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1965 26: 91-99

Unbound Amino Acids in the Plasma and Erythrocytes of Children with Various Anemias and with Erythroblastosis Fetalis
MARIA NICOLAIDOU, CHARLES C. LUND and RAPIER H. MCMENAMY

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Unbound of

Amino Children

Acids with

in the Various

Plasma Anemias

and

Erythrocytes and with

Erythroblastosis
By
MARIA NICOLAIDOU, CHARLES

Fetalis
C.
LUND AND RAPIER

H. MCMENAMY in the been acid plasma published values from whether peculiar to and

STUDY
erythrocytes

of

the

unbound
of normal

amino children In the

acid current

concentrations of age amino report

as a function

has

in

an

earlier

communication.1

bloods

of children with amino acid abnormalities any one particular type

different anemias characteristic

of anemia could
METhODS

were studied of anemia in be detected.

to determine general or

The

analyses

were

conducted

in

the

same

manner

as

those

reported

for

normal

chil-

dren.1 Amino salt-saturated

of the intensity
quantities

acids were developing

and size Aspartic

separated by system. After

of the acid, zones cysteine

one-dimensional staining the with standard

present.

tions
as were

were
also

not

determined.
serine plus

Leucine
glycine. Each

and value

( or cystine) isoleucine were

solution was

paper chromatograms using a chromatograms, visual comparison zones were made to obtain the and plasma tryptophan concentra-

determined
in

as combined
triplicate, i.e., at

zones
three

analyzed

different method
covery

concentrations,
averaged

and

a mean

taken.

The

coefficient

of amino

essentially

7 per cent, but varied somewhat for acids added to plasma and erythrocytes complete.2
RESULTS

with this different amino acids.2 The rewas found by this method to be

of variation

In

earlier

studies

it

was

found for the of same

are and

that expressing normal

the the

logarithmic concentration In was the

of patients

normal of the current

distribution amino In acids studies, figure acid condeficiency 1

was
in

reasonably the plasma children ranges and

for sickle (In is

satisfactory and as
the

erythrocytes patients,
values erythrocytes

children.
for

with the

distribution
reported plasma

employed. amino with iron

median

unbound

centrations, anemia, fetalis. mean

the the

cell anemia, logarithmic median in this 12 years values

the level

hereditary spherocytosis, and erythroblastosis normal distribution the antilog of the logarithmic the the linearized median for cord data. values bloods. or
as

for figure

)
for the 0.05)

For

comparison,

there

have

been to
t over

included 1 year, from

taking test,

normal cord
are

children bloods identified

aged differing (evaluated with

4 days signifby letters

1 to the

and
of

Concentrations

icantly

of normal

children

significance

the line graphs

according

to the legend

in the footnote.

Table

1 list 6 pa-

From the Departments of Pediatrics, Biological Chemistry and Surgery, Harvard Medical School, The Childrens Hospital Medical Center and the Fifth Surgical [Harvard] Service of the Boston City Hospital, Boston, Massachusetts. Support for this work was received from Grant #C-2497 of the National Institute of

Health,
Submitted

Bethesda,
Oct.

Maryland.
2, 1964; accepted

for

publication
91

Nov.

20, 1964.

BLOOD,

VOL.

26,

No.

(JuLY),

1965

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92
I. .

NICOLAIDOU,

LUND

AND

MCMENAMY

{ g; I
I
ALANN

I- r

ii!I!fillI!
I
480 a-ANINO-#{248}-SUTYRIC ACID 20

flJj JJ
ik11l
LYSPIt METNIONIIC

I
ISO
-

I
ARGIMN

I-

PI#{128}NYLALANNC
#{149}

I
4p
I:__:_
2400
1500

I
:
;
-

::
+
-#{149}

.
-

_ _

#{149}
--

i.

4t
NIThS#{128}

1.

.&+

0L__
480 360

S.UTAMIC

ACID I

I2001

1 FAN

1I

cs

240

#{176}:Lk
1600

20 SLUTAMl

400
300P

HISTIoII t40
.

flE0NI 450h1

isoI:J;
Izoft+4A*

360F

Fig.

1.-Concentrations
substances in the

(M./Kg.
erythrocytes

water-vertical
(continuous

axis)
lines)

of unbound
and plasma

amino
(dotted

acids
lines)

median concentrations are noted by a the range of concentrations. An A over and erythroblastosis fetalis patients inchdifferent from the median of 10 normal in the fifth column). A B over the graph spherocytosis patients indicates that the value was significantly different from the median value of 15 normal children aged 1 to 12 years (reported in the sixth column). A C over the graph of the erythroblastosis fetalis patients indicates that the value was significantly different from the medium values of cord bloods (reported in seventh column). The normal values and cord blood values are taken from reference 1. The letters H and L identify
the high and low median values, respectively.

with different anemias. The horizontal bar on the lines. The lines mark the graphs for the iron deficiency anemia cates that the median value was significantly children aged 4 days to 1 year (reported for the sickle cell anemia and hereditary

and other of subjects

Glutamic

acid,

ergothioneine,

and

taurine

concentrations

were

very

low

or absent

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UNBOUND

AMINO

ACIDS

IN PLASMA

AND

ERYTHROCYTES

93

v...
).

I
-

1110

4 R
I-

rz
-

z v.

o1l

Itilt!
60 FAH

!II I!
TRtPTOPHAN 64E1 48 321

;
-

d
IAH

;.,

SERINE+OLYCINE

x IO

4#{176}E1

+ 1
200 150L1 I00F,

I6Li 200 900 600 AH BH

j
TAURINE

TYROSINE

: i 5#{176}L.I.
VALINE ERGOTHIONEINE

1
AH UREA xIO

300 96 72 48 2:

_i1

SlOE 4201 280T I40

4
6

k
RETICULOCYTES (S)

4
3OO

+
LEUCWIE
ISOLEUCINE +

T
+
(Fig.
1.-Contd.)

I2

+1 TI
32 (GRAMS

AN 1

__i
HEMOOLO&N PER lOOmI. BLOOD)

OH

.+
200

+.

.k

OL 0

!
plasma and

!
erythrocytes,

I 1
erythrocytes. fetalis
respec-

in

the

plasma.

Arginine

concentrations
in two ,M./Kg.

Ethanolamine average levels lively.

was present of 42 and 74

of

were the

very low or absent in the babies with erythroblastosis

in the

at

water

Histories
Iron

of Patients
deficiency

anemia. The patients were 6-19 months old, weighed 5.6 to 11.3 Kg., pallid, but in fair to good nutritional state. The serum iron averaged 24 ,g./100 ml. blood (range 18-36) . Analyses were conducted on 6 patients before Fe+ + treatment with Imferon#{174}. The other 2 patients had treatment with Imferon#{174} for 10 days and 4 months prior to the analyses, but with slow response. Sickle cell anemia. The subjects were 10-12 years old, pallid, but in fair to good nutritional state. A hemolytic crisis preceded the analyses by 1 to 4 days. Hereditary spherocytosis. The subjects were nine months to 6.5 years old, pallid, but in good nutritional state. Analyses were taken 1 to 5 days after hemolytic crises. Erythroblastosis fetalis. The subjects were 1 to 45 hours old, jaundiced, Coombs positive. Two babies were full term (4.5 and 3.6 Kg.), two were premature (1.4 and 1.9 Kg.).

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94
Table
Plasma TMI TMA UHGS

NICOLAIDOU,

LUND

AND

MCMENAMY

1.-Patients
(SM/Kg. AHA Water)

with

Other

Anemias
Erythrocytes (MM/Kg. Water)

AIHA

SFHb

TMI

TMA

UHGS

AHA 1

AIHA 1

SFHb 1

Alanine a-Amino butyric Arginine

Glutamic Glutamine Histidine

t
nacid
acid

t
I

1
I I

t
I I

I
pius

Leucine isoleucine Lysine Methionine Phenylalanine Ornithine Proline Threonine Tryptophan Tyrosine Valine

I t t C C C I I I I I C C

I I I C

I I I I I I C I

I I C C C C I I I I C I I
36.2 9.7 age group. median; median. 1.3 6.4 The the The I

Ergothioneine Ethanolamine Serine plus glycine Taurine

Urea

I
I (/ of erythrocytes (Gm./100 were ml. blood) against indicates to
female,

Reticulocytes Hemoglobin The symbol

symbol

1.5 9.7 no:mal that that age 7

6

5.4 5.7 nearest lower higher

0.3 11.8 corresponding than than the the state,

age 6

17.1 9.1

patients

evaluated the value value

children it it is

of significantly

the

I I
is
:

following following chosen

normal normal

median
TMI: TMA

the according
minor; major;

indicates the nearest

male, age age

is significantly group. pallid.

extremely

Thalassemia Thalassemia

years.
years,

good
pale,

nutritional
hepatosphenomegaly. ; male,

hepatosphenomegaly.
U HGS : Unstable

reduced state, lead fair

tremities,

glutathione recent poisoning) nutritional

menorrhagia

(Family to age SFHb:

history naphthalene. 4 years, age 13 S +

of
good years,

naphthalene

poisoning) hemolytic
recent

years,
to
AIHA fever,

good
: on

nutritional and
Autoimmune prednisone,

exposure ; male, female, state.

AHA : Acquired nutritional state, jaundice, hemoglobinopathy;

Coombs

anemia
lead poisoning. rheumatic

(due

naphthalene

hemolytic

anemia;

(+ female,

+ + ), age

16

years,

pallid,

pains

in

ex-

aortic

insufficiency.

tients
umns children Except

where
identify

only
amino

1 patient
acids

per
whose

anemia

type

was

studied.
differ from

Arrows
those

in the
of

col-

concentrations

normal

by
for

more the plasma

than two standard deviations. babies with erythroblastosis amino Five of acid the when when the concentrations plasma compared the plasma substances

fetalis of the were

and

several high in

patients subjects the were erythro-

in

table essentially

1, the

anemia children compared were

normal.

blastosis fetalis subjects to 1 year; 4 were high

plasmas, against which

with normal values were fetalis subjects

aged 4 days with cord also evaluated concentrahemotime

at moderate

erythroblastosis

because
tions were

of the
found

very

young age in the plasma patient

copies of

of these subjects. of a 13-year-old was

the

High amino acid girl with autoimmune stress

may be

lytic

anemia.
or

( This
microfilm

under
data for

considerable
each patient

at
obtained

the

of

#{176}Photostat

from the 8420, 11 pages.

cost

American

Documentation

Institute,

Library

of

Congress,

Document

No.

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UNBOUND

AMINO due to

ACIDS

IN PLASMA

AND

ERYTHIIOCYTES

95 It seems probable other subjects amino subjects. age in in evaluated major 17 were groups, sickle erythroblasagainst anemia, high in a high high acid that had conWhen 15 cell the a

analysis,

the related

advanced to this

state stress

of state.

anemia.

high few
In

values high
contrast

are and in with were

to

Some

of the unbound from the 10 were 6 were latter were

low

plasma the plasma patients

amino acid values. values the erythrocyte differed of deficiency high when iron were markedly approximately anemia, the spherocytosis,

centrations compared substances anemia, tosis fetalis

most normal high high

normal same

children in in hereditary

2 were (4

substances

cord erythrocytes , 7 were high in a patient with 11 were high in a patient with acquired hemolytic patient with autoimmune with S & F hemoglobinopathy. observed and in the erythrocytes minor. amino also hemoglobin either the unbound acid thalassemia The unbound were the blood between or each the index level the hemolytic No of anemia, abnormally with and

thalassemia anemia,

3 were high in the patient high amino acid values were unstable reduced of glutathione the anemia count

patients

concentrations for relationships Preliminary amino acid acid

in the

erythrocytes the

patients and tion counts, when where function better 2. The to the
these

investigated

between

reticulocyte

content. unbound amino was acid count

checks showed poor correlaconcentrations and reticulocyte and hemoglobin content An analysis expressed content of the data as a multiple gave a much in table relating that amino

concentrations

set of variables unbound amino of the reticulocyte of correlation. of significance

compared separately. concentrations were and the hemoglobin

The results of the evaluation of the regression coefficients

are shown b1 and b2 indicated individual

reticulocyte two parameters the

count and hemoglobin have a significant

content respectively) effect on most of the

referred

acid concentrations 15 per cent of count and 26 per relationship to analytic of the
analyses (2)

( see
total cent negative. patients

several

exceptions

variation the With

could approximately

be

to below attributed to The cent of amino average is 0.0025,

On

the

the average reticulocyte for the latter due variation

to

hemoglobin average as

content. 6 per the variance

b term of the acids variance or

was anemia

uncertainties using the

( the

concentrations in 6 per to other dietary duplicate cent), the factors. variawere

from the

is taken logarithmic

0.04, distribution

remaining variance, These presumably tions,

only

roughly 50 per would be disease differences. acid,

whose

cent, is to be attributed categories, age variations, plus glycine

differed

or simple
substances

individual

Taurine,

glutamic

serine
concentrations

and

ergothioneine

the
other

appreciably

amino serine the and have their

than

acids plus hemoglobin

with glycine

respect showed content

to hemoglobin a somewhat than the other

content. higher amino acids For were amino studies. in the They

Taurine, concentration acids (b2

glutamic acid dependence values -182, to

and on -75 bebasis

-79, respectively in a common concentrations

any of

These three manner in other

.

have been observed example, on a relative of to leukemia be elevated found

increased
other amino

more acids.3

erythrocytes

patients in the

the

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96 Table
2.-Effect of Reticulocyte Content and

NICOLAIDOU,

LUND

AND

MCMENAMY

Hemoglobin

Content

on

the

Unbound
Proportion

Amino

Acid

Concentrations
Regression Equation
(x 1000)

in the
Constants*

Erythrocytes

of Anemic
Level of

Children
of Variances

Sig.(%) Anal. of

%
b2 95
99 99.9 95 95

SE.

SE.

a Alanine Amino Glutamate

Glutamine Histidine

b 13
14 -1 13 3

b, 4
5 5 4 4

b -34
-53 -79 -29 -29

b 16
19 16 13 15

Var.t 0.99
0.99 0.99 0.995 0.90

b1 99
98 10 99 50

bi 24
24 10 34 2

b2 17
20 59 14 17

2990 n1979 3558 2986 2330

butyrate

Leucine isoleucine Lysine Methionine Phenylalanine Ornithine Proline

Threonine

& 2813 2804 1974 2275 2701 2657

2807

8 14 16 9 6 8
11

-44

16

0.975

90

99

13

24

4 6 5 5 4
5

-60 -52 -48 -41 -41

-62

15 20 17 19 16
17

0.995 0.995 0.975 0.90 0.975

0.995

99 99 90 70 90
95

99.9 98 99 95 98
99

29 20 13 7 13
16

30 22 24
iS

21
34

Tryptophan Tyrosine Valine Ergothioneine Serine & glycine Taurine

Urea

1806 2214 2665 1267 3921 3527

3818

2 11 7 16 8 38
2

4 5 4 8 5 14
4

-26 -41 -36 106 -75 -182

-11 Log the Y

15 18 14 27 19 50
14

0.90 0.975 0.975 0.995 0.995 0.995

low

30 95 90 95 85 99
30

90 95 98 99.9 99.9 99.9

50
x2b2

1 20 14 11 7 22
1

13 15 21 45 41 30
3

5ViLju unbound blood. bloods were used S.E. and

relate amino

to
acid

the

regression

equation.
x, is

a
count,

xibi

and

S.E.
x is

+
the

S.E.,

where
in

is the
ml.

concentration,

reticulocyte

hemoglobin

Gm./i00

is the standard error. All subjects in figure 1 and table erythroblastosis fetalis patients were included in this study performed by the IBM 7094 computer at the Computer Center was
BIMED 29, of the UCLA Medical Center.

I except the normal (20 patients total) of this University. that (bi, distribution te)

children, cord . Computations The program (0,0). a

tAnalysis The level transformation

The

of variance: of significance for the

of
V8JUS

the

values of bi

in and

this b2
only

column give were obtained

in the case

the probability from the

of multiple

using
One would

standard
dif

coefficient.
variance apply regression. obtain

proportions

ferent were

b, each

and

b2

if

the

regressions as one

with independent

the

reticulocyte variable.

counts

and

blood

hemoglobin

contents

determined

separately

erythrocytes iments in that This intake been

dialysis.

of
.

a patient
acid

with
and

a severe
serine plus

kidney
glycine

infection
were also

( unreported
somewhat

experunique

Glutamic

they latter would noted

were than may be that in

the adults.1 be

only

amino Ergothioneine of be

acids the greater binds

found had in to dietary

to be a positive origin patients erythrocyte

higher of less

in the (b2 ergothioneine, seriously protein, be be Another to in

erythrocytes value ill. broken expected point be patients lower concentrafound than corIt 106). as its has in to of in with presumably

of children

b2 value

a reflection to type
larger

expected ergothioneine a loose

with

hemoglobin,
Patients

complex
amounts

which would of hemoglobin on this was leukemia the blood ergothioneine

probably would basis. found than hemoglobin

have interest

higher

concentrations in of an patients earlier

of with are not leukemia patients. acid

ergothioneine study granulocytic Although available, patients With

is that lymphocytic of the

concentration chronic tions that relations

leukemia.3

these patients granulocytic leukemia with amino

it is nevertheless frequently have lower hemoglobin levels regard were to not the reticulocyte as good as the counts,

lymphocytic

concentrations

hemoglobin

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UNBOUND

AMINO

ACIDS

IN PLASMA

AND

ERYTHBOCYTES

97

levels.
for regression

These histidine, reticulocyte

correlations ornithine,
studies since it

were and or

negligible tryptophan.

for Urea
no

glutamic functions
concentration

acid as

and

rather in

poor the with

a control
correlation

demonstrated

the

levels

hemoglobin

content. DIscussIoN

In
ship tions ficiency

none
between in

of
either anemia

the the
the counts

studies disease
plasma usually characteristically

did
or not

there the

appear and had the low erythrocytes.

to

be unbound

any The

distinguishing amino patients

patients,

relationconcentrairon
the

category

acid with

dewith

hemoglobin
In other

concentrations counts
these

reticulocyte

elevated

much.

hemo-

globin
than anemia tent or

content
those found

was
in

usually
iron

not
deficiency

so low identified

but by
was

the

reticulocyte
Despite

were

higher conby high supuncounts

anemia

subjects.

differences,

per
elevated

se,

that
of

is anemia the amino

either

low
always

blood This

hemoglobin latter
reticulocyte

reticulocyte

counts,

almost

accompanied

concentrations ported bound have some

acids

in the studies
hemoglobin

erythrocytes. where
content,

is well between were

by
amino been time

the

multiple
acid

regression The unbound

relationships
and

concentrations, to

evaluated. ago effect

amino

acid

concentrations

reported

be
on

adult
hemoglobin

erythrocytes.4

elevated in reticulocytes, There does not appear the unbound that elevated, This acid
major,

when compared to be a previous acid concentrations amino the individual from deficiency
anemia, for every

with normal report of blood in the erythroin conb cell

nu-

amino the almost

cytes. It is also the

values in figure anemia, hemolytic merically lion cipal of

to be the 1 and

noted were

when trend for

unbound all

acid the

concentratons amino acids in the evident sickle

autoimmune acid was

erythrocytes to
of

tributed

increase. amino table

patients, than elevation found

is evident evaluations the iron

hemolytic value

similarities
2.

the

regression 1 where,
the acquired median

in table

It is also
and

anemia,
amino

thalassemia anemia higher uniform difference

the
of between

median the
our

value erythrocyte
results

for
and

normal amino
those

children. acids
of Iyer5

This
or

observathe prinThe and fetalis,

is perhaps increases for leucine,

Allen.6 glycine, isoleucine,

former, rocyte
serine

studying concentrations
to the

iron

deficiency of alanine,

anemia

patients, acid, but sickle

reported glutamine,

in eryth-

glutamic patient
found methionine,

extent Allen, major,

of two studying thalassemia

hemolytic

to sevenfold, one minor,

anemia,

no increases each cell that with

and
Coombs

valine.

erythroblastosis

thalassemia amino
were

negative

anemia, Coombs concentrations

isoleucine,

positive, and of only certain

and valine)

acids
elevated

( glycine,
in the

alanine, presence of those and

leucine,

The
considerably

blood

amino
different of

acid
from lysine

pattens

high reticulocyte counts. of the erythroblastosis fetalis

of the typical were anemia patients.

subjects The for

to

were plasma and

chil-

concentrations fetalis ornithine amino acid patients, in cord

ornithine

high values

for the
in

the found

erythroblastosis lysine unbound

normal

which
plasma. concentrations

was

similar
Furthermore, were

to

the

high
although elevated

erythrocyte
relation

somewhat

From bloodjournal.hematologylibrary.org by guest on April 18, 2013. For personal use only.

98 dren mostly disturbance haps

probably

NICOLAIDOU,

LUND

AND

MCMENAM1

aged

4 days On in the logical

normal

to

1 year, the basis

the amino that

erythrocyte-plasma information concentrations the taken

babies

concentration it seems of these amino 1 to

day

ratios there was

were little of the

normal. more

of this

that

unbound to assume subjects,

newborn

patients. concentrations after

life.

It is perbirth, were

blood within
the first

acid 45
or

erythroblastosis

fetalis
for

hours
2 of

SUMMARY

The of 24 method 8 subjects; jects;

minor,

unbound children of sickle

thalassemia

amino with

acid anemias paper anemia-3 fetalis-4

concentrations of various subjects; subjects;

unstable reduced

in types

the have

plasma been

and determined deficiency

erythrocytes by anemiasubthalassemia
hemolytic

the

salt-saturated cell

chromatography hereditary and

( iron
1 subject
glutathione,

spherocytosis-3 each for

acquired

erythroblastosis

major,

anemia, autoimmune per se was found

hemolytic to have little

anemia, S & F hemoglobinopathy effect on the plasma amino acid

Anemia

concentrations, every amino analyses, found to

although a marked increase acid determined was found. the unbound amino acid the he a function of both tent, with a somewhat
distinction as to the

in the erythrocyte concentrations of In multiple regression and correlation concentrations in the erythrocytes and the blood on the latter.
evident in the

were

reticulocyte counts greater dependence

category was

hemoglobin conNo recognizable

unbound amino

disease

acid acid the

patterns elevations

of

the

anemia erythrocytes erythroblastosis

patients. was

The largely

extent

of

the of

unbound the severity

amino of from probabirth.

in the with
as those

a reflection in a category concentrations

first few the

anemia. The subjects with

the same

fetalis amino
during
IN

were acid

different were
hours after

those
l)ly

anemia.

Their
of

erythrocyte
normal babies

SUMMARIO

INTERLINCUA

Esseva plasma methodo includeva e

determinate le esseva erythrocytos

le

concentrationes de 24 juveniles a papiro anemia a carentia

de con post de

non-ligate anemias saturation ferro,

amino-acidos de
vane

in
typos.

le
Le

chromatographia con 3 subjectos e 1 subjecto

salin. 3 subjectos

Le con

casuistica anemia

8 subjectos

a cellulas falciforme, erythroblastosis fetal, major, immun instabilitate anemia

con spherocytosis hereditari, cata-un con thalassemia acquirite anemia a hemoglobina

4 subjectos con minor, thalassemia hemolytic, S e F. autoEsseva

de reducite glutathiona, hemolytic, e hemoglobinopathia anemia ben per que se ha pauc un marcate de omne

constatate que de amino-acido, centrationes

Analyses de

effecto super augmento le vane

monstrava que

le concentrationes esseva constatate includite

le concentrationes

plasmatic in le conin le studio.

de non-

erythrocytic
regression

amino-acidos

e correlation

ligate amino-acidos in le erythrocytos reticulocytic como etiam del contento superioritate del secunde de iste duo correlation differential inter

esseva un sanguinee dependentias.

function tanto del numeration de hemoglobina, con un certe Esseva recognoscite nulle de non-ligate amino-acidos e

le configurationes

From bloodjournal.hematologylibrary.org by guest on April 18, 2013. For personal use only.

UNBOUND

AMINO

ACIDS

IN

PLASMA

AND

ERYTHEOCYTES

99

le vane
non-ligate fiexion Le

typos del

de

anemia.
in

Le le anemia.

grado

del

elevationes esseva
formava

in in
un

le

concentration mesura
separate

del tin reab

amino-acidos

erythrocytos
fetal

grande
categoria

severitate
con

del

subjectos

erythroblastosis

illo del subjectos con esseva probabilemente horas post nato.

anemia. identic

Lor concentrationes con illos de normal

erythrocytic infantes

de amino-acido durante Ic prime

ACKNOWLEDGMENT

J. L. Oncley, Harvard Medical School, Dr. H. ShwachChildrens Hospital Medical Center for their interest and encouragement in this work. Appreciation is also expressed to Dr. F. X. Fellers, Childrens Hospital Medical Center, and Dr. J. F. Des Forges, Boston City Hospital, for assistance in recruiting children for this study. Acknowledgment is also expressed to Mr. Joseph van Marcke for technical assistance and to Mr. Roger Canipbell and Mr. I)avid I)idising of the Computer Center for assistance in programing.
The authors wish to thank Dr.

man

and

Dr.

C.

A.

Janeway,

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