blood 1965 nicolaidou 91 9

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1965 26: 91-99

MARIA NICOLAIDOU, CHARLES C. LUND and RAPIER H. MCMENAMYVarious Anemias and with Erythroblastosis FetalisUnbound Amino Acids in the Plasma and Erythrocytes of Children with

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U nbound A m ino A cid s in the P lasm a and Ery throcyteso f C h ildren w ith V ar ious A n em ias and w ithE ry th ro b las tos is F e ta lis

B y M A R IA N IC O L A ID O U , C H A R L E S C . L U N D A N D R A PIE R H . M C M E N A M Y

S T U D Y of the unb oun d am ino ac id co ncen tratio ns in th e p lasm a andery th rocy tes o f n orm al ch ild ren as a fu nc tion of age has b een pu b lished

in an ea rlier com m un ica tion .1 In the cu rren t rep o rt am in o ac id va lues frombloo ds o f ch ild ren w ith d iffe ren t anem ias w ere stud ied to d ete rm ine w h eth era m i n o ac id abn orm a litie s cha rac te ris tic o f anem ia in g en e ra l o r p ecu lia r toan y on e particu la r type of anem ia cou ld be de te cte d.

METhODS

T he a nalyse s w ere con du cted in the sam e m ann er as tho se rep orted fo r n orm al ch il-dren .1 A m ino ac ids w ere separa ted by one-d im en siona l paper chrom atogram s us in g asalt-sa tu ra ted d ev elop ing sys tem . A fte r s tain ing th e ch rom atogram s , v isua l com pariso no f th e in te ns ity an d size of th e zon es w ith stand ard zones w ere m ade to ob ta in thequan tit ies p re sen t. A sp artic a cid , cy ste ine or cyst ine) and p lasm a try p tophan concen tra -tion s w ere n o t de te rm ined . L euc ine an d iso leu cin e w ere de te rm in ed as com bined zo nesa s w e re also ser ine p lu s g ly cin e. E a ch so lu t ion w as an aly zed in trip lic ate , i.e ., a t th re ed iffe ren t concen tratio ns, and a m ean v alu e taken . T he coe ffic ien t o f va ria tio n w ith th ism et h o d av eraged 7 per cen t, bu t v aried som ew h at fo r d iffe ren t am in o ac ids.2 T he re -covery of am ino ac ids add ed to p lasm a an d ery th ro cy tes w as found by th is m ethod to beessen tia lly com ple te .2

R E S U L T SIn earlie r stud ies it w as fo und th at the log arithm ic n orm a l d is tribu tion

w as reaso nab ly satis fac to ry fo r exp re ssing the co ncen tra tion o f am ino ac idsin th e p la sm a and e ry th rocy te s o f no rm al ch ild ren . In the cu rren t s tu d ie s ,w ith ch ild ren as pa tien ts , the sam e d is tr ibu tion w as em p loyed . In f igu re 1the ranges and m ed ian v alues a re repo rted fo r the un bou nd am ino ac id co n-cen trations , fo r the e ry th rocy tes and p lasm a of pa tien ts w ith iron de fic iencyanem ia , sick le ce ll anem ia , h e red ita ry sph e rocy to sis , and e ry th rob las tosisfe ta lis. In the loga rith m ic n orm a l d istr ibu tio n the an tilog o f the lo ga rith m icm ean is the m ed ian fo r the linea rized da ta . ) F or co m parison , th ere haveb een inc lud ed in th is f igu re the m ed ian va lues fo r no rm a l ch ild ren aged 4 daysto 1 yea r, 1 to 12 yea rs an d fo r co rd b loo ds. C o ncen tra tion s d iffe rin g sign if-ican tly from th e va lues o f no rm al ch ild ren o r th e co rd b lood s ev a lua ted by t test, tak ing the leve l o f sign ificance as 0 .05 ) are id en tified w ith lette rsover the line graphs according to the legend in the footnote Table 1 list 6 pa

F rom the D epar tm en ts o f P ed ia tr ic s , B io lo g ica l Chemis try a nd Surgery , HarvardM edica l Schoo l, Th e C h ild ren s H o sp ita l M ed ica l C en ter and the F ifth S urg ica l [H a r-vard ] Serv ice o f the B os to n C ity H o sp ita l, B os to n , M assachusetts.

Sup port fo r th is w ork w as rece ived from G ran t C -2497 o f the N a tion a l In stitu te o fH ea lth , B e the sda , M ary la nd .

Su bm itted O c t. 2 , 196 4 ; accep ted fo r p ub lica tio n N o v. 2 0 , 1 964 .91

B L O O D , V O L . 26 , N o . 1 J uLY , 1 9 6 5

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92 N ICOL A ID O U , L U N D A N D MCM E N A M Y

_ I. .{ I I- *g ; r ;ii I fillI flJ jJ J jI AL ANNI 48 0 L Y S P I t

k 1 1 la-AN INO - {248}-SU TY RIC A C I DI I 20 M E T NIONIIC

ISO A R G IM N I- PI#{ 128} NYL AL ANNC I I :: {149}4p {149} : ; + #{ 149} + i. 4t 1 . .&+: _ _ _ _ -- - 0 L _ _

2400 S UTAMIC A C ID 4 8 0 N IThS # { 1 2 8 }1 5 0 0 1 36 0

FA N I .I200 1I c I 24 0 { # { 1 7 6 } :Lk 20

1 6 0 0 - SL U T A M l 40 0 300P

HIS TIoII flE 0 N I

t 40 . 4 5 0 h 1iso I:J; : : 360FIzoft+4A

Fig . 1 .-C oncentratio ns (M ./K g . w ater-vertical ax is ) o f unbound am ino ac idsand other substances in the erythrocytes (continuous lines) and plasm a (do tted lines)o f subjects w ith different anem ias. The m edian co ncentratio ns are noted by ahorizo ntal bar o n the lines. The lines m ark the range of concentrations . A n overthe g raphs for the iron defic iency anem ia and erythroblasto sis fe talis patients inch-cate s that the m edian value w as signif icantly different fro m the m edian o f 10 norm alchildren aged 4 day s to 1 year (repo rted in the fifth co lum n). A ov er the g raphfor the sickle ce ll anem ia and hereditary sphero cy tosis patients indicate s that thev alue w as s ignificantly differe nt from the m edian v alue of 1 5 norm al children ag ed1 to 12 y ears (repo rted in the six th co lum n). A C ov er the graph o f the ery thro-blastosis fe talis patients indicate s that the value w as sig nif icantly different fro m them edium values o f cord bloo ds (reported in seventh co lum n). h norm al valuesand cord blood v alues are taken from reference 1 . The le tters H and L identifythe high and low m edian values, respective ly .

G lutam ic ac id, erg othio ne ine , and taurine co ncentrations w ere v ery lo w or absent



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v ...

Itilt II IT R t P T O P H A N

1 +T YROS INE

V A L I N E

E R GOT HI ON E I N E

+ TL E UCWIE +

I SOL E U C I N E

OH. +. .k +

I 1 1 1 0 rz z. ; 4 v.R o1;. , Id -

S ER IN E+ OL YC IN E x IO

T AURINE

BH1A H_ 1

U RE A xIO

4R ETIC UL OC YT ES (S )

+ 1 TI AN1- __ i

j

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32 HE M OOL O& N(GR A M S PE R lOO m I. B LOO D )

L

(Fig. 1. C ontd.in the plasm a. A rg inine concentratio ns w ere v ery lo w or absent in the ery thro cyte s.Ethanolam ine w as present in tw o of the babie s with ery throblas tosis fe talis ataverage leve ls o f 4 2 and 74 ,M ./K g . w ater in the plasm a and erythrocytes , respec-l ive ly .H istories of Patients

Iron deficiency anem ia. The patients w ere 6-19 m onths o ld, w e ighed 5 .6 to11 .3 K g ., pallid, but in fair to goo d nutritional state . The serum iro n averaged2 4 ,g ./1 00 m l. blood (range 1 8-36) . A nalyse s w ere co nducte d on 6 patients be fo reFe + + treatm ent w ith Im fero n#{ 174 } . The o ther 2 patients had treatm ent w ith Im fero n#{ 174 }fo r 10 days and 4 m onths prior to the analyses , but w ith s lo w re spo ns e.

Sickle cell anem ia. The subjec ts w ere 10 -12 y ears o ld, pallid, but in fair to go odnutritio nal state . A hem oly tic cris is preceded the analyses by 1 to 4 days.H ereditary spherocytosis. The subjec ts w ere nine m onths to 6 .5 years o ld, pallid,but in g ood nutritio nal state . A nalyses w ere taken 1 to 5 day s after hem o ly tic crise s .

Erythroblastosis fetalis. The subjects w ere 1 to 45 hours o ld, jaundiced, Co om bspositive . Tw o babies w ere full term (4 .5 and 3 .6 K g .) , tw o w ere prem ature (1 .4and 1 .9 Kg .) .

U N B O U N D A M IN O A CID S IN PLA S M A A ND E RY THRO CY TE S 93

64E160 F A H 4 8 IA H

4 # { 1 7 6 } E 1 32I 6L i

200 2001 5 0 L 1 9 0 0I00 600 A H

5 # { 1 7 6 } L . I . 300SlO E 964201 72280 48I40 2:

4 63 O O I2

2000



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# { 1 76} Photostat or m icrofi lm copies o f the data for e ach patient may be o btained at m ode ratecost from the A m erican D ocum entation Institute , Library of Congress , D ocum ent N o.8420 , 1 1 pag es .

94 N ICO L A ID O U , L U N D A N D M CM E N A M Y

with O th er Anemia sable 1 .-Pa t ien tsP lasm a S M /K g . W at er )

T M I T M A U H G S A H AErythrocytes M M /K g . W at er )

A I H A S F H b T M I T M A U H G S A H A A I H A S F H bt t 1 1 1

I I I I I tI I

II

tt

I II

I

I

I

IICC

II

I

I

I

IIIII

IIII

I

II

II

Alaninea-A mino n-

butyric ac idArginineG lu tam ic acidG l u t a m i n eHis t id ineL eucine pius

isoleucineLysineMethioninePhenylalanineOrnithineProlineThreonineTryptophanTyrosineValineErgothioneineEthanolamineS e rine plus

glycineTaurineUreaRetic uloc ytes (/ o f ery throcy tes 1 .5 5 .4 0 .3 1 7 .1 3 6 .2 1 .3He mog lobin (G m./10 0 ml. b lood) 9 .7 5 .7 1 1 .8 9 .1 9 .7 6 .4

T h e p a t ien t s w er e ev a lu a t e d ag a in s t n o :m a l ch ild r en o f t h e n e ar e st cor r e sp o n d in g ag e g r ou p . T h esymb ol I fo llow ing the value indicates that it is s ignific antly low e r than the normal m edian; thes y m bo l I fo llow ing the value indic ate s that it is s ignific antly hig her than the normal me dian. T hem edian is c hosen ac co rding to the ne are st age g roup.

T M I : T h a lassem ia m in or ; m ale , ag e 7 y ears . pallid. g ood n u t r it io n a l s ta te , h e p a t osp h e n om eg a ly .T M A : T h a lasse m ia m aj or ; f e m ale , age 6 years , e x t r em e ly p a le, h e p a tosp h en om eg a ly . U H G S : U n st ab lere duc ed g lutathione (Fam ily history o f naphthalene poisoning ) ; m ale , age 6 years, g oo d nutri ti ona lstate , rece nt ex posure to naphthale ne. A H A : A cquired hemo lytic ane mia (due to naphthale ne andl ead po is oni ng ) m ale, ag e 4 y e ars , g o o d nutritional state, r e ce n t lead p o ison in g . A I H A : A u t o im m u n ehemo lytic ane mia; fem ale , age 13 years , jaundice, C o o m bs (+ + + ), r h e u m at ic f ev e r , o n p r ed n ison e,fair nutritional state . S FHb: S + F hemo globinopathy ; female, age 16 ye ars , pallid, pains in ex-t r e m it ies , m en or r h ag ia aortic i n s u f f i c i e nc y .

tients w here only 1 patient per anem ia ty pe w as s tudied. A rrow s in the co l-um ns identify am ino ac ids w hose concentrations differ from those o f norm alchildren by m ore than tw o standard dev iations.

Except for the babie s w ith erythroblas tosis fetalis and several patients intable 1 , the plasm a am ino acid concentrations o f the anem ia subjec ts w ereessentially norm al. Five o f the plasm a substances w ere hig h in the ery thro-blastosis fe talis subjec ts w hen co m pared w ith norm al children aged 4 daysto 1 year; 4 w ere high w hen the plasm a v alues w ere com pared w ith co rdplasm as, against w hich the ery thro blastosis fetalis subjec ts w ere also ev aluatedbecause o f the very young ag e of these subjects . Hig h am ino ac id concentra-tions w ere found in the plasm a of a 13-year-o ld g irl w ith auto im m une hem o-ly tic anem ia. This patient w as under co nsiderable stre ss at the tim e of



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U N B O U N D A M IN O A CID S IN PLA SM A A N D E RYT HIIOCYT E S 95

analysis , due to the advanced state o f anem ia. It s eem s probable that thehigh v alues are related to this stress state . S om e of the other subjec ts had afew hig h and low plasm a am ino ac id values.

In contrast to the plasm a values the ery throcyte unbound am ino ac id co n-centratio ns in m os t patients differed m arkedly fro m norm al subjects . W hencom pared w ith norm al children of appro x im ate ly the sam e age g ro ups, 1 5substances w ere hig h in iro n defic iency anem ia, 10 w ere high in sickle ce llanem ia, 2 w ere high in hereditary spherocytosis , 6 w ere high in ery thro blas-to sis fe talis (4 substances w ere high w hen the latter w ere ev aluated ag ainstc ord e ry thro cy te s , 7 w ere high in a patient w ith thalassem ia m ajor anem ia,11 w ere high in a patient w ith acquired hem oly tic anem ia, 17 w ere high in apatient w ith auto im m une hem o ly tic anem ia, and 3 w ere high in the patientw ith S & F hem o globinopathy . N o abno rm ally high am ino ac id values w ereo bserved in the ery throcyte s o f patients w ith unstable reduced g lutathioneand thalassem ia m inor.

The unbo und am ino acid concentrations in the ery throcyte s o f the anem iapatients w ere also investig ated for relationships be tw een the re ticulo cy te co untand the blood hem o globin co ntent. Pre lim inary checks show ed poor co rre la-tio n betw een e ither the unbound am ino ac id concentrations and reticulo cy teco unts, o r the unbound am ino ac id concentrations and hem oglo bin contentw hen each se t o f variable s w as co m pared separate ly . A n analys is o f the dataw here the unbo und am ino ac id concentrations w ere expressed as a m ultiplefunc tion of the re ticulocy te count and the hem og lobin co ntent g av e a m uchbetter index of corre lation. The results o f the evaluation are sho w n in table2 . The leve l o f sig nificance of the reg ressio n coe ff ic ients b1 and b2 relatingto the re ticulocy te count and hem o globin content respectiv ely ) indicated thatthese tw o param eters have a s ignif icant e ffec t o n m os t o f the indiv idual am inoa ci d c o nc e ntrati on s see several exceptions referred to belo w On the averag e15 per cent o f the to tal v ariation could be attributed to the reticulo cy teco unt and 26 per cent to the hem og lobin co ntent. The b term for the latterre latio nship w as negative . W ith appro x im ate ly 6 per cent o f the v ariation dueto analy tic uncertaintie s the average variance o f am ino ac ids co ncentrationso f the anem ia patients is taken as 0 .04 , the averag e variance in duplicateanalyses (2 ) using the logarithm ic distributio n is 0 .002 5 , or 6 per cent) , therem aining variance , roug hly 5 0 per cent, is to be attributed to other fac tors .These presum ably w o uld be disease categorie s , ag e variatio ns, dietary varia-tions, or sim ple indiv idual differences .

Taurine , g lutam ic acid, serine plus g ly cine and ergothione ine w ere theo nly substances w ho se concentratio ns differed apprec iably from the otheram ino acids w ith respect to hem o globin content. Taurine , g lutam ic acid andserine plus g ly cine show ed a som ew hat hig her concentratio n dependence onthe hem oglo bin content than the other am ino ac ids (b2 v alues -18 2 , -75and -79 , re spec tive ly These three am ino ac ids have been o bserv ed to be -have in a co m m o n m anner in other studies. For ex am ple , on a re lative basistheir concentrations increased m o re in the ery throcyte s o f leukem ia patientsthan any o f the o ther am ino ac ids.3 They w ere fo und to be e lev ated in the



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96 N IC O L A I D O U , L U N D A N D M C M E N A M Y

Table 2 .-E ffec t o f R eticu locy te C on ten t and H em og lob in C on ten t on the U nbou ndAm ino A c id C oncen tra tions in the E ry th ro cy te s o f A nem ic C h ild ren

Re gress ion E quation Constants* Proportion(x 1 0 0 0 ) L e v el o f o f V a r ia n c es

Si g . ( ) %SE . S E . A n a l. o fa b b, b b V ar.t b1 b2 bi b2

A lanine 2 990 13 4 -34 16 0 .99 99 9 5 24 17A mino n-

butyrate 1979 14 5 53 19 0 99 98 99 24 20Glutamate 3 5 5 8 -1 5 -7 9 1 6 0 .9 9 1 0 9 9 .9 1 0 5 9Glutamine 2986 13 4 29 13 0 995 99 95 34 14H ist id in e 2 3 3 0 3 4 -2 9 1 5 0 .9 0 5 0 9 5 2 1 7L eucine &

isole ucine 281 3 8 16 0 975 90 99 13 24L ysine 2 804 14 4 -60 15 0.995 99 9 9.9 29 30M ethionine 197 4 16 6 -52 20 0 .995 99 9 8 20 22Phe nylalanine 2 275 9 5 -4 8 17 0.975 90 99 13 24O rnithine 270 1 6 5 -41 19 0.9 0 70 95 7 iSProline 26 57 8 4 -4 1 16 0.975 90 98 13 2 1T h r eo n in e 2 8 0 7 1 1 5 -6 2 1 7 0 .9 9 5 9 5 9 9 1 6 3 4T rypto phan 180 6 2 4 -2 6 15 0.90 30 9 0 1 13T yrosine 22 14 11 5 -4 1 18 0 .975 95 9 5 20 15V aline 2 665 7 4 -3 6 14 0.9 75 90 98 14 2 1E rg othio neine 1 267 16 8 10 6 27 0 .995 95 99 .9 11 45

S erine &g lycine 39 21 8 5 -7 5 19 0 .995 85 9 9.9 7 4 1

T aurine 35 27 3 8 14 -182 50 0.995 99 9 9.9 22 30Urea 3818 2 4 11 14 low 30 50 1 3

5V iL ju relate to the re gression equatio n. L o g Y = a x ib i S .E . + x 2 b 2 S .E ., w here Y is theunbound am ino a cid c o n c en t r a t io n , x , is t h e r et icu lo c y t e co u n t , a n d x is t h e h em o g lo b in in G m ./i0 0 m l.bloo d. S .E . is the standard e rro r. A ll subje cts in figure 1 and table I ex ce pt the normal c hildre n, c ordbloods a n d e r y t h r ob la s t os is fet a lis patie nts w ere inc lude d in this study (20 patients total) . Computatio nswere performed by the IB M 709 4 com pute r at the Compute r Center of this U nivers ity . T he prog ramuse d w as B I M E D 2 9 , o f t h e U C L A M e d ic a l C en t e r .

tA nalys is of varianc e: the values in this co lumn giv e the probabil ity that (bi, te ) (0 ,0).T h e le vel of s ign ific a n ce of b i a n d b 2 w e r e o b t a in e d fr o m t h e t d is t r ib u t io n u s in g a s ta n d a r d

transformation for t h e coe fficien t. T h e p r o p o r t io n s o f v a r ia n c e a p p ly o n ly in t h e c a se o f m u lt ip le r eg r e ss io n . O n e w o u ld o b t a in d if

fer e n t b , a n d b 2 V 8 J U S if the regre ss ions w ith the re ticulocy te c ounts and blood hemo globin c onte ntsw e r e e a c h d e t er m in ed sep a r a t e ly a s on e in d ep e n d en t va r ia b le.

ery throcyte s o f a patient w ith a sev ere kidney infec tion unreported exper-iments ) G lutam ic ac id and serine plus g lyc ine w ere also so m ew hat uniquein that they w ere the o nly am ino ac ids found to be higher in the ery throcyte so f children than adults .1 Ergo thioneine had a po sitive b2 v alue (b2 value 10 6).This latter m ay be a re flec tion of the die tary o rig in of erg othio ne ine , as itsintake w ould be expec ted to be greater in patients less serio usly ill. It hasbeen noted that ergothione ine binds to ery thro cy te pro tein, presum ablyhem og lobin, in a loose ty pe co m plex w hich w ould pro bably be bro ken indialys is. Patients w ith larger am ounts of hem o globin w o uld be expec ted tohave higher concentrations o f erg othio ne ine o n this bas is . A nother po int o finterest is that in an earlier s tudy ergothione ine w as found to be lo w er inco ncentration of patients w ith granulo cy tic leukem ia than in patients w ithchronic lym pho cytic leukem ia.3 A lthoug h the blo od hem og lobin concentra-tio ns o f these patients are not available, it is neverthele ss frequently foundthat the g ranulocytic leukem ia patients have low er hem og lobin leve ls thanlym phocytic leukem ia patients . W ith reg ard to the reticulo cy te co unts, cor-re lations w ith am ino ac id concentrations w ere not as goo d as the hem oglo bin



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U N B O U N D A M IN O A CID S IN PLA S M A A N D E RYT HB OCYT E S 97

leve ls . These corre lations w ere neg lig ible for g lutam ic ac id and rather poorfo r histidine , ornithine , and tryptophan. U rea func tions as a contro l in theregression studies since it dem onstrated no concentration correlation withthe re ticulocy te leve ls o r hem og lobin co ntent.

DIscussIoNIn none of the studie s did there appear to be any disting uishing re latio n-

ship betw een the disease categ ory and the unbo und am ino acid concentra-tions in either the plasm a or the ery throcyte s. The patients w ith iro n de-fic iency anem ia characteris tically had lo w hem o globin concentratio ns w ithreticulocyte counts usually no t elevated m uch. In o ther patients, the hem o -g lobin co ntent w as usually not so lo w but the re ticulocy te co unts w ere higherthan those found in iron defic iency anem ia subjects. D espite these differences,anemia per se , that is anem ia identif ied by either lo w bloo d hem oglo bin con-tent or e levated reticulocyte counts , w as alm ost alw ays acco m panied by hig hconcentrations of the am ino acids in the ery thro cy tes . This latter is w ell sup-ported by the m ultiple regress ion studies w here re lationships be tw een un-bound am ino ac id co ncentrations, hem o globin content, and reticulocyte countshave been evaluated. The unbo und am ino acid concentratio ns w ere repo rtedsom e tim e ag o to be elevated in re ticulocy te s, w hen com pared w ith norm aladult ery throcyte s.4 There do es not appear to be a prev ious repo rt o f blo odhem oglobin effec t on the unbound am ino ac id concentrations in the ery thro-cy tes.

It is also to be no ted that w hen the unbound am ino ac id co ncentratons inthe ery throcyte s w ere e levated, alm ost all the indiv idual am ino ac ids con-tributed to the increase . This trend is ev ident fro m the s im ilaritie s in the bvalues o f the am ino ac id reg ressio n evaluations in table 2. It is also ev identin figure 1 and table 1 w here , for the iron deficiency anem ia, sickle cellanem ia, thalassem ia m ajor, acquired hem olytic anem ia, and auto im m unehem olytic anem ia patients, the m edian value for every am ino ac id w as nu-m erically higher than the m edian v alue fo r norm al children. This observa-lio n of unifo rm elevation of the ery thro cyte am ino ac ids is perhaps the prin-c ipal difference found betw een our results and those of Iy er5 or A llen.6 Theform er, study ing iro n defic iency anem ia patients , reported increases in ery th-ro cy te co ncentrations o f alanine , g lutam ic ac id, g lutam ine, g ly cine , andserine to the ex tent o f tw o to sev enfo ld, but no increases fo r leuc ine , iso leuc ine ,and v aline . A llen, study ing one patient each w ith erythro blastosis fetalis ,thalas sem ia m ajor, thalassem ia m ino r, sickle ce ll anem ia, Coo m bs pos itive , andCoom bs negativ e hem o ly tic anem ia, found that concentratio ns o f only certainam ino ac ids g lyc ine , alanine , m ethionine , leucine, iso leuc ine , and valine)w ere e levated in the presence o f high re ticulocy te counts.

The blo od am ino ac id pattens o f the ery throblastos is fetalis subjec ts w ereco nsiderably different from tho se of the typical anem ia patients . The plasm aconcentrations of ly sine and ornithine w ere hig h fo r the ery thro blastosisfe talis patients , w hich w as sim ilar to the high v alues found for ly sine andornithine in cord plasm a. Furtherm ore , althoug h the ery throcyte unbounda mi n o a c i d c o n c e n t r a t i on s we r e som ew hat e levated in re lation to no rm al chil-



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dren aged 4 day s to 1 year, the ery throcy te-plasm a concentratio n ratio s w erem os tly norm al. On the basis o f this inform ation it s eem s that there w as littledisturbance in the unbound am ino co ncentrations o f these patients . It is per-haps m ore log ical to assum e that the bloo d am ino acid concentrations o f theery throblastos is fetalis subjects , taken w ithin 1 to 45 hours after birth, w erepro bably norm al for new born babies the firs t day or 2 of life .

S U M M A R YThe unbound am ino ac id concentrations in the plasm a and erythrocy tes

o f 24 children w ith anem ias o f v arious types have been determ ined by them ethod of salt-saturated paper chro m atography iron de fic iency anem ia-8 subjec ts ; sickle ce ll anem ia-3 subjects ; hereditary sphero cy tosis-3 sub-jec ts; ery throblas tosis fe talis-4 subjec ts ; and 1 subject each for thalassem iam inor, thalassem ia m ajor, unstable reduced g lutathione , acquired hem olyticanem ia, auto im m une hem oly tic anem ia, S & F hem o globinopathy A nemiaper se w as found to have little e ffec t on the plasm a am ino acid concentrations,although a m arked increase in the ery thro cy te concentratio ns o f ev ery am inoac id de term ined w as fo und. In m ultiple reg ression and corre latio n analyses,the unbound am ino acid concentratio ns in the ery throcyte s w ere found tohe a func tion of bo th the re ticulocy te counts and the blo od hem o globin co n-tent, w ith a som ew hat greater dependence on the latter. N o reco gnizabledistinction as to the disease category w as ev ident in the unbound am inoac id patterns o f the anem ia patients . The extent o f the unbound am inoacid e lev atio ns in the ery thro cyte s w as larg ely a reflectio n of the sev erity o fthe anem ia.

The subjects w ith erythroblasto sis fe talis w ere in a catego ry different fromthose w ith anem ia. The ir ery thro cy te am ino ac id co ncentrations w ere proba-l)ly the sam e as those of norm al babies during the firs t few hours after birth.

S U M M A R I O IN IN T E R L IN C U AEsseva de term inate le concentrationes de non-lig ate am ino -ac idos in le

plasm a e le ery throcyto s de 24 juv eniles co n anem ias de vane ty pos . Lem etho do esseva chrom atog raphia a papiro post saturation salin. Le casuisticainc ludeva 8 subjec tos con anem ia a carentia de ferro , 3 subjecto s con anem iaa ce llulas falc iform e , 3 subjecto s con sphero cytosis hereditari, 4 subjec tos co nery throblastos is fe tal, e 1 subjec to cata-un con thalas sem ia m inor, thalassem iam ajo r, instabilitate de reducite g lutathio na, acquirite anem ia hem o ly tic , auto -im m un anem ia hem o ly tic , e hem o globinopathia a hem o globina S e F. Essevaco nstatate que anem ia per se ha pauc e ffec to super le concentrationes plasm aticde am ino -ac ido , ben que un m arcate aug m ento esseva cons tatate in le con-centratio nes ery thro cy tic de o m ne le vane am ino-ac idos includite in le studio .A nalyses de regression e corre lation m onstrav a que le co ncentrationes de non-lig ate am ino -ac idos in le ery throcytos esseva un func tion tanto de l num erationre ticulocy tic co m o etiam de l contento sang uinee de hem og lobina, con un certesuperio ritate de l secunde de is te duo dependentias . Essev a reco gno sc ite nullecorre lation differential inter le co nfiguratio nes de non-lig ate am ino-ac ido s e



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le vane ty pos de an em ia. L e grad o de l eleva tion es in le co ncen tra tion d e lnon-liga te am in o-ac id os in le e ry th rocy to s e sseva in g ran de m esura tin re-f iex ion de l sev erita te d el anem ia .

L e su b jectos con ery th rob las to sis fe ta l fo rm ava un ca tegoria separa te abillo de l sub jec tos con anem ia . L or concen tra tio nes e ry th rocy tic d e am ino-acid oes sev a p ro bab ilem en te iden tic con illo s de no rm a l in fan te s du ran te Ic p rim eho ras po st na to .

CKNOWLE DGMENTT he a uth ors w ish to tha nk D r. J. L . O nc ley , H arvard M edica l S choo l, D r. H . S hw ach-

m an and D r. C . A . Janew ay , C h ild ren s H osp ita l M edica l C en te r fo r th e ir in ter es t anden cou ra gem en t in th is w ork . A pprec ia tio n is a lso expressed to D r . F . X . Fe lle rs, C h ild ren sH osp ita l M edical C en ter , and D r. J . F . D e s F org es, B oston C ity H osp ita l, fo r ass ista nce

in rec ru i ting c h ild ren for th is s tud y . A ck now led gm en t is a lso e xpressed to M r. Jo se ph vanM arck e fo r tech n ica l assis tan ce an d to M r. R og er C an ipbe ll and M r. I )av id I)id is ing ofth e C om pu ter C en ter for assis tan ce in program ing .

REFERENCES1 . N ico laid an ,M .,\lc M e nm y, R . H ., an d

L u nd , C . C . : U nbo un d a m ino acidco nce n tratio ns in p lasm a an d e ry thro -cy tes o f no rm al ch i ld re n and co rdb lo od . A rch . B ioc hen i. B ioph ys . 96 :61 3 , 1 962 .

2 . M cM en am y, R . H ., L und , C . C ., N eville ,C . J. , an d \V allac h , D . F . H . : S tu d iesof u nbo un d am ino acid d is trib u tionsin p lasm a, e ry thro cye s, leuk oc y te s,an d ur ine of no rm a l hu m a n su bjec ts.J. C lin . Invest. 3 9 :1675 , 1960 .

3. W allach , D . F . H .: U n boun d

am ino acid c onc en trat ion s in pIasi ia,ery tb rocy tes , leu koc y te s an d urine o fpat ient s w ith leu kem ia . J. C lin . In ves t.39:1688 1960

5 . Iye r, C . Y . : F ree lev el a m in o acid s inno rm al an d an em i a ery th roc y te s in re-la t ion to po tas siu m c on ten t and g lyc o l-ys is ra te . A rc h . B ioc hem . B io ph ys. 7 4 :24 , 19 58 .

6 . A llen , D .: A m ino ac id a ccu m u lat ion byh u m an re ticu locy tes . B lo od 16 : 164 ,1960 .

M ar ia Nicolaidou M .D . L ed le F el l ow D epar tment of Pedi -atr i cs H ar var d M edical School and the Chi ldr ens H ospi talM edical C enter Boston M ass. Pr esent Addr ess 122 Pati sson

Street Athens Gr eece.

Char les C . L und Pr ofessor of Sur ger y H ar i xi r d M edicalSchool and the F i fth Sur gi cal H ar var d Ser vi ce of the BostonCi ty H ospi tal Boston M ass. Pr esent Addr ess Pr otein F ounda-

tion Boston Mass.

Rapier H . M cM enamy Resear ch Associate D epar tment ofBiological Chemistr y H ar var d M edical School Boston M ass.Pr esent Addr ess D epar tment of B iochemistr y the M edicaland D ental Schools State U niver si ty of New Yor k at Buffalo

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