABLE 3 
______________________________________ 
Brain Contents of Polyols in Control, 
Salt-Loaded, and Water-Deprived (-H.sub.2 O) Rats 
Brain Contents (nmol/mg protein) 
Polyol Control Salt-Loaded 
-H.sub.2 O 
______________________________________ 
Myo-inositol 
65.7 .+-. 5.1 
89.5 .+-. 8.3.sup.1 
57.8 .+-. 2.0 
Sorbitol 0.40 .+-. 0.10 
0.47 .+-. 0.07 
0.39 .+-. 0.05 
Total Polyols 
66.1 .+-. 5.1 
90.0 .+-. 8.3.sup.3 
58.2 .+-. 2.0 
______________________________________ 
Contents of myoinositol and sorbitol were measured in PCA extracts of 
brain with .sup.1 H NMR spectroscopy (myoinositol) or a biochemical assay 
(sorbitol). .sup.1 p &lt; 0.02, .sup.2 p &lt; 0.05. 
Total amino acids, methylamines, and polyols: The sum of the brain contents 
of the major methylamines, polyols, and amino acids in the three groups of 
rats is shown in FIG. 13. The total of these solutes in the control rats 
averaged 282.+-.22 nmol/mg whereas the salt-loaded rats contained 
377.+-.23 nmol/mg indicating that salt loading was associated with a net 
increase of 95 nmol/mg or 34% in the content of these solutes. Amino acids 
constituted the largest fraction (+58%) of this organic solute change 
exhibiting a net increase of 55 nmol/mg. The net change in polyols (24 
nmol/mg) was 25% of the total change in these solutes and this was due 
entirely to an increase in myo-inositol. The increase in methylamines (16 
nmol/mg) represented 17% of the total change. Three days of water 
deprivation did not produce an elevation in individual or total (257 
nmol/mg) organic solutes. 
D. DISCUSSION 
In the present study, .sup.1 H NMR spectroscopy and biochemical assays were 
used to identify and quantify the major organic solutes in brains of 
normal, salt-loaded, and water-deprived rats. Chronic salt loading (5 
days) caused an increase in P.sub.Na and an increase in organic solute 
contents in brain. Three-day water-deprived rats had an elevated P.sub.Na 
compared to controls; however, the degree of hypernatremia was less than 
that in salt-loaded animals, and they failed to show a significant change 
in brain organic solutes. The organic solutes which accumulated in the 
brains of salt-loaded rats belonged to three chemical classes, amino 
acids, methylamines and polyols. In particular, myo-inositol, glutamine, 
glutamate, PCr+Cr and GPC were considerably higher in salt-loaded rats 
compared with controls. Interestingly, these three classes of solutes are 
known to be involved in cell volume regulation in numerous mammalian and 
nonmammalian systems, including the hypertonic renal IM (see Background). 
Qualitatively, the brain .sup.1 H NMR spectra were identical in all three 
groups of rats. The most prominent peaks visible in the .sup.1 H NMR 
spectrum were identified as glutamate, myo-inositol, NAA, glutamine, 
PCr+Cr, GABA, and GPC, in decreasing order of content. 
It is important to note that no new organic compounds were detected in 
extracts from either salt-loaded or water-deprived rats. Rather, 
adaptation to salt loading was associated with elevation in the amounts of 
individual amino acids, methylamines, and polyols which were also present 
in the brain of control and water-deprived animals. 
The major amino acids in brain were elevated by 31% with salt loading (235 
vs. 180 nmol/mg protein; Table 1) due to selective elevation of two amino 
acids, glutamine and glutamate. Brain glutamine content was 65% or 22.8 
nmol/mg protein higher in saltloaded rats than in control rats. Glutamate, 
a stimulatory neurotransmitter, was elevated by 27%, or 21.4 nmol/mg 
protein. Brain NAA content, which is known to be very stable under various 
physiological and non-physiological conditions, was unchanged. Similarly, 
GABA, a known inhibitory neurotransmitter, was unchanged in the present 
study. Therefore, of the four major amino acids detected in brain, only 
glutamine and glutamate were significantly elevated with salt loading. 
Other less abundant amino acids were also identified in the NMR spectra; 
however, these were not quantitated because they were generally present in 
unmeasurable quantities and/or had overlapping peaks. These amino acids 
included aspartate, alanine, glycine, serine, and taurine. With the 
exception of taurine, the contents of these amino acids are known to be 
low in the brain such that their combined contents account for only 11% of 
the total amino acids. Furthermore, these amino acids account for only 14% 
of the elevation in brain total amino acids with salt-loading. Although we 
were unable to measure the contribution of these amino acids to the 
osmolyte adaptation to hypernatremia, the apparent low quantities of these 
less abundant amino acids in both control and experimental rats suggests 
that, at most, they play only a minor osmoregulatory role. 
Methylamines are known to play a prominent osmoregulatory role in marine 
vertebrates and invertebrates as well as bacteria. In the present study, 
brains of control, salt-loaded, and water-deprived rats all contained the 
same five methylamines: PCr+Cr, GPC, PCholine, betaine, and choline, in 
decreasing order of content. Total brain content of these methylamines was 
elevated by 16 nmol/mg protein, or 45% with salt loading. The methylamine 
accumulation with salt loading was very selective whereby PCr+Cr increased 
by 6.3 nmol/mg or 32%, GPC increased by 5.6 nmol/mg or 75%, and choline 
increased by 2.5 nmol/mg or 114%. In contrast, three-day water-deprived 
animals showed no change compared to controls. To our knowledge, changes 
in GPC have not previously been documented in the brain; however, GPC 
accumulates in the renal IM in water-deprived animals (see Example I), in 
cultured renal epithelial cells exposed to hyperosmolar medium (Nakamishi 
et al. (1988), supra), and in some tumors (Daly, P. F. et al., FASEB J. 
2:2596-2604 (1988)). Betaine was unchanged in the brain in the two 
experimental groups, a finding which suggested it was not performing an 
osmoregulatory role. This was a notable difference from its described role 
as an osmolyte in the renal IM. The cellular basis for the 114% rise in 
choline is unknown but this may relate to its acting as a precursor for 
GPC inside the cell. 
Total polyols (myo-inositol+sorbitol) were elevated by 36% in brain 
extracts of salt-loaded rats (90.0 vs. 66.1 nmol/mg protein in controls) 
related entirely to the accumulation of myo-inositol. There was no change 
in polyols noted in water-deprived rats. Myo-inositol was the second most 
abundant organic solute measured in the brain, was elevated 36% in 
salt-loaded rats, and accounted for 25% of the 95 nmol/mg protein increase 
in brain organic solutes observed with salt loading. In contrast, brain 
sorbitol content (0.4 nmol/mg protein) was less than 1% of the 
myo-inositol content and failed to respond to either salt loading or water 
deprivation. In a recent report, Lohr and coworkers (supra) observed 
similar polyol responses in brain extracts of salt-loaded, water-deprived 
animals: myo-inositol was elevated 53% and sorbitol was unchanged. 
Conversely, Prockop et al. (supra), studying hyperglycemia in the dog, 
found that both myoinositol and sorbitol were elevated in brain. These 
findings suggest brain is able to accumulate organic solutes in both 
salt-loading and hyperglycemia, but can utilize different solutes to 
achieve this endpoint. It is also notable that a previous study of polyols 
in the renal IM of water-deprived rats showed that when myo-inositol 
accumulated, sorbitol was unchanged. Therefore, polyols can behave as 
osmolytes in brain as well as in renal IM. 
The failure of water-deprived rats to accumulate osmolytes was somewhat 
unexpected since previous studies of hyperosmolar states have suggested 
that hypernatremia is associated with formation of idiogenic osmoles in 
the brain. However, this hypothesis is based primarily, if not 
exclusively, on studies of salt-loaded animals. Formation of idiogenic 
osmoles in brain of animals submitted solely to water deprivation (without 
salt loading) has not, to our knowledge, been reported. Based on findings 
in the present study, it seems unlikely that changes in food intake or 
body weight were important since saltloaded rats exhibited similar 
changes. Furthermore, although the shorter period of hypernatremia (i.e., 
3 days) may have been important, 3 days is sufficient for detectable, if 
not maximal, accumulation of brain idiogenic osmoles with salt loading, 
and for accumulation of osmolytes in the renal IM of water-deprived rats. 
The degree of hypernatremia could have been an important determinant of 
osmolyte accumulation since the P.sub.Na was significantly higher in 
salt-loaded rats (165 meq/l) than in water-deprived rats (151 meq/l). In 
conclusion, using .sup.1 H NMR spectroscopy, a number of specific amino 
acids, methylamines, and polyols were shown to exist and accumulate in 
brain extracts of salt-loaded rats. No accumulation of organic solutes was 
observed in a 3-day water deprivation protocol. The major brain organic 
osmolytes which accumulated in salt loading were glutamine, myo-inositol, 
glutamate, PCr+Cr, and GPC. Furthermore, the previously recognized role of 
amino acids, methylamines, and polyols in osmoregulation of other organs 
and other species suggests that these organic solutes accumulate to 
protect brain cells from the deleterious effects of cellular dehydration 
and/or accumulation of inorganic ions. 
EXAMPLE III 
Methylamines and Polyols in Kidney, Urinary Bladder, Urine, Liver, Brain, 
and Plasma 
The purpose of the present study was to use .sup.1 H nuclear magnetic 
resonance (NMR) spectroscopy to identify specific methylamines and polyols 
in several tissues of normal rats. 
A. MATERIALS AND METHODS 
Male Sprague-Dawley rats were used, as describe in Example 11. Perchloric 
acid extracts of renal IM, urinary bladder, liver, and brain were prepared 
as described in Examples I and II. Urine and plasma were prepared as 
described in Example IV, and extracted essentially as described in 
Examples I and II. NMR spectroscopy was performed as described in Examples 
I and II. 
B. RESULTS 
FIG. 14 is a typical .sup.1 H NMR spectrum of normal rat renal IM which 
shows characteristic osmolyte peaks including methylamines, polyols, and 
lactate. In particular, methyl protons of both GPC and betaine are evident 
at 3.23 and 3.27 ppm, respectively. A companion GPC resonance is evident 
at 4.32 ppm whereas a companion betaine resonance is apparent at 3.91 ppm. 
Peaks characteristic of myoinositol (4.06, 3.61, and 3.65 ppm) and 
sorbitol (3.85 ppm) can be detected. Upfield from the methylamines and 
polyols are a variety of smaller peaks including resonances characteristic 
of lactate. Since NH.sub.3 protons can freely exchange with the deuterium 
in D.sub.2, there is no signal from urea. 
FIG. 15a is a typical .sup.1 H NMR spectrum of a urinary bladder and FIG. 
15b provides an expanded view of the region from 2.9 to 3.4 ppm which is 
known to contain methylamines. These spectra show distinct differences in 
methylamine content of the bladder and the IM. Unlike the IM, the bladder 
has multiple resonances in the region of 3.0 ppm, most of which were 
unidentified. This spectrum also indicates that neither GPC (3.23 ppm) nor 
betaine (3.27 ppm) was the most prominent compound in this region of the 
spectrum. Characteristic resonances for myo-inositol, but not sorbitol, 
were also detected in the spectra. 
A typical .sup.1 H NMR spectrum of rat urine (FIG. 16) indicates the 
presence of several methylamines including betaine, GPC and choline. 
Neither myo-inositol nor sorbitol was apparent but other unidentified 
organic solutes were detected. 
A typical .sup.1 H NMR spectrum of liver is shown in FIG. 17. Numerous 
organic solutes were present in this extract; however, due to extensive 
overlapping of peaks in the 3.3-4.5 ppm region, it was not possible to 
identify most of the compounds. Characteristic GPC and betaine methylamine 
peaks were discernible at 3.23 and 3.27 ppm. 
A typical .sup.1 H NMR spectrum of a brain extract is shown in FIG. 18. The 
largest peaks represent phosphocreatine and creatine (PCr+Cr) and N-acetyl 
aspartate (NAA). The major classes of compounds which are apparent in this 
spectrum included amino acids, methylamines, and polyols. Major amino 
acids which were detected included glutamine, glutamate, 
.gamma.-aminobutyric acid (GABA), and NAA. Other amino acids which were 
present but were relatively less abundant included taurine (TAU), alanine, 
and serine. Methylamines included GPC and betaine; polyols included 
myo-inositol. 
FIG. 19 is a typical .sup.1 H NMR spectrum of a PCA extract of plasma which 
shows only the methylamine region (2.9-3.3 ppm). Several methylamines 
including betaine, GPC, choline, and PCr+Cr were detected. Neither 
myo-inositol nor sorbitol was detected. 
C. DISCUSSION 
The present investigation confirmed the presence of significant glutamine 
and glutamate in the brain of normal animals. In addition, we observed a 
variety of methylamines including GPC, betaine, phosphorylcholine, and 
choline. Several strong resonances representing myo-inositol were also 
observed in the brain spectra suggesting a role for this polyol in 
osmoregulation in the brain. 
Analysis of plasma showed that several methylamines, including GPC, choline 
and PCr+Cr, circulate in the blood and offer a potential source of 
osmolytes for delivery to any organ in the body. 
In conclusion, this investigation showed that solutes which are known to 
act as organic osmolytes in the renal IM, including methylamines, polyols 
and amino acids, are also present in a variety of extra-renal locations. 
EXAMPLE IV 
Methylamine and Polyol Responses to Salt Loading in Renal IM 
This study examined the responses of IM methylamines, polyols, and total 
osmolytes in a diuretic state, salt loading. Using a model of salt loading 
modified from Arieff et al. (1977, supra), we tested the hypothesis that 
IM methylamines, polyols, and total osmolytes would respond uniformly, and 
in a parallel fashion, to a decrease in urine osmolality (U.sub.osmol). 
The results indicated that salt loading promoted a 42% decrease in 
U.sub.osmol but the total pool of methylamines and polyols was unchanged. 
In fact, betaine and sorbitol were elevated with salt loading, indicating 
a dissociation in their levels from U.sub.osmol and demonstrating that 
U.sub.osmol was not the predominant stimulus for accumulation of these 
solutes. Finally, GPC was directly correlated with U.sub.osmol, and this 
was the only organic osmolyte demonstrated to have this association. 
A. MATERIALS AND METHODS 
Animals were male Sprague-Dawley rats, as described in Example II. Tissue 
extracts were prepared as described in Examples I and II. Plasma sodium 
concentration was measured in blood collected from the tail at the 
beginning of the experiment and from trunk blood collected at the time of 
death. Sequential, 24 hour urine samples were collected under mineral oil 
for daily analyses of volume, sodium, and osmolality. Sodium 
concentrations were measured by flame photometry (Instrumentation 
Laboratories), and osmolality was measured with a vapor pressure osmometer 
(Wescor, No. 5100, Logan, UT). Osmolytes were measured with .sup.1 H-NMR 
spectroscopy or by biochemical assays as described in Examples I and II. 
Statistical analyses were performed as described in Examples I and II. 
B. RESULTS 
Metabolic parameters. As shown in Table 4, salt loading resulted in a 
significant increase in plasma sodium concentration (i.e., hypernatremia) 
from 144.+-.3 to 163.+-.4 meq/l, whereas control rats exhibited no 
significant change in plasma sodium concentration from their initial level 
of 143.+-.1 meq/l. It is important to note that plasma sodium values in 
salt-loaded rats were determined in samples obtained 24 h after NaCl 
gavage and presumably reflect minimum or "trough" values. In addition, 
salt-loaded rats reduced their food intake by 81% and lost 22% of their 
body weight. 
FIG. 20A-D show time-dependent changes in urine volume, sodium 
concentration, sodium excretion, and osmolality in salt-loaded and control 
animals. Control and salt-loaded (FIG. 20A) rats excreted 12.0.+-.1.4 and 
12.6.+-.1.2 ml (p&gt;0.5) of urine, respectively, on day 0. Though urine 
volume of controls did not change significantly throughout the protocol, 
the salt-loaded rats developed marked saline diuresis that resulted in a 
4- to 5-fold increase in urine volume on days 1-5. Urine sodium 
concentration (FIG. 20B) of control rats was 155.+-.24 meq/l on day 0 and 
did not change significantly throughout the protocol. In contrast, urine 
sodium concentration in salt-loaded rats rose significantly from 186.+-.18 
meq/l on day 0 (p&lt;0.5 vs. control) to 426.+-.15 meq/l on day 2 (p&lt;0.001), 
and plateaued at this level throughout the remainder of the protocol. 
Urine sodium excretion (FIG. 20C) in control rats and salt-loaded rats was 
similar on day 0 (1.79.+-.0.20 and 2.20.+-.0.22 meq sodium/day, 
respectively, p&lt;0.5). Sodium excretion in controls did not change 
significantly from the initial rate on any day throughout the protocol 
(final day 2.27.+-.0.27 meq sodium/day). In contrast, urine sodium 
excretion in salt-loaded rats increased some 15-fold to a maximum of 
4.6.+-.3.9 meq sodium/day on day 2 and plateaued at about 30 meq 
sodium/day for the remainder of the protocol. Sodium excretion of 
salt-loaded rats at the end of the protocol (day 5) was 30.9.+-.7.1 
meq/day, significantly higher than their level on day 0 (p&lt;0.01) and 
nearly 13-fold higher than control rats on the final day (p&lt;0.01). 
TABLE 4 
______________________________________ 
Metabolic Parameters 
Control Salt-Loaded 
Initial Final Initial Final 
______________________________________ 
Plasma Na.sup.+, 
143 .+-. 1 
139 .+-. 3 
144 .+-. 3 
163 .+-. 4* 
meq/l 
Body wt, g 
347 .+-. 8 
369 .+-. 7** 
342 .+-. 3 
263 .+-. 6* 
Food intake, 
27.2 .+-. 2.0 
26.0 .+-. 2.0 
24.3 .+-. 2.0 
4.7 .+-. 2.0* 
g/day 
______________________________________ 
Values are means .+-. SE. Plasma sodium concentration, body weight, and 
food intake were measured at the beginning (day 0) and end (day 4 for 
controls, day 5 for saltloaded ) of eaxh protocol. The saltloaded rats 
exhibited an increased plasma sodium concentration, decreased body weight 
and decreased food intake. * p &lt; 0.001 vs. initial: **p &lt; 0.05 vs. 
initial. 
Despite the significant rise in urine sodium concentration (FIG. 20B), 
salt-loaded rats exhibited a 34% fall in U.sub.osmol, from 1,885.+-.105 
mosmol/kgH.sub.2 O on day 0 to 1,246.+-.115 mosmol/kgH.sub.2 O (p&lt;0.001) 
on day 5 (FIG. 20D). The control rats experienced a slight rise in urine 
osmolality from 1,849.+-.62 to 2,147.+-.95 mosmol/kgH20 (p&lt;0.05). At the 
end of the protocols the control and salt-loaded groups of rats differed 
significantly in urine output, sodium concentration, sodium excretion, and 
osmolality. Furthermore, within 2 days the salt-loaded rats achieved a 
steady state in urinary excretion of volume, sodium, and osmolality. 
FIG. 21 compares the IM contents of methylamines and polyols observed in 
control and salt-loaded rats. The IM methylamines, GPC and betaine, were 
both altered significantly with salt loading. GPC was 41% lower in 
salt-loaded rats (200.+-.17 nmol/g wet wt). In striking contrast, betaine 
was 286% higher in salt-loaded rats (251.+-.26 nmol/mg protein; 
22.6.+-.2.7 .mu.mol/g wet wt) than in control rats (65.0.+-.10.0 nmol/mg 
protein; 6.5.+-.1.4 .mu.mol/g wet wt). Because these methylamines changed 
in opposite directions with salt loading, the total methylamine pool 
(GPC+betaine) of the salt-loaded animals (451 .+-.25 nmol/mg protein; 
43.6.+-.3.5 .mu.mol/g wet wt) was not significantly different from the 
methylamine pool of the control rats (406.+-.32 nmol/mg protein; 
39.2.+-.4.1 .mu.mol/g wet wt, FIG. 22). 
IM sorbitol content in control rats was 129.+-.7 nmol/mg protein 
(12.6.+-.1.3 .mu.mol/g wet wt) and it was 33% higher in salt-loaded rats 
at 171.+-.6 nmol/mg protein (16.7.+-.1.3 .mu.mol/g wet wt). In contrast, 
the myo-inositol content of salt-loaded rats (276.+-.40 nmol/mg protein; 
26.9.+-.4.4 .mu.mol/g wet wt) was not significantly different from that of 
control rats (302.+-.27 nmol/mg protein; 29.2.+-.3.1 .mu.mol/g wet wt). 
Total polyols (sorbitol+myo-inositol) in controls were 431 .+-.31 nmol/mg 
protein (41.7.+-.4.0 .mu.mol/g wet wt) vs. 447.+-.45 nmol/mg protein in 
salt-loaded animals (43.6.+-.5.5 .mu.mol/g wet wt), demonstrating no 
significant change (FIG. 22). 
FIG. 22 compares the total IM organic osmolyte contents (i.e., 
GPC+betaine+myo-inositol+sorbitol) in the two groups of rats. Control rats 
had a total of 837.+-.nmol/mg protein (81.0.+-.6.6 .mu.mol/g wet wt) 
compared with 898.+-.53 nmol/mg protein (87.1.+-.8.1 .mu.mol/g wet wt, 
p&gt;0.5), in salt-loaded rats, demonstrating that chronic salt loading did 
not alter significantly the total organic osmolyte pool of the IM. 
Furthermore, neither total methylamines nor total polyols were 
significantly affected by salt loading (FIG. 22) in spite of the 
significant changes observed in GPC, betaine, and sorbitol (FIG. 21). 
C. DISCUSSION 
This study examined changes in IM methylamines and polyols, which occur in 
response to the saline diuresis associated with salt loading. Salt loading 
produced a fall in urine osmolality and marked increases in urine volume 
and sodium excretion. Specifically, urine osmolality in salt-loaded 
animals (1,246.+-.115 mosmol/kgH.sub.2 O) was 42% less than in controls. 
Urine volume and sodium excretion were 427 and 1262% higher, respectively, 
in salt-loaded rats. Consequently, salt loading produced sustained saline 
diuresis and decreased urine osmolality. 
The most striking finding in this study was that GPC was the only IM 
organic osmolyte that changed in parallel with urine osmolality. IM GPC 
content in salt-loaded rats was 41% lower than controls compared to 
U.sub.osmol which was 42% lower. 
To address whether there was a direct relationship between GPC content and 
U.sub.osmol we compared the results of this study with those in Example I, 
above, with other data obtained in our laboratory using different rat 
strains and water states. FIG. 23 indicates that there is a direct and 
highly significant correlation (p&lt;0.001) between IM GPC content and urine 
osmolality. The slope of the regression line is -0.137 nmol GPC.mg 
protein.sup.-1. Analysis of all the individual data from the present study 
also showed a significant correlation (p&lt;0.001) between GPC and 
U.sub.osmol and the slope of the regression line (0.151 nmol GPC-mg 
protein.sup.-1.mosmol.sup.-1, r=0.72) was comparable to that shown in FIG. 
4. 
In summary, significant changes in individual IM methylamines and polyols 
were induced with salt loading without a change in total osmolytes. Only 
GPC content was directly correlated with urine osmolality, and this 
correlation was confirmed in three separate studies in our laboratory. IM 
myo-inositol content was similar in control and salt-loaded rats, 
appearing not to participate in an osmoregulatory response. In addition, 
with salt loading, IM betaine and sorbitol levels were dissociated from 
urine osmolality but paralleled elevations in urine and plasma sodium, 
which might facilitate accumulation of these solutes. Urine osmolality, 
however, could not be implicated in the accumulation of betaine and 
sorbitol. 
EXAMPLE V 
Hypertonicity-Induced Myo-Inositol Accumulation In C6 Glimoa Cells: A Model 
of Brain Osmoregulation 
To characterize the mechanisms of neural cell organic osmolyte regulation, 
rat C6 glioma cells were cultured in a medium made hypertonic (440 mOsm) 
by addition of 90 mM NaCl. Partially confluent (30%) C6 cultures exposed 
to gradual increases in NaCl concentration (30 mM every other day) 
followed by 6 days of maintenance in 440 mOsm medium showed normal growth 
and survival. 
.sup.1 H NMR revealed that the major organic osmolyte accumulated in C6 
cells during exposure to hypertonic medium was myo-inositol. Enzymatic 
assays demonstrated that cell myo-inositol content increased significantly 
from 45.+-.5 nmol/mg prot. (n=4) in control medium to 216 .+-.25 nmol/mg 
prot (n=4) in 440 mOsm medium. Total cellular myoinositol content 
increased rapidly from 78.+-.11 nmol/mg protein in control medium to 
243.+-.24 nmol/mg protein (n=4) after 10 hrs of exposure to 440 mOsm 
medium and remained constant for an additional 62 hrs. 
These results indicate that myo-inositol plays an important role in C6 cell 
volume regulation. C6 cells provide a useful model for understanding brain 
organic osmolyte regulation during disturbance in extracellular fluid 
osmolality. 
To examine the mechanism responsible for myo-inositol accumulation by C6 
glioma cells, myo-inositol transport studies were performed. The results 
indicated that C6 cells possess a phlorizininhibitable myo-inositol 
transport pathway which is responsible for accumulation of myo-inositol by 
these cells under hyperosmolar conditions. 
Confluent C6 cells were treated for 4, 10, and 24 hours with control medium 
or hyperosmolar (+NaCl) medium. At each time point, radioactive 
myo-inositol uptake by the cells was measured as follows. Cells were 
washed twice with PBS of the appropriate osmolality and 3 ml of control or 
hyperosmolar medium containing .sup.3 H-inositol was added to each plate. 
After 1 minute the medium was rapidly removed and the cells were washed 
three times with ice-cold "stop" solution (0.1 M MgCl.sub.2 +0.1 mM 
phlorizin for control cells and 0.15 M MgCl.sub.2 +phlorizin for 
hyperosmolar cells). The cells were scraped from the dish in the presence 
of 1 ml perchloric acid. The .sup.3 H-inositol content of the PCA extract 
was determined by scintillation spectroscopy. Parallel experiments 
determined the .sup.3 H-inositol uptake in the presence of phlorizin, an 
inhibitor of Na+-inositol cotransport. The results indicated that the C6 
cells possess an myo-inositol uptake mechanism which is inhibited 80 to 
90% by phlorizin. The transport rate of cells under control conditions was 
77.+-.4 pmol/min/mg protein. Moreover, there was an up-regulation of the 
phlorizin-inhibitable myoinositol transport under hyperosmolar conditions 
(FIG. 24). The increase in myo-inositol transport corresponded exactly 
with the time course of myo-inositol accumulation by these cells. In 
addition, the uptake rate was sufficient to account for all of the 
myo-inositol accumulated by the cells under hyperosmolar conditions. In 
conclusion, C 6 glioma cells, a model of brain glial cells, accumulate 
myo-inositol under hyperosmolar conditions by uptake from the 
extracellular environment. 
EXAMPLE VI 
Restoration of Brain Tissue Water Levels Under Hyperosmolar Conditions with 
Osmolytes 
The loss of water from brain tissue which accompanies hypernatremic 
conditions was modeled in a system of water loss from rabbit brain tissue 
slices induced by high salt. The ability of two organic osmolytes, 
myo-inositol and glutamine, to correct this dehydration was assessed. 
Rabbits were anesthetized with ketamine plus ether and the cranium was 
surgically opened. Brain tissue was removed and placed in ice-cold serum. 
Brain tissue was sliced using a Stadie-Riggs tissue slicer. The slices 
(0.2 to 0.5 g wet weight) were placed in capped polycarbonate flasks 
containing the appropriate experimental medium, gassed with a mixture of 
95% O.sub.2 and 5% CO.sub.2, and incubated in a shaking water bath at 
37.degree. C. The experimental medium was either rabbit serum, (Control), 
serum+100 mM NaCl (hyperosmolar), or serum+100 mM NaCl +myo-inositol (2 
mM) and/or glutamine (2 mM). After 3 hours of incubation, the tissue was 
blotted with filter paper and wet weight was determined with an analytical 
balance. The tissue was dried for 18-24 hours at 100.degree. C. and dry 
weight was then determined. The % Tissue Water was calculated as: [(wet 
wt. -dry wt.) / wet wt.].times.100. 
As shown in FIG. 25, brain slices incubated in serum contained 83.3% water, 
whereas the tissue water of slices exposed to hyperosmolar NaCl, to mimic 
hypernatremia, was reduced markedly, to 80.2%. This water loss was almost 
completely reversed by addition of 2 mM myo-inositol (82.6%), 2 mM 
glutamine (82.7%), or a mixture of myoinositol and glutamine (85%). 
EXAMPLE VII 
The Effect of Hyponatremia on Brain Organic Osmolytes 
Sprague-Dawley rats were made hyponatremic as described previously 
(Verbalis, J. G. et al., Kidney International 34:351-360 (1988)). Briefly, 
rats were placed on a nutritionally balanced liquid diet (AIN-76, 
Bio-Serv, Frenchtown, NJ) formulated as follows: 258 g powered formula+520 
ml of a solution of 14% dextrose. Each day the rats received 40 ml of the 
liquid diet. Control rats also had access to tap water ad lib. 
Subcutaneous osmotic minipumps were implanted to enable continuous 
infusion of DDAVP, a vasopressin analogue, to hyponatremic rats or 
infusion of saline to control rats. Plasma Na.sup.+ concentrations were 
142 and 102 meq/l in control and hyponatremic rats, respectively. At 0, 2, 
7, and 14 days rats were sacrificed and the brains were rapidly removed, 
bisected, and weighed. One hemisphere was used to prepare a perchloric 
acid (PCA) extract for measurement of organic osmolytes as described 
previously (Heilig, C. W. et al Am. J. Physiol. 257:F1108-F1116 (1989). 
Neutralized PCA extracts were analyzed by high performance liquid 
chromatography (HPLC) as described previously (Wolff, S. D. et al., Am. J. 
Physiol. 256:F954-F956 (1989). At days 2, 7 and 14, the levels of 
myoinositol, glutamate, glutamine, glycerophosphorylcholine, taurine and 
creatine were lower in brains of hyponatremic rats than in control rat 
brains. (FIG. 26A-26F). These results indicate that as the serum sodium 
concentration decreases, brain organic osmolytes become depleted. 
Having now fully described this invention, it will be appreciated by those 
skilled in the art that the same can be performed within a wide range of 
equivalent parameters, concentrations, and conditions without departing 
from the spirit and scope of the invention and without undue 
experimentation. 
While this invention has been described in connection with specific 
embodiments thereof, it will be understood that it is capable of further 
modifications. This application is intended to cover any variations, uses, 
or adaptations of the inventions following, in general, the principles of 
the invention and including such departures from the present disclosure as 
come within known or customary practice within the art to which the 
invention pertains and as may be applied to the essential features 
hereinbefore set forth as follows in the scope of the appended claims.