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The N eurotoxicity o -Valine Deficiency in Rats1PATRICK K . CU SICK,2 KATHLEEN M . K OEHLER,3 4BARBARA FERRIER ANDBETTY E. HASKELL 6D epa rtm ent o f F ood Science U niversity of Illino isUrbana Illinois 618 1

ABSTRACT W hen valine, an essential am ino acid, was withdrawnfrom the diet of weanling rats, the anim als rapidly developed a uniquepattern of neurological symptoms characterized by head retraction, staggering and aimless circling. At necropsy degenerative changes were mostprom inent in the neurons of the red nuclei, brain stem structures whichm odulate m otor function. To explore the pathogenesis of the neurotoxicityassociated with valine deficiency, we fed rats purified diets deficient invaline alone or in valine plus other branched chain and neutral am inoacids, and we examined brain tissues by light microscopy. M otor disfunction and red nuclei damage occurred only in rats fed diets lacking valinealone and not in rats fed diets lacking all three branched chain aminoacids. These results suggest that the neurotoxicity of valine deficiencyresults from amino acid imbalance rather than from lack of dietary valineper se. J. Nutr. 108: 1200-1206, 1978.INDEXING KEY W ORDS valine amino acid deficiency aminoacid imbalance bran ched chain am ino a cidsValine and isoleucine are unique amongthe essential am ino acids in that deficiencyproduces neurological signs. The signs arespecies specific. In rats, valine deficiency

1), but not isoleucine deficiency 2), produces staggering, head retraction and aimless circling. In hum an infants, isoleucinedeficiency 3), but not valine deficiency 4), results in tremors of the arms andlegs and episodes of tw itching. N eurological signs associated w ith valine deficiencyor isoleucine deficiency disappear whenthe missing am ino acid is restored to thediet 1, 3).To our knowledge, the cause of the central nervous system damage in isoleucinedeficiency has not been explored. However, Scott 5) has identified sites of braindamage in valine-deprived rats. He observed severe cytoplasm ic vacuolizationand chrom atolysis in the neurons of thered nuclei and, to a lesser degree, of themotor facial and deep cerebellar nuclei.Chromatolysis as seen via the light

m icroscope im plies the dissolution of N isslsubstance and suggests a change in them ajor protein synthe sizing organelle of thecellspecific ally, the detach ment of ribo-som es from the endoplasm ic reticulum andthe loss of characteristic parallel stacks ofm em branes. Such essential changes m ightbe expected to occur in deficiency of anyamino acid, since withdrawal of an essential amino acid from the diet promptlydepresses protein synthesis 6). One wonders, however, w hy valine deficiency differs from a deficiency of other essentialamino acids in that it apparently results indamage prim arily to the red nuclei. Is

R eceived for publication January 4, 1978.1 Supported by grants from T he N utrition F oundation No. 499) and from the National Institutes ofH ea lt h A M -1 93 03 ).2 D ep artm en t o f P ath olo gy , C olle ge o f V ete rin aryM ed icin e, M ic hig an S tate U niv ers ity , E ast L an sin g,M i ch ig a n 4 X 8 24 .3 Based in part on data In Ph.D. thesis submittedto the U niv ersity o f Illin ois, U rb an a, Illino is, In M ay , r se nta dd re ss : D ep t. o f N utr iti on al S ci en ce s,U ni ve rs it y o f W is co ns in , M ad is on , W is co ns in 5 37 00 . o w ho m c or re sp on de nc e s ho ul d b e a dd re ss ed .1200

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VALINE DEFIC IENCY IN RATS 1201there a unique but presently unrecognizedrole for valine in the maintenance of thesebra in cel ls?This paper describes a nutritional approach to the problem of the neurotoxicityof valine deficiency. The results suggestthat the neu ro logical d isorder s cha racte ristic of valine-deficient rats are due to am inoacid imbalance rather than lack of dietaryvaline per se.

MATERIALS AND METHODSWeanling, male Sprague-Dawley rats were housed individua lly in wire -bottomedcages in an air-conditioned laboratorymaintained at 23 to 24and fed either acomplete purified diet or a diet lackingone or more of the essential amino acids.The complete purified diet had the following composition (g/100 g diet): Dextrin, 75.23; am ino acids, 12.26; salts, 4.00;vitam in m ix, 1.5 0; corn oil, 4.00; cellulos e,2.00; and sodium bicarbonate, 1.00.The amino acid mixture was that ofRanhotra and Johnson' (7) Diet C III.Amino acid s were purchased.8The salt mix was that of Draper et al.(8). This formulation supplied at 4 ofthe diet provides 60 of the NationalResearch Council requirement for phosphorus, the assumption being that additional phosphorus will be supplied by dietary protein (9). For example, diets containing 20 casein or soy protein wouldprovide an additional 140 mg P/100 g diet.Since crystalline amino acids were substituted for protein in our diet, we addedadditional phosphorus ( 1 00 mg/100 g diet )as dibasic potassium phosphate .The vitamin mixture w as that describedby Parsons, Shrader and Zeman9 ( 10 ) withminor modifications.The amino acid-free diet was preparedby substituting an equal w eight of dextrinfor the missing amino acid mixture. Inamino acid-deficient diets, the nitrogencontent of the diet was maintained at aconstant level by appropriate increases inthe L-glutamic acid content of the diet andby appropriate decreases in the dextrincontent.Unless indicated otherw ise, the diet w assupplied as a gel prepared as follows: 40g agar 10was dissolved in 1 liter hot water.

One kg of the purified diet mixture wasadded with vigorous stirring. The slurrywas ladled into plastic trays, cooled overnight at 3 and stored frozen.Fresh food and water were supplieddaily. Diet was supplied ad libitum exceptto a group (pair-fed controls) whose foodintake was restricted to that of the aminoacid-deficient rats. Food intake values arereported for dry weight of diet. Suitablecorrection was made for a weight lossaveraging 10 which occurred during exposure of the gel diet to air for 24 hours.Rats were assigned randomly to a complete or experimental diet according to theplan indicated in table 1. Data for weightand food intake are reported only for survivors. D eaths occurred among the valine-deprived groups: 1 in experiment 2 and 4

in experim ent 3. In anticipation of a higherdeath rate among valine-deprived rats,we assigned additional animals to thisgroup. Differences in growth were evaluated statistically with Student's i-test(2-tailed).Tissue samples were collected from ratsanesthetized with pentobarbital sodiumand killed by vascular perfusion. Immediately before perfusion, the chest wasopened, the heart exposed and 1 ml ofheparin (1,000 units/ml) and 2.5 ml of sodium nitrite solution w ere injectedinto the left ventricle. The right ventricleslit to permit escape of blood andasperfusion fluid, the descending aortaclamped with a hemostat and hypertonicperfusion fluid introduced into the leftven tric le . The perfu sion mix tu re con ta ined :2.5 r eagent grad e glutaraldehyde and2.0 p araformaldehyde in 0.08 M sodium Sprague-D awley D ivision of M ogul Corporation,Mad ison , Wiscons in .7 It supplied (mg/100 g diet): L-arginlne-HCl,500; L-histidine-HCl-HzO, 338; L-lsoleuclne, 550;L-leuelne, 730; L-lysine-H Cl, 1,184; L-methlonine,539 ; L-phenylalantne, 763 ; L-threonlne, 500 ; L-tryp-tonhnn, 150; DL-alanlne, 230; i.-anpartic acid, 230;L-glutumlc acid, 3,290; glycine, 1,394; L-prollne,230; L-cystlne, 230; DL-serlne, 230; L-tyroslne, 230;L-asparaglne, 395 ; and L-vallne, 550 mg.Ajlnom oto Company, Tokyo, Japan.'t supplied per 100 g diet : chollne chloride, 7.5

mg ; inosltol, 37.5 mg ; ascorbic acid, 1.75 mg ; calcium pantothenate, 3.75 mg ; nlacln, 2.25 mg;thiamin-HCl, 2.25 mg ; menadione, 1.87 mg; p-amlno-benzoic acid, 750 ng; riboflavin, 750 wg ; folie acid.45 tig; blotin, 19 ne : vitamin B-12, 2 us ; DL-atocopheryl acetate, 9 IU, cholecalciferol, 112 ICU ;retinyl acetate, 1136 IU ; pyrldoxlne-HCl, 2.25 mg.10Bacto Agar, D lfco, D etroit, M ichigan. Eastman Kodak, Rochester, New York.

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1202 C US IC K, K OE HL ER , FE RR IE R A ND H ASK EL LTABLE 1

f fec t o f d iet de fic ie nt in on e o r more o f th e b r n ch edch in m ino cids on grow th nd food int kein we n ling r tsDietExpt.

1CompleteStock

dietVal-freeExpt.

2CompleteVal-freePair-fedExpt.

3*CompleteVal-freePair-fedvai-,

ile-,leu-freeExpt.4 0 .0 5) f ro m t ha t i nva line-deprived rats expt. 3). W eight los s significantlyd iffe re nt a s c om pa re d t o th at i n r ats fe d v ai -, i le -, l eu -f re e d ie t Expt. 4); for rats fed the vai-, ile-, leu-, try-, phe-, tyr-freediet and for rats fe d the try-free diet, P < 0.01 ; for rats fe d t hea mi no a ci d- fr ee d ie t a nd f or r at s f ed t he i le -f re e d ie t, P < 0 .0 01 .

phosphate buffer, pH 7.4. The final os-molarity of the solution was 1,080 to 1,100milliosmoles per liter. It was stored at 4no longer than 72 hours before use andwas filtered by gentle suction early on theday it was to be used. After the rat hadbeen perfused for 30 m inutes, the brainwas removed and stored at 4n the sameso lution. B rain tissue w as sliced, processedand embedded in paraffin by standardmethods. The brain tissues in paraffinblocks w ere sectioned at 6 /A, deparaffin-ized, stained w ith hem atoxylin-eosin, ande xa min ed v ia lig ht m ic ro sc op y.RESULTSW hen weanling rats were fed a purifieddiet lacking valine, food intake droppedsharply and the animals lost weight stead

ily. After 25 days of valine-depletion, ratstypically weighed about 10 g less than atweaning. Growth and food intake data areshown in table 1.Clear signs of central nervous systemdamage were apparent after rats hadeaten the valine-free diet for 18 to 20 days.A characteristic early sign was inability ofthe rat to cling to the wire mesh side ofthe cage without falling. By 23 to 25 days,severe m otor incoordination w as ap parent.The rats walked with a staggering gaitand frequently fell. They appeared disoriented and circled aim lessly. H ead m ovem ents characteristic of n orm al exp loratorybehavior became grossly exaggerated inthat extrem e retraction of the head occurred. The rats appeared unusually sensitive to touch. They failed to groom themselves. Their paws and nose became cakedwith diet. Death was common in rats feda valine-free diet more than 25 days. Inthe terminal stages, rats huddled in thecorner of the cage and m oved only whengently prodded. These signs were not apparent in pair-fed controls or in the controls fed a valine-supplemented diet adlibitum.O n lig ht m ic ro sc op ic e xa min atio n, b ra in sof valine-deprived rats had cellular damage apparently confined to the neurons ofthe red nuclei. Neuronal cell bodies wereswollen with large round eccentricallyplaced nuclei. Neuronal cytoplasm waspale , devo id of N iss l clum ps, and contained

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VALINE DEFICIENCY IN RATS 1203prominent empty spaces, particularly adjacent to cell borders (fig. 1A). Whitem atter changes were not noted. H owever,specific staining techniques to detect earlymyelin degeneration were not used. Exam ination of tissues from liver, kidney, heart,and skeletal m uscle were unremarkable.Pair-fed con tro ls and valine-supplementedcontrols fed ad libitum had no apparents ignificant changes in b rain o r other organs .Light micrographs of red nuclei from controls fed ad libitum ( fig. IB ) and pair-fedcontrols (fig. 1C) are shown.When rats were fed a diet lacking allthree branched-chain am ino acids (valine,isoleucin e and leuc ine), the charac teristicneurological signs of valine deficiency didnot appear. Although such a diet resultedin growth depression as severe as that produced by valine deficiency (see table 1,expt. 3), it produced no signs of motor incoordination. W hen the red nuclei of ratsdeprived 25 days of all three branched-chain amino acids were examined by lightmicroscopy, these neurons were indistinguishable from those of controls. A lightmicrograph of red nuclei from rats fed adiet deficient in valine, isoleucine and leu-cine is shown in figure ID.No neurologic signs characteristic ofvaline deficiency were observed when ratswere fed diets lacking (1) leucine alone,(2) isoleucine alone, (3) tryptophan alone,(4) valine, isoleucine, leucine, and tryptophan, (5) valine, isoleucine, leucine, tryptophan, phenylalanine, and tyrosine, and(6) all amino acids. All of these diets produced severe growth retardation (table 1,expt. 4) but none produced changes in thered nuclei detectable by histolog ical exam ination. Of these experimental diets, onlythe tryptophan-free diet produced an indication of mild nerve disfunction. Tryptophan deficient rats appeared hyperirri-table but did not display the motor incoordination and other neurologic signscha racte ris tic o f valine defic iency.

DISCUSSIONThe first report of the neurological disorders characteristic of valine deficiencywas that of Rose and Eppstein (1) whonoted peculiar symptoms unlike any we

have encountered in other types of amino

acid deficiencies including severe lack ofcoordinatio n, staggering g ait, he ad retraction and a rotary motion resembling thatof a dog chasing his tail. These observations have been confirmed (5, 11).Scott (5) traced the apparent cause toneuronal deterioration and myelin degeneration in nerve pathways related to coordination and orientation. In young ratsdepleted of valine for 22 days, severe damage occurred in the cell bodies of the rednuclei, and the myelin of the vestibularnerve showed clear signs of deterioration.Ih adult rats which tolerated 70 days ofdepletion, myelin damage was even moreextensive in the facial and vestibularnerves and was apparent, to a lesser degree, in the medial longitudin al fasciculus.Scott reported that these changes werespecific for valine deficiency and were notobserved in rats dep leted o f phenylalanine,threonine, histid ine, tryptophan and isoleucine. An obvious extension of Scott s workwas to determine whether the neurotox-icity of valine deficiency is due to lack ofvaline per se or due to amino acid imbalance. Specifically, it seemed important todetermine whether the signs of valineneurotoxicity could be observed not onlyin rats fed a diet lacking valine alone butalso in rats fed a diet lacking all threebranched-ch ain am ino acids.Our experiments demonstrate that theneurologic al symptoms attributed to valin edeficiency can be reproduced w ith rats fedan improved purified diet which permits asatisfactory rate of growth in controls(4.3 0.50 to 6.4 0.13 (see table 1) ascompared to 3.0 g/day in Scott s experiments) (5).The nature of the cellular changes observed in the red nuclei in valine deficiencysuggests that valine may be the rate-limiting amino acid for protein synthesis inthese cells. W ithdrawal of valine from thediet resulted in a deterioration of Nisslsubstancethat is, in a disruption of thechief protein synthesizing machinery ofthe cell. S im ilar cellular ch anges have beenobserved in liver of rats fed a tryptophan-free diet (12). Removal of tryptophanfrom the diet caused an abrupt decreasein the rate of protein synthesis. The depression of p rotein synthe sis is manifeste d

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12 4 C US IC K K OE HL ER F ER RIE R A ND H AS KE LL

B ..

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VALINE DEFIC IENCY IN RATS 1205

r

Fig. 1 Neurons of the red nuclei from rats fed the following diets for 25 days: (A)valine-free; (B) control, ad libitum; (C) control, pair-fed to valine deficients; (D) iso-leucine-, leucine-, and valine-free. Cell sw elling, chrom atolysis and nuclear displacem ent areapparent in neurons from valine-deprived rats but not in the neurons of controls or of ratsdeprived of all three branched chain amino acids. Photographed at a magnification of 450X.Stain: hematoxylin-eosin.

at the cellular level as a disaggregation ofpolysom es. The cellular response of liverwas characteristic o f tryptophan deficiencyand not of a deficiency of other essentialamino acids, apparently because tryptophan is the rate-limiting amino acid forprotein synthesis in this organ (12). Thatbranched-chain am ino acids lim it the rateof protein synthesis in muscle has beendemonstrated ( 1 3 ). Our data are consistentwith the possibility that valine may limitthe rate of protein synthesis in the rednucleiand perhaps, judging from Scott swork, in other parts of brain.We observed that neurological signs didnot occur and the red nuclei appearednormal when rats were fed diets lackingall three branched-chain amino acids.These data strongly suggest that the nervetissue dam age characteristic of valine deficiency actually is a consequence of am inoacid imbalance. In other words, it apparently was not simply a lack of valinewhich caused the nerve damage but thepresence in the v line free diet of isoleucine and leucine.Branched-chain amino acids competewith one another and with other neutral

amino acids for transport into brain (14).The essential amino acid supply for protein synthesis comes both from diet andfrom tissue breakdown (15). In proteindeficiency, am ino acids from muscle (andpresumably from other extracerebral tissues) are salvaged to maintain brain protein ( 16 ). In an animal fed a diet lackingall three branched-chain amino acids, onemight anticipate that a sm all but balancedsupply of isoleucine, leucine and valinefrom breakdown of extracerebral tissueswould compete for entry into brain. In therat fed a diet deficient in valine alone, therelatively high concentrations of leucineand isoleucine in the diet m ight inhibit thetransport into brain of valine from tissuebreakdown. Thus, because of its uniquetransport system which limits entry ofamino acids, brain m ight suffer m ore fromvaline deficiency than other organs. Suitable experiments to test this hypothesisa re in p rogress.

LITERATURE CITED1. Rose, W. C. Eppstein, S. H. (1939) Thedietary indispensability of valine. J. Biol.C hem . 127, 677-684.

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120G CUSICK, KOEHLER, FERRIER AND HASKELL2. Womack, M. Rose, W. C. 1936) Therelation of leucine, isoleucine and norleucineto growth. J. Biol. Chem. 116, 381-391.3. Snyderman, S. E., Boyer, A., Norton, P. M.,Roitman, E. Holt, L. E., Jr. 1964) Theessential amino acid requirem ents of infants.IX Isoleucine. Am. J. Clin. Nutr. 15, 313-3214. Snyderman, S. E., Holt, L. E., Jr., Smellie,F., Boyer, A. Westall, R. G. 1959) Theessential amino acid requirements of infants.Valine. Am. J. Dis. Child. 97, 186-191.5. Scott, E. B. 1964) Histopathology ofam ino acid deficiencies. VII. Valine. Experimental and M olecular Pathology 3, 610-621.6. Wannemacher, R. W., Jr. Allison, J. B.1 968 ) Plasma amino acid concentrations inrelation to protein synthesis. In: Protein Nutrition and Free Amino Acid PatternsLeathern, J. H., ed.), Rutgers UniversityPress, New Brunsw ick, N.J., pp. 206-227.7. Ranhotra, G. S. Johnson, B. C. 1965)Effect of feeding different am ino acid diets ongrowth rate and nitrogen retention of weanling rats. Proc. Soc. Exp. Biol. Med. 118,1197-1201.8. Draper, H. H., Bergan, J. G., Chi, M.,Csallany, A. S. Boaro, A. V. 1964) Afurther study of the specificity of the vitam inE requirement for reproduction. J. Nutr. 84,395-400.

9. Greenfield, H. Briggs, G. M. 1971) Nutritional methodology in metabolic researchwith rats. Ann. Rev. Biochem. 40, 549-572.10. Parsons, P. L., Shrader, R. E. Zeman, F. J.1976) Adrenal function in young of protein-deprived pregnant rats. J. Nutr. 106,392-404.11. Ferrare, A., Roizin, L. Givner, I. 1947)Ocular changes in rats on diets deficient inamino acids. II. Corneal dystrophy due tovaline deficiency. Arch. Ophth. 38, 342-352.12. Munro, H. N. 1968) Role of amino acidsupply in regulating ribosome function. Fed.P roc. 27, 1231-1237 .13. Fulks, R. M., Li, J. B. Goldberg, A. L.1975 ) Effect of insulin, glucose, and aminoacids on protein turnover in rat diaphragm.J. Biol. C hem . 250, 290-298.14. Oldendorf, W. H. Szabo, J. 1976)Amino acid assignment to one of three blood-brain barrier amino acid carriers. Am. J.P hy sio l. 2 30 , 9 4-9 8.15. Wannemacher, R. W., Jr. 1967) Proteinsynthesis in various tissues from animals atd iff er en t s tage s o f p ro te in -c al or ie malnutr it ion.Seventh Int. Cong. Nutr. 5, 205-210.16. Dallman, P. R. Spirito, R. A. 1972)Brain response to protein undemutrition:M echanism of preferential protein retention.J. C lin. Invest. 51, 2175-2180.

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