CHANGES
IN
RED
CELL
ENZYME
ACTIVITY
IN
RELATION
TO
RED CELL
SURVIVAL
IN INFANCY
Eugene Kaplan, M.D., and J. Tyson Tildon, B.S.
With the technical assistance of
Joseph Stevenson and Claire Fluharty
Deport nents of Pediatrics’ of the Sinai hospital of Baltimore and the Johns hopkins- School of \Ie(licine,
Baltimore, Maryland
O-FILL T(RN
44
0 40
0 36
32
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00
28
24 ---.--- 0
0 20 8
0
8
8(Submitted Jauiuarv 28; acc-d1)ted for pu1)1ic11ti11 April 25, 196.3.)
This studs’ ‘sVaS sUppOrtc(l I)\ Ilc’searc-h Crant A\I-01387 and Training Grant 2A5271 from ti’t’ National
Institutes of Health, United States Public health Service.
ADDRESS: (E.K.) Sinai Hospital of Baltimore, Belvedere Avenue at Greenspring, Baltimore 15, Maryland.
PEDIATRICS, September 1963 371
T
HE ACTiVITY of many red cell enzymesdecreases with advancing erythrocyte
age. This phenomenon of senescence has been measured in vivo by enzyme assays in serial samplings of blood following tagged
red cell transfusion,’- ‘ and in vitro by the separation of a single blood specimen into older and younger erythrocyte fractions
uti-lizing gravimetric’ or differential osmotic
Ilemolysis techniques. In this fashion it has
been demonstrated that
glucose-6-phos-phate dehdrogenase (G6PDH), an enzyme
present in the stroma-free lysate of red cells,
is significantly reduced in fractions
contain-ing older erythrocytes as compared to
VOtlIl cell fractions. ‘ Similarly, it has been
shown that acetylcholinesterase (AChE), an
enzyme present in the stromal portion of
the erythrocyte, is also redtlced in activity in tile older erythrocyte fractions and in-creased in younger red cells.’
The enzyme characteristics of
erythro-cvtes in the newborn period differ from
those of older children and
Ery-throcyte G6PDH activity in the newborn
exceeds that in later life, and gradually de-creases to adult levels in tile first year of
life.1 Erythrocyte AC11E activity is less in
the newborn than in older subjects, and
gradually increases to adult levels in the
first year of life.’10 Studies of these two
enzymes in normal infants have not
in-eluded sufficient data within the first 4 months of life to reveal any consistent pat-tern in this time period.
Red cell life span has 1)eeIl measured in 6
2
8
4
O-5DA’rs 3-9WKS l0-l7WKs
FIG. 1. CI-” erythrocvte half-life, birth to 17 weeks.
Horizontal dashed line represents lower limit of normal adult values. Each circle represents a single
determination on a single full-term infant. (Data
is taken from Kaplan, E., and Hsu, K. S.: PEDI-ATISICS, 27:354, 1961.)
normal full-term infants during the first four months of life.” While at birth the survival of erythrocytes appears the same,
in most instances, as the survival of erythro-cytes from normal adults, between the third and ninth weeks of life there is a significant
the apparent reduction in red cell survival in the second and third months of life re-fleets the decreased erythrocyte life span of an older red cell population, which would
result from the period of decreased eryth-ropoiesis characteristic of the first weeks of life.”
Since red cell activity with respect to
G6PDH and ACiiE decreases with cell
aging, a systematic comparison of tile
ac-tivity of these enzymes in erythrocytes of
infants at successive months of life might support the hypothesis for the shortened red cell life span observed in the second
and third months of life. The present study represents such a comparison with respect to erythrocvte G6PDH and AChE activity.
SUBJECTS
Studies are carried out at three age pe-nods corresponding to the prior groupings
in the study of Cr” red cell survival,” Group A, the first three days of life, Group B, the third to ninth weeks of life, Group C, the tenth to seventeenth weeks of life. The
newborn subjects are chosen at random
from the normal full-term infants in the hospital nursery. Excluded are infants with
ABO or Rh hemolytic disease of the new-born, and infants suspect of an inherited
deficiency of glucose-6-phosphate dehydrog-enase because of extremely low red cell activity of this enzyme.” The older infants represent random subjects appearing in the well baby clinic for routine check-up or in the hospital ward convalescent from minor medical or surgical illness, and without significant anemia. The sex distribution of the infants is approximately equal, and approximately two-thirds are Negro. The adult subjects are normal healthy volunteers of whom approximately half are Negro, and approximately three-fourths male.
METHODS
The blood samples represent venous
blood! from adults, and skin capillary blood from infants. These red cells are washed three times by centrifugation and
resuspen-sion in 0.85% saline SOltltiofl and then made
up to a 20% cell suspension in saline. A further dilution in 20 parts of distilled
water results in lysates which are then
assayed for enzyme activity.
Erythrocyte G6PDH activity is
deter-mined by the method of Zinkham” with
the minor modification that there is no
in-cubation or centrifugation of the lysate
prior to the assay. All assays are performed
in duplicate, and the final value represents the average of the duplicates, which does not vary more than ± 3%. It is found that enzyme activity decreases in lysate prepara-tion after one hour, but is stable in cells suspended in saline solution up to 6 hours.
Activity is expressed as units per gram
hemoglobin in lysate. One unit of enzyme activity is defined as that activity resulting in reduction of 1 .m TPN/minute.
Erythrocyte AChE activity is determined
by
Frawley’s’ modification of Michel’smethod. Variations between duplicates are
less than 5%. Data are expressed as units
per gram hemoglobin. One unit-decrease of one pH unit/hour.
The blood of several volunteers ilas been assayed for enzyme activity repeatedly over
a period of 12 months. Variations for
one individual are usually less than 1,
both for G6PDH and AChE. In one adult
they vary as much as 18%.
In order to demonstrate the suitability of
these methods to detect differences in
en-zyme activity between young and old eryth-rocytes, comparisons are made between the enzyme activity of the top and bottom fractions of several cord and adult blood
samples after centrifugation. In Table I it
is seen that the top fraction, the young
erythrocytes, has activity of both enzymes
which is approximately 150% of tile original, while the bottom fraction, or older
erythro-cytes, has activity approximately 80% of the original.
RESU LTS
The results of comparisons of red cell
G6PDH and AChE activity between adults and infants in the first months of life are
A O-3YS B. 3-I(CKS C. IO-ITSttKS UNITS ‘4 ‘3 #{149}2 I S 6 5 4 6VAN1S .‘lduit Top Botto,n #1 #3 #4 #5 #7 #8 #1 #3
S Each pair represents activity of top and bottom fractions of one blood sample. Activity is expressed as
(frat’t ion act ivity - original activity) X 100.
data represent the mean and standard
de-viation for the adult subjects, and for each of the three infant subgroups. Erythrocyte G6PDH activity is greater in the infants
lip to 17 weeks of age than in adults,
with ou’ne overlap 11 tile enzyme activity of adults and infailts audi i)etWeen infants
of the three age categories. Tile mean
en-zyme activity for the infant groups is
sig-nificantly different, however, and is
de-creased between 3 and 9 weeks as compared to that at birth and after the ninth week. Erythrocyte AChE activity is less in the first months of life than in adult red cells,
with some overlap, but tile mean enzyme
activity for the three infant groups form a
pattern with a decrease in activity
be-tween 3 and 9 weeks of life. G6PDH
ac-tivity at birth is 144%, at 3-9 weeks 108%, and at 10-17 weeks 1ZS% of tile mean adult value. AChE activity at birth is 68%, at 3-9 weeks 55%, at 10-17 weeks 89% of the mean adult value. No significant differences are
observed between male and female, or
white and Negro subjects.
TABLE I
EIIYTII RO(’YTE ENZYME ACTI VITY :* COMPARISONS OF To ANI) B0TT0SI FRA(-TI0NS
Cord
Top 1?i’)ttom
#1 1H 87
ft-i 18 100
#3 151 73
156 81
#5 144 88
#6 187 79
#7 116 9
Aeetyl- t’hohill-esterase
#1 116 97
1 95
#3 136
8-#4 150 77
fc
LTS
‘“‘AMY.
G6PDH AChE
Fic. 2. Erythrocyte G6PDH and AChE activity in
adults and full-term infants up to 17 weeks.
C6PI)H is measured in units/gm of hemoglobin; AChE is measured in units/gm of hemoglobin x 10’. Vertical lines indicate mean, and standard
deviation.
COMMENT
The red cell G6PDH and AChE activity in our studies conform to results previously reported in comparison between the red
cells of adults and newborn full term
in-fants. And like others we have demonstrated
an overall trend toward adult enzyme
ac-tivity values by the fourth month of life.
Our special interest, however, was to ex-amine more closely successive chronologic periods immediately following birth. In this regard our data indicate a significant
de-crease in red cell G6PDH and AChE
ac-tivity between birth and nine weeks of age,
130
::
and an increase in activity at 10-17 weeks19 108 of age. With respect to red cell AChE
ac-16-2 85 tivity, where the trend is from a decreased
130 95 birth level toward a higher level
approxi-108 105 mating the adult, the further decrease in
:
:
activity from birth to 9 weeks of ageap-pears especially significant. With respect to red cell G6PDH activity, where the trend is from an increased level at birth to a
1’)0 96 lesser level approaching the activity in red
I9 cells of adults, the transient reversal of this
108 74 trend is noteworthy.
Since the completion of our study addi-tional data concerning red cell G6PDH ac-tivity in normal infants have been reported
TABLE II
ERYTHROCYTE ENZYME ACTIVITY IN ADULTS AND Fuu-TERu INFANTS*
Pnzymef (6P1)hl Number Activity .-lduht.s’ Infa,,t.s 0-3 days (17) 7 7±0.8
.J-9 neek.c 10-1? weeks
(21) AChE Number Activity (24) 6.3± 1.0 (30)
4.3 ± 0.9
(15) 3.5±0.5 (8) 5.6±0.7 (‘3) (II) 8.3±.() 9.7±1.9 )<O.0Ol 1)0.05 0.001 P<O.OOl
* Activity is expressed as mean and standard deviation. Data represent single determination on single subjects.
t G6PDII
=
Glucose 6 phosphate dehydrogenase (units/gm hemoglobin); AChE= Acetylchohinesterase (unit s/ gus hemoglobinx
102).did not observe the same marked decrease in enzyme activity as we did in comparing the first week and the next 8 weeks of life.
Nevertheless they did demonstrate a
sig-nificant deceleration at the fourth month in the earlier trend toward decreased G6PDH
activity.
These patterns of red cell enzyme activity might reflect differences in tile age distribu-tion of the erythrocyte population at sue-cessive weeks following birth. A decrease in erythropoiesis immediately after birth would result in an older red cell population, with decreased G6PDH and AChE activity and decreased mean red cell life span.
With
a return of erythropoietic activity several weeks after birth there would follow a relative increase in red cell enzymeac-tivity, and in mean red cell life span. The
observed parallelism between red cell life
span and red cell enzyme activity in our
studies adds additional weight to this
hy-pothesis.
CONCLUSIONS
The change in activity of red cell G6PDH and AChE in normal full term infants forms a pattern which parallels that of Cr5’ red cell life span in the first months of life.
These two phenomena, one relating to
erythrocyte biochemistry and the other to erythrocyte function, are in keeping with
the hypothesis that reduced erythropoiesis after birth results in an older red cell popu-lation.
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