Brain Uptake of Myoinositol after Exogenous Administration
Stephen M. Silver,
Barbara M. Schroeder and
Richard H. Sterns
Department of Medicine, Rochester General Hospital, University of Rochester School of Medicine, Rochester, New York.
Correspondence to: Dr. Stephen M. Silver, Department of Medicine, Rochester General Hospital, 1425 Portland Avenue, Rochester, NY 14621. Phone: 716–922–4707; Fax: 716–922–5223; E–mail: stephen.silver{at}viahealth.org
ABSTRACT. An acute increase in plasma tonicity results in anadaptive increase in brain organic osmolyte content, but thisprocess requires several days to occur. Slow reaccumulationof brain organic osmolytes may contribute to osmotic demyelination.It was investigated whether administration of intravenous myoinositolin rats could speed entry of the osmolyte into the brain. Twogroups of animals were studied: normonatremic animals and animalswith hyponatremia (105 mmol/L) of 3–d duration. Animalswere intravenously administered either 1 M NaCl to induce a25 to 28 mM increase in serum sodium concentration over 200min or an infusate that maintained serum sodium concentration.In some animals, myoinositol was administered intravenouslyover the same time period to raise plasma myoinositol levelsby 5 to 10 mM. Brain myoinositol, electrolyte, and water contentswere determined at the end of the infusions. In both normonatremicand hyponatremic rats, infusion of hypertonic saline withoutmyoinositol or infusion of myoinositol without hypertonic salinedid not increase brain myoinositol levels above control levels.In normonatremic animals, concurrent infusion of hypertonicsaline and myoinositol increased brain myoinositol levels byabout 50% above control levels. Brain myoinositol content inanimals with uncorrected hyponatremia was about 50% of thatfound in normonatremic controls; concurrent infusion of hypertonicsaline and myoinositol increased brain myoinositol to levelssimilar to those found in normonatremic controls. Intravenousinfusion of myoinositol did not alter brain water content comparedwith animals not infused with myoinositol. In conclusion, systemicinfusion of myoinositol can rapidly increase brain myoinositolcontent, but only when plasma tonicity is concomitantly increased.
Maintenance of brain volume in response to osmotic stress iscrucial for survival. After acute hypernatremia or the acutecorrection of chronic hyponatremia, the brain initially gainsinorganic solutes within minutes to hours and then accumulatesorganic osmolytes over hours to days (1–5). This relativelyslow increase in brain organic osmolytes has been attributedto the time required for production of transporters requiredto bring organic osmolytes into the cell (6). Delayed reaccumulationof organic osmolytes after an acute increase in plasma tonicitymay contribute to development of osmotic demyelination (7).This hypothesis is supported by preliminary evidence that uremia,which protects against osmotic demyelination, also induces anearly increase in brain myoinositol content (8,9). We questionedwhether exogenous infusion of organic osmolytes could directlyincrease their content in brain. In preliminary studies (10),we compared brain uptake of myoinositol and taurine, two majorbrain organic osmolytes that have safely been given to humans(11–14). In these studies, infusion of taurine did notaffect brain taurine content; thus, only the effect of myoinositolis now described. We explored whether systemic infusion of myoinositolcould increase the brain contents of this solute in normal andacutely hypernatremic animals. We observed that myoinositolentered brain after infusion, but only during concomitant inductionof hypernatremia. We then investigated the effect of myoinositolinfusion on brain metabolism in hyponatremia and its acute correction,and we observed that infused myoinositol entered brain onlywhen hyponatremia was being acutely corrected.
For all studies, male Sprague–Dawley rats (Harlan, Indianapolis,IN) weighing 275 to 350 g were used. Rats were housed in ananimal facility according to National Institutes of Health andinstitutional animal care and use guidelines.
Surgical Insertion of Indwelling Jugular Dual Catheter
Animals were weighed and anesthetized with pentobarbital (55mg/kg intraperitoneally). An incision was made between the shoulderblades. The external jugular vein was exposed through a 2.5–cmtransverse incision in the right anterior neck region. A dualcatheter (Dow Corning Silastic; Helix Medical, Inc., Carpenteria,CA) with a silastic button was drawn subcutaneously from theincision to the exposed vein. The silastic button was positionedbehind the right ear and secured with two 5–0 Prolenesutures. The vein was ligated 2 cm proximal to the heart, andthe dual catheter was placed through it and inserted into theatrium. Sutures were firmly tied around the catheter and vein.Efficacy was confirmed by withdrawing blood into both catheterswith a syringe. The incision at the neck was closed with 5–0Prolene sutures. The animal was placed in a prone position,and the dorsal end of the dual catheter was drawn through astainless steel anchor button and protective tether (Kent ScientificCorporation, Litchfield, CT). The anchor button was attachedunder the skin, and the dorsal incision was closed with 5–0Prolene sutures. The tether was attached to a Precision 22 gaugesingle channel fluid swivel (Kent Scientific Corporation) connectedto a stand. Four hours after surgery, catheters were flushedwith saline and locked with 100 U/ml heparin. Animals were housedindividually with free access to chow and water and were alloweda 2–d recovery period. This technique allowed continuousintravenous infusion and blood sampling in unanesthetized, unrestrainedrats.
Experimental Protocols
Two experiments were performed. Experiment I consisted of rapidinduction of hypernatremia in normonatremic animals, with orwithout concomitant myoinositol infusion. In experiment II,the plasma sodium concentration in chronically hyponatremicanimals was rapidly corrected with or without concomitant myoinositolinfusion.
For all infusions in both experiments, an initial rapid infusionof fluid at a rate of 1.67 ml/100 g body wt for 20 min was followedby a slower infusion rate of 0.67 ml/100 g body wt for 180 min,resulting in a total infusion time of 200 min. When myoinositolwas added to the infusion, 3.33 mmol/kg body wt was infusedover the first 20 min to increase plasma concentration of myoinositol,and then 3.33 mmol/kg per h was administered for 180 min. Atthe end of the infusion, animals were killed and brain was obtainedfor analyses as described below. Mannitol served as a controlsolute in experiments I and II and was infused at a rate determinedin preliminary studies in normonatremic animals to approximatethe increase in plasma osmolality obtained by myoinositol infusion.Mannitol (0.94 mmol/kg body wt) was infused over 20 min andmaintained at a rate of 0.94 mmol/kg per hr.
Experiment I: Effect of Myoinositol Infusion on Brain Adaptation to Acute Hypernatremia.
Rats were divided into four experimental groups:
Group 1: Normonatremia,surgical control (n = 6).
Group 2: Infusion of myoinositolin water (n = 6).
Group 3: Infusion of myoinositol and hypertonic(1 M) saline(n = 6).
Group 4: Infusion of mannitol and hypertonic(1 M) saline (n= 6).
All animals were awake, unrestrained, and had free access towater and standard rat chow until infusions were begun. Indwellingcatheters (described above) were placed in infused animals (groups2 to 4) at least 2 d before infusion. Before infusion, plasmawas obtained from the catheter for measurement of sodium, glucose,and urea concentration. Plasma was sampled again to obtain thesevalues and myoinositol and mannitol levels at 20 min (immediatelyafter completion of the rapid infusion of solute), 80 min, and200 min after beginning the infusion. Then, at 200 min, animalswere killed by decapitation, and brain was immediately removedand processed for analyses as described previously (16).
Experiment II. Effect of Myoinositol Infusion on Brain Adaptation to Hyponatremia and Its Rapid Correction.
On day 1, indwelling dual catheters were inserted into ratsas described previously. Rats were allowed a 6–h recoveryperiod before induction of hyponatremia. Chronic hyponatremiawas induced by continuous infusion of synthetic vasopressinvia subcutaneous osmotic pumps (Alzet Corporation, MountainView, CA) at a rate of 20 mU/hr and intravenous infusion of2.5% glucose. The 2.5% glucose was administered at the rateof 15 ml/100 g body wt over 12 h on day 1, 12 ml/100 g bodywt over 12 h on day 2, and 8 ml/100 g body wt over 12 h on days3 and 4. Rats were given free access to a low–potassium,low–sodium diet (ICN Biomedicals, Inc., Costa Mesa, CA)prepared in 10% dextrose at 0.36 g/ml supplemented with 2% vitamin–free,salt–free casein hydrolysate (ICN Biomedicals). On day4, 2–ml blood samples were taken from all hyponatremicrats for measurement of plasma sodium by flame photometry andplasma myoinositol concentration by HPLC. The rats were randomizedinto one of four experimental groups, which are listed below.Myoinositol and mannitol were infused in the same amount andrate used in experiment I.
Group 1: Chronic hyponatremia (surgical control).
Group 2.Chronic hyponatremia infused with myoinositol and hypotonic(105 mM) saline.
Group 3: Rapid correction of chronic hyponatremiaby infusionwith hypertonic (1 M) saline and myoinositol.
Group4: Rapid correction of chronic hyponatremia by infusionwithhypertonic (1 M) saline and mannitol.
In groups 2 to 4, plasma was obtained for measurement of serumsodium, urea, glucose, myoinositol, and mannitol at the endof the 200-min infusion (just before the animals were killed).
Analytic Procedures Plasma Sodium.
Plasma sodium was measured by flame photometry (943, InstrumentationLaboratory, Boston, MA).
Brain Water and Electrolytes.
A hemisphere of the brain was dried at 100°C for 48 h andreweighed to determine water content. The dried tissue was thencrushed and dissolved in 0.75 N HNO3 for sodium and potassiumanalyses by flame photometry.
Brain Organic Osmolyte Content.
The method for measurement of organic osmolyte content in thebrain has been described previously (16). Frozen brain tissuewas crushed under liquid nitrogen and lyophilized. Amino acidcontent of lyophilized tissue was then determined by HPLC afterderivitization with phenylisothiocyanate (Pico Tag, Waters Corporation,Milford, MA). Methionine sulfone served as the internal standard.The contents of myoinositol, mannitol, and creatine were determinedby HPLC using a Sugar Pak I column (Waters). Maltose servedas the internal standard. Levels of organic osmolytes were quantifiedon a Waters Millenium 2001 chromatography workstation.
Plasma Organic Osmolyte and Mannitol Concentration.
The method for measurement of organic osmolyte concentrationin plasma has been previously described (16). Plasma was extractedby addition of an equal volume of 6% perchloric acid containing2 mM maltose as internal standard. The concentrations of myoinositolglucose and mannitol urea were determined by HPLC using a SugarPak I column.
Statistical Methods
Data are expressed as mean values ± SE. Differences inplasma and brain analyses between the groups in experimentsI and II were assessed by a one–factor ANOVA with significancedetermined by Scheffe F–test (StatView 512+ Brain Power,Calabasas, CA). Significance was accepted at the P < 0.05level.
Experiment I Plasma Analyses.
Before infusion, plasma sodium was equivalent in all groups.Twenty minutes after initiation of the infusion, the time atwhich the rapid infusion of solute was complete, plasma sodiumhad decreased to 141 mM in group 2 (myoinositol and water infused)and remained at this level at 80 and 200 min postinfusion. Thisdecrease in plasma sodium was partially offset by an increasein plasma myoinositol; in animals infused with myoinositol (groups2 and 3), plasma myoinositol was significantly elevated at alltime points after initiation of the infusion. In animals infusedwith hypertonic saline (groups 3 and 4), plasma sodium was significantlyincreased 20 min after initiation of the infusion and remainedat about this level throughout the infusion. At 200 min, plasmasodium was slightly but not significantly increased in group3 compared with group 4. The plasma mannitol concentration achievedin group 4 was 2.5 mM less than myoinositol concentrations ingroup 3 (Table 1).
Brain Tissue Analyses.
Brain myoinositol content increased significantly in animalsinfused with myoinositol and hypertonic saline (group 3) incomparison with other groups. However, this increase in myoinositoldid not prevent brain shrinkage, and an initial increase inbrain electrolytes occurred as has been observed after acutehypernatremia without myoinositol infusion: brain water decreasedsignificantly and brain sodium content increased in both hypernatremicgroups. Brain potassium content increased only in group 3. Thesum of other major brain organic osmolytes was equivalent after200 min in all groups. No brain mannitol was detected in mannitol–infusedanimals (Table 2; Figure 1).
Figure 1. Brain myoinositol content in experiment I. *P < 0.01 versus other groups.
Experiment II Plasma Values.
Significant hyponatremia was induced after 24 h and maintainedfor 3 d, and all groups had an equivalent degree of hyponatremiabefore initiation of the experimental protocol. In animals infusedwith myoinositol and 105 mM saline (group 2), serum sodium didnot change during the experiment, but myoinositol concentrationincreased significantly. In groups 3 and 4, plasma sodium increasedby 28 and 25 mM respectively over the 200–min experimentalperiod. Plasma myoinositol and mannitol increased in the plasmaof the groups infused with these solutes. Plasma myoinositollevels appeared substantially higher than those measured inexperiment I (Table 3).
Table 3. Plasma analysis, experimentc 2(all values in mM)
Brain Analyses.
There was no difference in brain water, electrolyte, or organicosmolyte content (including myoinositol) between uninfused hyponatremicanimals and those infused with myoinositol. In chronically hyponatremicanimals that were acutely corrected with hypertonic saline,there was no difference in brain water or electrolyte contentbetween animals infused with myoinositol or with mannitol asa control. However, there was a substantial increase in brainmyoinositol content in animals infused with myoinositol andhypertonic saline compared with all other groups (Figure 2).The sum of other major brain organic osmolytes was equivalentafter 200 min in all groups. No brain mannitol was detectedin mannitol–infused animals (Table 4; Figure 2).
Systemic administration of myoinositol in rats significantlyincreased the brain myoinositol content in 200 min when plasmasodium was simultaneously increased; it occurred in normonatremicanimals made acutely hypernatremic and in hyponatremic animalsin which plasma sodium was rapidly corrected. Myoinositol infusiondid not affect brain myoinositol content when plasma sodiumwas not increased—in both normonatremic and hyponatremicanimals. There was no increase in brain myoinositol within 200min of hypernatremia or after correction of hyponatremia inanimals without myoinositol infusion. It cannot be determinedon the basis of our results whether an increase in plasma sodiumor a less specific increase in plasma tonicity or osmolalityis required for infused myoinositol to increase brain myoinositolcontent.
Plasma myoinositol levels in hyponatremic animals (experimentII) were significantly higher than in normonatremic animals(experiment I) infused with equivalent amounts of myoinositol.The cause for this is unclear, but a difference in the volumeof distribution or metabolism of myoinositol in hyponatremicanimals is implied. Plasma myoinositol was substantially higherthan mannitol levels in corrected hyponatremic animals. It isunlikely that this difference in tonicity contributed to thedifference in brain myoinositol content between the two groupsin light of other studies that have also failed to demonstratechanges in brain myoinositol after acute correction of hyponatremia(1,17).
Most likely, the increase in brain myoinositol content aftermyoinositol and hypertonic saline infusion was at least partiallydue to increased uptake by brain cells; if myoinositol had beenretained in the extracellular fluid only, extracellular myoinositollevels would have to have been significantly higher than inplasma. When studied in rabbits, myoinositol entrance into thecentral nervous system appears to be regulated by a saturabletransport system that may serve to maintain cerebrospinal fluidand brain myoinositol concentration despite fluctuation of plasmamyoinositol (18). Other studies in animals have demonstratedan increase in brain myoinositol after much larger infusionsof myoinositol (30 to 60 mmol/kg), but no measure of plasmatonicity was performed (19–20).
Our results imply that there is a mechanism for rapid myoinositoluptake into brain cells that is dependent on substrate availabilityand thus can be enhanced by administration of myoinositol. Moreover,this process appears to be dependent on an increase in plasmatonicity. Brain cell myoinositol content increases after chronichypernatremia; this process is mediated by gene expression ofmRNA encoding for the Na+ myoinositol transporter (SMIT), whichleads to increased synthesis and membrane insertion of the transportproteins (21–22). An increase in SMIT mRNA level occurs4 to 6 h before activation of the transporter; therefore, thisprocess is unlikely to account for the increase in brain myoinositolwe observed after only 200 min (23). Increased exposure to substrateleading to increased uptake by existing transporters is an unsatisfactoryexplanation because elevation of plasma myoinositol did notincrease brain myoinositol in animals infused with myoinositoland water. In our study, an increase in plasma sodium was necessaryfor myoinositol to enter brain, but hypertonicity does not changethe Km of the myoinositol transporter in isolated glial cells(24). Possibly, a more rapid posttranslational process for organicosmolyte intake, akin to that observed in cultured kidney cells,may be stimulated by hypertonicity (25).
Organic osmolytes could potentially be therapeutically usedto aid in brain adaptation and prevent osmotic demyelinationduring correction of hyponatremia or after a rapid onset ofhypernatremia. The increase in brain myoinositol we observedafter myoinositol infusion in conjunction with an increase inplasma sodium would be expected to militate against the braindehydration and electrolyte accumulation observed in animalsafter acute hypertonicity without myoinositol. However, brainwater and sodium content in the two groups were equivalent.One explanation is that the increase in brain myoinositol representsonly about a 1% increase in total brain solute, and thus anyresultant changes in brain water or electrolytes may be toosmall to detect by our methods. It might then be reasonablyargued that the increase in brain myoinositol, though statisticallysignificant, would have no real influence on brain metabolismor adaptation to hypertonicity. On the other hand, there areregional differences in organic osmolyte content in brain thatcorrelate to susceptibility to osmotic demyelination after rapidincrease in plasma osmolality, and these regions might be moreaffected by the observed increase in brain myoinositol content(26). Endothelial cell shrinkage may be central to the pathogenesisof osmotic demyelination, and an increase in brain myoinositolcould have a disproportionate influence on this process (27–28).
In support of the possibility that an increase in brain myoinositolmay ameliorate osmotic demyelination is the observation thatazotemic rats appear to be protected from osmotic demyelinationafter acute correction of hyponatremia (8). Recently, in conjunctionwith our laboratory, Soupart et al. (9) recently observed thatwhen azotemic hyponatremic animals were treated with hypertonicsaline, brain water content 2 h postcorrection did not differfrom controls, but there was a significant increase in brainmyoinositol content. This pattern is similar to the findingsof this report, in which myoinositol content of brain increasedafter 3 h in animals infused with myoinositol, but brain waterwas equivalent to controls. Of interest, nonazotemic rapidlycorrected animals developed cerebral edema 24 h after correction,and azotemic rapidly corrected animals did not. The increasein brain myoinositol may have played a role in the protectiveeffect of azotemia on postcorrection demyelination and cerebraledema.
The current studies demonstrate that administration of myoinositolin modest doses can increase brain content of myoinositol butonly when plasma tonicity is concomitantly increased. Furtherstudies are needed to explore the therapeutic use of myoinositolto prevent the osmotic demyelination syndrome.
Acknowledgments
This work was supported in part by a grant from the UpstateNew York Chapter of the National Kidney Foundation. We alsothank Drs. Donald Kamm and Ruth Kouides for their critical reviewof the manuscript.
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Received for publication August 3, 2001.
Accepted for publication December 22, 2001.
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Abstract
Introduction
