Hemolytic Anemia in Hereditary Pyrimidine 5

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Hemolytic
Deficiency:
Anemia
in Hereditary
Nucleotide
Inhibition
Phosphate
Akio
By
We
evaluated
the
itary
pyrimidine
ings
included
erythrocytes
an
ascorbate
cyanide
pentose
red
cells
‘ac-i
-glucose
cyte
measured
was
controls.
(G6PD)
pH
and
7.1
43%
50%
CTP
.
by
activity
the
release
compared
by 5.5
was
mM
find-
a
of the
of
to
14C02
mM
control
from
reticulo-
uridine
a competitive
inhibitor
(UTP)
for
G6P
(K
at
1.7
=
E
RYTHROCYTE
PYRIMIDINE
5’-nucleotidase
deficiency
is characterized
clinically
by a chronic
nonspherocytic
hemolytic
anemia
and splenomegaly)2
In their intial description
of pyrimidine
5’-nucleotidase
deficiency,
Valentine
et al.’ demonstrated
that these
erythrocytes
contain
increased
concentrations
of
pynimidine
5’-nucleotides
and increased
reduced
glutaIn
thione.
observed
incubating
his preliminary
investigations,
Valentine
an increase
in Heinz
body formation
after
pynimidine
5’-nucleotidase
deficient
red
cells
with
acetylphenylhydrazine
despite
“normal”
pentose
phosphate
shunt activity.3
Buc et al. observed
that unstimulated
pentose
shunt activity
was similar
to
that seen in normal
reticulocyte
controls.4
The activities ofG6PD,
6PGD,
glutathione
reductase,
and glutathione
peroxidase
have been shown
to be normal
in
hemolysates
from pynimidine
5’-nucleotidase
deficient
erythrocytes.56
Thus,
the mechanisms
for the chronic
hemolysis
formation
This
metabolic
and the increased
tendency
have remained
unclear.
report
data
presents
pentose
on erythrocytes
to Heinz
control
red
similar
degree
concentrations
G6PD.
activity
body
to
pyrimidine
shunt
patients
and
with
mother
3.59
Case
for
and
noma
white
5’-nucleotidase
history
splenectomy
at
36%,
and
control
KmS
of
phosphate
deficiency.
pathway
of hemolysis
and
control
5’-monophos-
heterozygous
(patient
and
I 3.6,
in
anemia.
uridine
of her
8.20
gram
not
PCV
includes:
l06/isl,
white
32%,
fi,
mother
I .96 and
1.78,
respectively).
7.10
and
6.05,
history
16
to pyrimidine
mo
Her
of
0.68,
hemo2.8 1 x
Pyrimidine
‘)with
hr
Palpable
recent
red cell count
reticulocytes.
.
age.
most
9.8 g’dl,
14.0%
is unremark-
secondary
time.
hemoglobin
and
past
at
at that
nucleotidase
activity
(Mmol
. gHb
substrates were: patient
I .35 and
control
whose
anemia
diagnosed
present
I 12
MCV
girl
hemolytic
deficiency
was
CMP
mother
3.34
and
and
5’UMP
3.86,
as
and
respectively.
MATERIALS
AND
informed
consent,
METHODS
Patients
After
obtaining
collected
with
heterozygous
immune
From
normal
the Department
School
two
samples
were
patients,
their
individuals
anemia
(sickle
with
cell
high
disease,
a-thalassemia).
ofMedicine.
ofMedicine.
in part
ofHealth
the
and
hemolytic
anemia,
blood
from
volunteers,
nonenzymatic
hemolytic
venous
as anticoagulant
heparin
mothers,
Supported
mean
corpuscular
I 2, and
current
volume
with
left
(MCV)
The peripheral
eli-Jolly
bodies,
blood smear shows
and Pappenheimer
5’-nucleotidase
activity
(smol
.
red
127
cell
fi, and
for
present)
breast
packed
count
19.2%
carci-
by Grant
andfrom
x
.
hr)
was
decreased
of
West
Carson
Medicine.
Presented
San
Section.
with
reprint
ment
ing.
reticulocytes.
April
Address
cell volume
2.82
coarse
basophilic
stippling,
Howbodies.
The patient’s
pyrimidine
gHb’
Submitted
Signifi-
(gallstones
includes:
g/dl,
reported
anemia.7
mastectomy
hemogram
12.4
previously
hemolytic
a cholecystectomy
age
hemoglobin
woman
deficiency
included
at age 39. Her
(PCV)
1212
their
Harbor-UCLA
Torrance.
AM-14898from
the Sickle
Medical
Cen-
Calif
Cell
the National
Disease
Research
InstiFounda-
tion, Los Angeles.
I is a 46-yr-old
past
and
chronic
5’-nucleotidase
tutes
pyrimidine
cant
8.73,
patient
2 is a 2.5-yr-old
except
splenomegaly
PRESENTATION
1
and
below
hemolytic
that
a
intracellu-
phosphate
(CMP)
to
2
Case
able
between
the
pathogenesis
while
in
inhibited
pentose
deficiency
by
concentrations
pentose
5’-monophosphate
ter. UCLA
Case
to the
intermediate
pynimidine
5’-nucleotidase
deficiency
and studies
on the
effect of pynimidine
nucleotides
on enzyme
kinetics.
CASE
contribute
as substrates,
activity
5’-nucleotidase
of the
(UMP)
Case
pyrimidine
affected
are
high
and
not
Since
depress
7.8
=
shunt
was
NADP
that
impairment
cytidine
phate
and
5’-nucleotidase
reticulocyte
phosphate
from two
in
7.1
or UTP.
suggest
5’-nucleotides
this
both
CTP
of G6P
data
pH
(K
reductase.
were
at
mM
NADP
phosphate
hemolysate
these
appears
was
dehydrogenase
by 5.5
lar
for
glutathione
Pentose
cell
for
inhibitor
peroxidase.
compounds.
Thus,
R. Tanaka
a noncompetitive
Glutathione
shunt
5’-triphosphate
5’-triphosphate
and Kouichi
pyrimidine
erythrocytes
cytidine
and
these
patients’
high
mM)
mM).
5’-Nucleotidase
and the Pentose
6-phosphogluconate
positive
dehydrogenase
from
5.5
hered-
intraerythrocytic
shunt
in hemolysates
inhibited
(CTP)
by
Neil A. Lachant,
content.
formation,
decreased
decreased
with
Significant
Glucose-6-phosphate
activity
was
and
A. Noble,
glutathione
body
phosphate
as
patients
reduced
Heinz
test.
Nancy
deficiency.
increased
incubated
The
of two
5’-nucleotidase
increased
pH.
Tomoda.
Pyrimidine
of G6PD
Shunt
if..
2. 1982;
accepted
requests
to Kouichi
Bin
400,
Torrance,
in part
at the American
American
Calif
30. 1982.
Tanaka.
M.D..
Medical
Center.
Depart1000
90509.
Society
Texas.
December
Federation
for
February
18. 1982.
(0 1 982 by Grune
& Stratton,
R.
Harbor-UCLA
Street.
Antonio.
June
8.
Clinical
of Hematology
1981.
and
Research,
the
Carmel,
meetWestern
Cal-
Inc.
0006-4971/82/6005-0021$01.00/0
Blood,
Vol. 60. No. 5 (November).
1982
From www.bloodjournal.org by guest on March 31, 2015. For personal use only.
PYRIMIDINE
5’-NUCLEOTIDASE
1213
DEFICIENCY
Materials
All
reagents
MO.),
were
for
except
Inc.,
N.Y.)
Mass.).
adenine
dinucleotide
NaOH
before
using
Chemical
pyrimidine
were
Louis,
England
and
All
Cell
cell
1N
a-cellulose
red
cell
and microcrystalline
adjusted
to
red
cell
were
and
the
by
and
at
The
counts
5’-nucleotidase
et al.’ The
Valentine
glycolytic
of
suspensions
were
cellulose
according
between
2.8
to the
was
determined
formation
6PGD,
Data
prepared
were
to Beutler
and
3.2
The
x
Of
incubated
for 4 hr in 2 ml of0.066
acetylphenylhydrazine
detected
pH
intracellular
Hilpert’2
using
were
reductase
cyanide
and
mg/dl
crystal
glucose.
and
perbody
of
40%)
with
Heinz
Whole
tubes
have
method
to
buffer
was
1 mg/mI
bodies
blood
by a modification
microhematocrit
gluta-
was
the
adjusted
violet.
measured
test
of
(PCV
and
Jandl.’#{176}Heinz
modification
ml
to
activities
M phosphate
30
with
a
0.1
and
by staining
were
and
of the
a Radiometer
red
cell
method
of
Phosphate
Pentose
tion
of the
from
shunt
method
of Davidson
‘4C- 1-glucose
and
ionization
that
the
build-up
are
as mean
given
performed
mined
by
was
buffer
NADP.
Fifty
packed
red cells
Effects
Enzymes
of the
of
with
distilled
blue
was
White
cell
water)
platelet-free
washed
3 times
were
added
to the
red
with
of 2 hr.
deter-
Krebs-Ringer
and
( I :5
to 400
2 mM
dilution
of
l of buffer.
system.
on Red
cells
obtained
isotonic
NaCI
1 . Red Cell Pentose
Table
RBC
Reduced
The
patients’
controls.
Shunt
(tmoles/GIucose
.
Oxidized
Intact
‘)
Student’s
t
Glutathione
and
Enzyme
Activity
Meyerhof
ductase,
pathway,
glutathione
transaldolase
from
solution
Phosph
G6PD,
6PGD,
glutathione
peroxidase,
transketolase,
was
their
mothers.
Heinz
Body
normal
for
both
reand
patients
and
Formation,
Phosphate
Ascorbate
Shunt
Heinz
body
in the patients’
Incubated
increased
cells
had
a markedly
control,
while
of case
between
the
result
normal
and
then
ate Shunt
Activit
( I 0%
and
I 9%)
and
the
0.005). Both patients’
<
positive
ascorbate
cyanide
1 had an ascorbate
cyanide
her daughter
and the normal
in the
mother
of case
2 was
high reticulothe patients’
RBC pentose
shunt activity
was the same as that
normal
controls
and was significantly
decreased
y in Pyrimidine
5’-Nucleotidas
1
of the
(p
Deficiency
High
Reticulocyte
Value
Value
Case
e (P5’N)
Deficient
Patients
ofp
and
normal.
The patients’
red cell pentose phosphate
shunt activity was compared
to that of normal
and high reticulocyte controls
(Table
I ). Before
new methylene
blue
stimulation,
the pentose shunt activity
of the patients’
red cells was higher
than that of the normal
controls
P5’N
33)
Test,
formation
was significantly
(36% and 24%)
ned cells
compared
to their
mothers
normal
controls
(1 1% ± 5%,p
red
Cyanide
Activity
Case
Controls
In -
ofp
2
15)
red cells
Beforestimulation
0.14
±
0.07
<0.05
0.19
0.32
NS
0.21
±
0.12
Afterstimulation
1.79
±
0.60
NS
1.43
2.78
<0.02
3.26
±
0.43
1.28
±
0.29
<0.001
3.71
4.64
NS
4.80
±
2.40
Hemolysate
‘Stimulation
by 1OeM
NS. not significant.
new methylene
blue.
tests
methods.’5
(p < 0.05) but was similar
to that ofthe
cyte controls.
However,
after stimulation,
Cell
Controls
In -
derivation.
statistical
(as high as 1 150 previously);
case 2, 959; and control,
607 ± 79. The activity
of the enzymes
of the Embden-
Reticulocyte
Activity
1O’#{176}RBC ‘ . hr
mea-
reduced
glutathione
(GSH)
content
of the
red cells was elevated
compared
to the normal
GSH values
(jzg/lO’#{176}RBC)
were: case I, 776
Normal
Pentose
the
were
concentrations
I standard
±
by standard
test. The mother
test intermediate
stimu-
was
ATP
hemolysate
5’-Nucleotides
and
were
cell
not added
ofPyrimidine
volunteers
red
7.4
B-4631).
a total
of Smith.’4
1 mM
Fifty
was
hemolysates
with
so
of pH
(Sigma
for
cell
method
was supplemented
microliters
methylene
in red
I ml
system
blue
monitored
activity
modified
system.
in
I hr. the
of ‘4CO2
electrometer
been
in a closed
methylene
continuously
shunt
has
suspended
After
new
release
reed
method
were
buffer.
M
by a modifica-
The
in a vibrating
original
cells
a modification
bicarbonate
Tanaka.’3
is measured
red
l0
phosphate
was determined
and
measured
‘4CO2
10 zl of
production
Pentose
activity
bicarbonate
with
14C02
of
for
under
RESULTS
Pentose
Activity
The
of packed
Krebs-Ringer
lated
was
chamber.
microliters
New
Shunt
phosphate
conditions
described
micro-pH
electrode.
Pentose
gluta-
activities.
for determining
transketolase
of Jacob
by
blood,
g. The
6PGD,
(GP)
detailed
are
membranes
10,000
method.
106
according
laboratory
ascorbate
method
Beutler.’1
whole
determined
glutathione
and
published.9
according
was
in this
transaldolase,
previously
formed
used
G6PD,
peroxidase,
been
activity
methods
enzyme,
thione
37#{176}C.The
hemoglobin
RBC/l.’
Pyrimidine
at
peroxidase
measurements
cyanmethemoglobin
red cell
20 mm
of G6PD,
glutathione
activity
figures.
The
measurement
performed
6PGD
to the
sured
for the
(GR),
water.
at 4#{176}C
for
Statistics
Methods
platelet-free
and
assays
distilled
by centrifugation
was used
legends
with
20 vol of ice-cold
reductase
G6PD
triphosphate
neutralized
removed
thione
nicotine
adenosine
prepared
with
were
supernatant
Nuclear,
5’-nucleotides,
(NADP),
freshly
(St.
Pharmaceuticals,
(New
‘4C-1-glucose
of the
Co.
use.
Red
White
Sigma
(ICN
phosphate
and Tris-HC1
and
and
Solutions
(ATP)
General
from
tert-butyl-hydroperoxide
Plainview,
Boston,
purchased
lysed
<
From www.bloodjournal.org by guest on March 31, 2015. For personal use only.
1214
TOMODA
ET AL.
0.02) compared
to the high reticulocyte
controls.
The
patients’
mothers
had normal
pentose
shunt
activity
(data
not shown).
Since
the patients
had lower than expected
pentose
phosphate
stimulation
shunt
activity
and because
functions
under
after
new
the pentose
restraint
severe
methylene
phosphate
blue
shunt
in the intact
red cell,
shunt
activity
was measured
in red
to determine
if the decrease
in shunt
pentose
phosphate
cell hemolysates
I
CMP
activity
in the intact erythrocyte
was due to increased
shunt suppression
or a loss of metabolic
capacity.
In
the red cell hemolysates,
the patients’
pentose
phosphate
shunt
activity
(
)
was
threefold
that
of the normal
controls
p < 0.001
and was similar
to that of the high
reticulocyte
controls
(Table
I ), suggesting
increased
shunt suppression
in the intact
RBC with pyrimidine
5’-nucleotidase
deficiency.
Intracellular
The
and
pH
intraerythrocytic
transmembrane
and
the
Although
pH
extracellular
(pHi),
for the patients,
pH
pyrimidine
5’-nucleotidase
intraerythrocytic
and
transmembrane
pH,
their
normal
controls
are shown
in
all 3 groups
had similar
plasma
patients
with
had a decreased
an
mothers,
Table
pHs,
Effects
ofPyrimidine
Cell
5’-Nucleotides
deficiency
increased
on the Activity
pHi
NADPatpH
on red cell enzyme
activity.
The effects
of
5’-nucleotides
(CMP,
CDP and CTP)
on the
of G6PD
in red cell hemolysates
at pH 7. 1 (the
of the
red
Table 3. These
ited the activity
cells
of case
I
) is
cytidine
nucleotides
of G6PD.
Most
shown
in Fig.
1 and
significantly
inhibeffective
was 5.5 mM
CTP,
which
decreased
G6PD
activity
by 42.8%.
The
activity
of G6PD
was decreased
by 28.2% and 14.6%,
respectively,
6PGD
CMP.
of 5.5 mM
in the presence
activity
at various
was
not
affected
by
CDP
or CMP.
CTP,
CDP,
2.
Intraerythrocytic
and Plasma
5’-Nucleotidase
P5’N
Case
Extracellular
pH
7.36
(P5’N)
1
Case
7.33
pH in Pyrimidine
Deficiency
Deficient
Mothers
2
Case
7.33
1
Controls
Case
7.33
2
Case
7.37
1
to be 1.78
7.1.
ofCTP
Various pHs
on the
Effects
2). The activity
mM
for
Activity
G6P
and
7.8
ofG6PD
and
The effects
of CTP
on G6PD
and 6PGD
were studied
at pHs ranging
from 6.6 to 7.8
shown
in
Fig.
4. G6PD
activity
declined
mM
for
6PGD
at
activity
and are
in a linear
fashion
as the pH decreased
(Fig. 4A). At any given
pH, there was a further
decline
in G6PD
activity
in the
presence of 5.5 mM CTP. Conversely,
even though
the
declined
with a decrease
in pH, there
suppression
of 6PGD
activity
in the
(Fig. 4B).
activity
of6PGD
was no further
presence
ofCTP
Effects
to confirm
the inhibitory
effects of CTP on
we studied
the effects
of this compound
on the
Table
(Fig.
or
In order
G6PD,
concentrations
of G6PD
decreased
with increasing
concentrations
of
CTP.
Lineweaver-Burk
plots of G6PD
activity
with G6P
and NADP
are shown
in Fig. 3 (A and B). CTP
was
shown
to inhibit
the enzyme
competitively
with G6P,
and noncompetitively
with NADP.
The K values were
estimated
Enzymes
Since the erythrocyte
in pyrimidine
5’-nucleotidase
deficiency
appears
to have a reversible
suppression
of
pentose
phosphate
shunt
activity,
we evaluated
the
effects
of increased
pyrimidine
5’-nucleotide
concentrations
cytidine
activity
Fig. 1 .
Effect
of cytidine
5’-nucleotides
on the activity
of G6PD
in red cell hemolysates.
The reaction
mixture
containing
red cell
hemolysate
(31 MM heme).
100 zM NADP.
and 0.1 M Tris-HCL
buffer at pH 7.1 was incubated
at 37’C for 10 mm in the presence
or absence
of 5.5 mM CMP. CDP. or CTP. Then G6P (0.45 mM final
concentration)
was added to the reaction
mixture.
The increase
of
absorbance
was measured
at 340 nm. The arrow
in the figure
shows the addition
of G6P.
enzyme
2.
the
pH.
of Red
Control
min-’-l
le-5
for
Case
7.34
lntraerythrocyticpH
7.10
7.16
7.18
7.22
7.20
7.21
TransmembranepH
0.26
0.17
0.15
0.11
0.17
0.13
G6PD
The
of Various
and
5’-Nucleotides
on
effects
of various
pyrimidine
and 6PGD
activity
Table
3. Triphosphate
at
(CTP,
TTP)
effective
UTP,
and
of G6PD
activity.
42.8%, 50%, and 52.5%
and TTP,
respectively.
inhibitory
triphosphate
were
the
(5
5’-nucleotides
mM)
on G6PD
summarized
in
tors
2
Pyrimidine
6PGD
most
pH 7.1 are
nucleotides
inhibi-
G6PD
activity
was inhibited
in the presence
of CTP,
UTP,
For all 3 nucleotide
bases, the
effect on G6PD
activity
was
than for the diphosphate
greater
for the
or monophos-
From www.bloodjournal.org by guest on March 31, 2015. For personal use only.
PYRIMIDINE
5’-NUCLEOTIDASE
1215
DEFICIENCY
Table
3.
Effects
of Various
Pyrimidine
5’-Nucleotides
on the A ctivity
of G6PD
an d 6PGD
6PGD
G6PD
.tmole
.
-‘
mm
.
‘
gHb
Percent
moIe
Inhibition
. min
‘
‘
gHb
‘
Percent
Inhibition
Control
9.60
-
2.72
-
CTP
5.49
42.8
2.80
0
COP
6.89
28.2
2.72
0
CMP
8.20
14.6
2.72
0
UTP
4.80
50.0
2.70
0
UDP
5.50
42.7
2.72
0
UMP
8.21
14.5
2.72
0
TIP
4.56
52.5
2.32
14.7
TDP
5.28
45.0
2.80
0
TMP
7.19
25.1
2.71
0
2.18
77.3
-
-
CTP
ATP
+
of reaction
The composition
phate compound.
The
not have a significant
activity
(Table
3).
mixture
and experimental
conditions
pyrimidine
inhibitory
5’-nucleotides
effect
on
5’-Nucleotides
Activity
on Pentose
are the same as in Fig. 1.
did
6PGD
600
Effects
ofPyrimidine
Phosphate
Shunt
1/v
Since
the pyrimidine
5’-nucleotides
do not cross the
intact
red cell membrane,
their
effects
on pentose
phosphate
shunt
activity
was determined
in normal
red
Peitose phosphate
shunt activity
was
in Krebs-Ringer
buffer
containing
2 mM
cell hemolysates.
determined
NADP
system
and I mM ATP. The final pH of the incubation
was adjusted
to pH 7.1 in all studies.
Pentose
shunt
activity
in Krebs-Ringer
buffer
zmoles
glucose
oxidized/1O’#{176}RBC
phosphate
was
0.85
There
mM
was
CTP
a 52%
and
decrease
40.7%
in shunt
decrease
when
.
alone
hr.
5.5
activity
when
5.5 mM
UTP was
1
2
1/G6P
3
(105M’)
300
10
‘-
1/V
.0
I
200
0)
C
0
E
____________
0
10
CTP
20
(mM)
Fig. 2.
Effect of various
final concentrations
of CTP on G6PD
activity.
The experimental
conditions
are the same as in Fig. 1.
except
that different
concentrations
of CTP were
added to the
reaction
mixture.
I
I
I
2
1/NADP
3
(105M1)
Fig. 3.
Inhibitory
effect
of CTP
on the activity
of G6PD
expressed
as Lineweaver-Burk
plots
for substrates
G6P and
NADP.
The initial velocity
of G6PD was measured
changing
the
concentrations
of G6P or NADP
under the same conditions
as in
Fig. 1 . The reaction
was started
by the addition
of 225 aM NADP or
225 MM G6P (final concentration).
(A) G6P. (B) NADP
(#{149}
5.5 mM
CTP. A no CTP).
From www.bloodjournal.org by guest on March 31, 2015. For personal use only.
1216
TOMODA
to
that
of
normal
0)
I-
I
Valentine’s3
E
formation
0
dant
E
and
(3)
Embden-Meyer-
observation
upon
stress.
In
the
of an increase
exposure
order
to
in Heinz
explain
this
body
cells
of affected
to oxi-
phenomenon,
pentose
phosphate
shunt
activity
was examined
intact
red cells before and after
new methylene
stimulation
and also in red cell hemolysates.
The
U
6.6
7.0
7.4
7.8
pH
4
0)
3.
E
2
with
compared
pyrimidine
to those
tions
with
both
counts,
shown
since pentose phosphate
shunt activity
to be a cell age-related
phenomenon.’6
normal
and
5’-nucleotidase
from control
elevated
defipopula-
reticulocyte
has been
Although
controls.
These
data
suggest
that
there
is increased
suppression
of pentose
phosphate
shunt activity
in the
intact
red cell in pyrimidine
5’-nucleotidase
deficiency,
1’
but that the suppressing
factor(s)
is lost or diluted
out
in tests performed
in red cell hemolysates.
This obserI
I
I
7.4
7.8
I
6.6
7.0
vation
activity
The
pH
Fig. 4.
Effct
of CTP on G6PD and 6PGD activities
at various
pHs. (A) G6PD activity
in the presence
or absence
of CTP. The
composition
of the reaction
mixture
is the same as in Fig. 1 . (no
CTP 0, CTP S). (B) 6PGD activity
in the presence
or absence
of
CTP. The composition
of the reaction
mixture
is the same as in Fig.
1 . except
that 0.46 mM 6-phosphogluconate
(final concentration)
was added to the reaction
mixture
in the place of G6P (no CTP 0
CTP#{149}).
added
to the Krebs-Ringer
values
are comparable
ATP-NADP
to the results
buffer.
in Table
nonspherocytic
hemolytic
case 2 was twice
that of case
range
of our high reticulocyte
may be related
to the patient’s
high
reticulocyte
controls
were
I and approached
the
controls.
This finding
young
age. All of our
adults
with
hemolytic
These
occurs
those from an adult
population.
The role of the spleen
must
also
be considered
when
comparing
these
2
patients.
Since
case
I had
previously
undergone
a
splenectomy,
a larger
number
of her most
severely
3.
anemia
may
explain
the normal
hemolysate
G6PD
noted in previous
reports.56
pentose
shunt
activity
of the erythrocytes
of
anemia.
Travis’7
has shown
increased
G6PD,
hexokir.ase, and pyruvate
kinase
activity
in the red cells of
1 1-1 2 mo old infants
compared
to adults.
Thus,
the red
cells of a 2.5-yr-old
child
might
be expected
to have
increased
pentose
shunt
activity
when compared
to
DISCUSSION
A chronic
patients
were
However,
there
was no signifcant
difference
between
the pentose shunt activities
of the red cell hemolysates
from
the patients
and
from
the high
reticulocyte
0
0-
from
ciency
in pyrimidine
5’-nucleotidase
deficiency,
the nature of
which has been obscure.
Associated
clinical
findings
include
splenomegaly
and exacerbation
of the anemia
affected cells might
with
infection,
stress,
or
vestigations
have
shown:
case 2.
Since red cell concentrations
of the pyrimidine
nucleotides
CTP,
CDP,
UTP,
and UDP
have
shown
to be increased
in hereditary
pyrimidine
nucleotidase
deficiency,’8
these
nucleotides
were
formation
after
phenylhydrazine,
unstimulated
in
blue
data
pentose
phosphate
shunt
activity
in pyrimidine
5’nucleotidase
deficiency
was shown to be similar
to that
of normal
reticulocyte
controls
after
new methylene
blue stimulation,
it was significantly
decreased
compared to that of high reticulocyte
controls
(p < 0.02).
5
E
controls,3’4
of the
pathway,
G6PD,
6PGD,
glutathione
reductase,
glutathione
peroxidase
in red cell hemolysates.5
The
current
investigations
of erythrocytes
with
pyrimidine
5’-nucleotidase
deficiency
have confirmed
I
I
of the enzymes
hof
and
.0
hO
reticulocyte
normal
activity
ET AL.
the
pregnancy.2
( 1 ) increased
Previous
Heinz
incubation
of red cells with
but not in fresh
preparations,3
pentose
phosphate
shunt activity
inbody
acetyl(2)
similar
tion, while cells with
would
be sequestered
remain
in her peripheral
circula-
a similar
degree of dysfunction
and destroyed
in the spleen
of
5’been
5’con-
From www.bloodjournal.org by guest on March 31, 2015. For personal use only.
PYRIMIDINE
5’-NUCLEOTIDASE
sidered
to be prime
pentose
phosphate
idine nucleotides
‘4C02
by
pentose
phosphate
inhibitors
of the
pyrimmembrane,
mM CTP and 5.5 mM
effect
on the generation
pentose
presented
for
Since phosphorylated
cross the red cell
that 5.5
inhibitory
the
hemolysates.
The data
candidates
shunt.
do not
we have shown
have a marked
1217
DEFICIENCY
phosphate
indicate
shunt
shunt
in
the
suppression
that
activity
in the
UTP
of
red
G6PD
decrease
cell
of
patients’
fact suggested
that the pHi of the patients’
red cells
should
be decreased,
which
was confirmed
in the
present
study.
The decrease
of pHi may induce
some
metabolic
changes
in the patients’
red cells including
suppression
of the pentose shunt.24 As shown
in Fig. 4,
in the presence
The
red
one
and Table
significantly
content,
chronic
CDP,
UTP,
UDP,
TTP,
and TDP
G6PD
activity.
The mode of inhi-
would
that
the
dependent
nucleotide,
phosphate.
total
the
activity
of 6PGD
inhibitor
however,
(Table
3 and
thione
reductase,
or gluatathione
Oda and Tanaka2#{176} have shown
5’-nucleotides
do not inhibit
of
did
Fig.
4B),
the activities
concentration
3.4-6.4
in affected
red
is in the
range
of
mM. The estimated
red cell concentration
of
is about
1 .tM and the Km of G6PD
for NADP
NADP
is about
5 j.tM.’9
Similarly,
the G6P
concentration
is
about 27 .tM and the Km about
50 j.tM.2122 Therefore,
in vivo red cell G6PD
should not be saturated
by these
substrates.
Furthermore,
G6P and NADP
mM,
respectively
cytic concentration
should
be above
of
since
the
K,s of the
CTP
for
are approximately
1 .7 mM and 7.8
(Fig. 3, A and B), the intraerythroof total pyrimidine
5’-nucleotides
the K1 for G6P and approaching
that
NADP.
Thus,
these
data
strongly
suggest
that
of G6PD
activity
in pyrimidine
5’-nucleotidase deficient
red cells should occur in vivo.
inhibition
Yoshida’9
indicated
that
G6PD
activity
inhibited
40% by physiologic
concentrations
( 1 .5 mM) in normal human red cells. Therefore,
cells
with
pyrimidine
5’-nucleotidase
will be
of ATP
in red
deficiency,
red cells is decreased
as organic
phosphates,
and CTP
that the
(Table
3).
intracelluar
a decrease
which
in reduced
would
serve
glutathione
the
5’since
glutathione
Finally,
In
the
addi-
and concomi-
pH
be remembered
glutathione
of impaired
potential
The
to increase
ion concentration
could shift
reduced
and oxidized
glutaincrease
in reduced
glutait should
reduced
presence
the ATPacross the
content.
intraerythrocytic
function.
in
5’-nucleotides
inhibit
of oxidized
glutathione
membrane,
content
pentose
reduced
that
does
not
phosphate
glutathione
con-
be even higher
than that attained
in pyrimidine
5’nucleotidase
deficiency,
if there was normal function
of the pentose
phosphate
shunt.
In conclusion,
the hemolysis
in pyrimidine
5’-nucleotidase
deficiency
appears
to be due,
in part,
to a
decrease
in G6PD
activity
with subsequent
suppression of pentose
phosphate
shunt
activity.
The mechanisms involved
include:
(I ) competitive
inhibition
of
G6P and noncompetitve
inhibition
of NADP
for G6PD
by the pyrimidine
5’-nucleotides
and (2) further
suppression
of G6PD
and 6PGD
activity
by a decrease
in
intraerythrocytic
pyrimidine
phosphate
pH due
5’-nucleotidase
oxidant
to the accumulation
Thus,
should
5’-nucleotides.
shunt
activity
deficient
stress,
Heinz
red
body
of acidic
the decreased
pentose
render
the pyrimidine
cell
more
formation,
susceptible
and
to
hemolysis.
ACKNOWLEDGMENT
where
the ATP
concentration
is normal,
G6PD
will be further suppressed
by the combined
effects of ATP and
pyrimidine
5’-nucleotides.
In our experimental
system,
G6PD
activity
was markedly
suppressed
in the presence of both ATP
Duhm23
showed
increase
tent of an erythrocyte
with impaired
oxidized
glutathione
transport
and decreased
intracellular
pH might
and Whittaker’t
and
pyrimidine
nucleotide
cells
CTP.
in hereditary
G6PD
deficiency
with
Kondo
et al.25 have demonstrated
pyrimidine
transport
content.
shunt
specific
for G6PD.
Based on reports
of Torrance
Valentine
et al.,1 the estimated
expect
an increased
preclude
the
by the
for G6PD
content
of the pyrimidine
red cell remains
enigmatic,
the decreased
thione
and
to be
are
inhibited
is accentuated
paradoxical
tant increase
in hydrogen
the equilibrium
between
thione,
favoring
a further
of the glyco-
lytic enzymes
hexokinase,
phosphofructokinase,
pyruvate
kinase,
their inhibitory
effect appears
the
intraerythrocytic
tion,
gluta-
peroxidase.
Since
these
pyrimidine
that
cell
red
glucose-6not affect
of
as is seen
hemolysis.
bition
is competitive
with G6P,
and noncompetitive
with NADP
Fig. 3A and 3B). This result is consistent
with the data of Yoshida’9
showing
that ATP,
another
is a competitive
These
nucleotides,
of 5.5 mM
nature
reduced
glutathione
nucleotidase
deficient
cells can be attributed
to the inhibition
of G6PD,
the
rate-limiting
enzyme
of the pentose
phosphate
shunt,
by pyrimidine
5’-nucleotides.
As shown
in Figs. 1-4
3, CTP,
inhibit
and
6PGD
activities
of pHi. This effect
The
for
study,
vibrating
Purcell
wish
authors
Dr.
reed
W.
to thank
Dr.
D. Davidson
electrometer
for technical
Nomie
for
Shore
use of the
apparati,
and
H.
DE,
Harris
for
referring
ionization
M.
Louie
case
and
E. K.
assistance.
REFERENCES
pH
(pHi)
when impermeable
anions,
accumulate
in the cells.
of
such
This
1. Valentine
Hereditary
5’-nucleotidase
WN,
hemolytic
Fink
K, Paglia
anemia
deficiency.
J Clin
with
human
Invest
SR.
erythrocyte
54:866,
1974
2
chamber-
Adams
WS:
pyrimidine
From www.bloodjournal.org by guest on March 31, 2015. For personal use only.
1218
TOMODA
2.
the
Paglia
DE,
Valentine
pyrimidine
Hematol
3:75,
3.
iT,
with
Hereditary
of
1980
Valentine
Paglia
WN:
nucleotidase
Bennett
Wakem
CJ:
red
cell adenine
and
ribosephosphate
ciency:
on two
Studies
red-cell
HA,
JC,
1979
5.
Beutler
PN,
M, Selby
glutathione
A: Study
Feagler
G, Singh
and
defi-
24:1 57,
1973
Chim
of
cases
J,
Matsumoto
P: Hemolytic
Report
Acta
anemia
of eight
F,
due
cases
hereditary
H,
Yunis
hemolytic
in a Japanese
M,
anemia
with
pyrimidine
family.
Hum
Genet
Travis
cell
K, Tanaka
51:107,
Genet
5’-
43:423,
19.
whole
Noble
Rattus
NA,
10.
with
Comp
Jacob
C, Blume
genetic
20.
studies
5’-nucleotidase.
Physiol
Jandl
JH:
A
dehydrogenase
and cyanide.
J Med
N EngI
Beutler
E,
primaquine.
VI.
primaquine.
J Lab
Hilpert
related
Dern
An
P,
RJ,
Clin
and
R,
in groups
of
in 2,3-diphosphoglycer-
simple
visual
AJ,
Oski
disease:
FA:
Effects
Boston,
Glucose
WG:
Abnormal
of NADP
Little,
reple-
Brown,
1974,
Kumar
1969,
SP,
Paez
alterations
in
and
metabolism
in
Methods
in Red
Biochemical
Academic,
I 4: 1 349,
human
Cell
27
PC,
Delivoria-Papadopoulos
postnatal
life
in
glucose-6-phosphate
M:
term
infants:
dehydrogenase.
1980
JD,
Whittaker
in pyrimidine
D:
Distribution
5’-nucleotidase
of
erythrocyte
Br J Haematol
deficiency.
1979
Yoshida
A: Hemolytic
and G6PD
anemia
deficiency.
Science
1973
Oda
5,
Tanaka
KR:
5’-nucleotidase
AS:
Kempe
screening
employing
WD,
Tanaka
KR:
diates
22.
studies
Biochemical
deficiency.
test
for
ascorbate
23.
The
hemolytic
Clin
effect
of
to
D, Bartels
Am
H:
The
J Physiol
Bohr
205:337,
Continuous
measurement
24.
in
Res
erythrocyte
24:441A,
Duhm
Top
Hematol
J: Effect
1976
J Biochem
1971
T,
58:543,
1965
erythrocyte
and
oxygen
Pflugers
of
glycolytic
function
pH
glucose-6-phos-
Arch
Dale
GL,
human
and
326:341,
H, Schultze
and
and
affinity
and
mutant
Beutler
other
Jacobasch
blue
on
E:
pH
G,
the
of
Glutathione
Proc
Rapoport
pathways
in erythrocytes
erythrocytes.
organic
intracellular
1971
M,
methylene
and lactate formation
inside-out
vesicles
from
USA 77:6359,
1980
on
interme-
in normal
of 2,3-diphosphoglycerate
V, Roigas
20:44,
Human
H: Studies
of the
I :1, 1978
on
erythrocytes.
Kondo
U:
Structure
compounds
Albrecht
T, Yoshikawa
J Biochem
Testa
5: The influence
glucose utilization
25.
of
L,
dehydrogenase:
Curr
C, Saito
I. Determination
erythrocytes.
Luzzatto
phosphate
of erythrocytes
5, Suzuki
glycolysis.
in human
human
sensitivity
pH.
Minakami
subjects.
1955
erythrocyte
21.
erythrocyte
phate
1966
for
45:40,
Fleishman
to blood
enzymes
1979
Alving
and
1976
I963
Davidson
SF,
pyrimidine
Hum
of leukocytes
88:328,
62B:81,
162,
test
Med
removal
Med
deficiency
274:1
in vitro
The
Clin
differences
glucose-6-phosphate
.
electrophoretic
Erythrocyte
KR:
Biochem
HS,
KG:
J Lab
blood.
Tanaka
norvegicus
ate levels.
and
ionization
(abstr)
E, West
from
9.
Kinetic
in pyrimidine
York,
Torrance
nucleotides
1979
Beutler
8.
platelets
KR:
deficient
An
1969
1979
JJ (ed):
enzymes
179:532,
erythrocytes
Gottlieb
Yasmineh
metabolic
Res
K:
37:361,
in erythrocytes.
73: 1 73,
in Medicine.
in Yunis
New
Pediatr
Nomura
NE,
6:313,
JJ,
Glycolytic
to pyrimi-
Med
in hepatic
T: Statistics
erythrocytes,
Red
Miro-
Kay
J Hematol
Genetics.
in six families.
Matsumoto
JR.
activity
Clin
pp 11-62,99-150
I 8.
K, Fujii
deficiency
7. Shinohara
I 3.
Am
16.
severe
pathway
J Lab
metabolism
I 5. Colton
baso-
of a case with
Clin
Smith
tion.
1980
5, Nakashima
of human
I 2.
method.
anaemia
I977
effect
phosphate
erythrocyte
(RPK)
deficiency.
PV,
deficiency:
56:251,
Miwa
nucleotidase
I I
pentose
chamber
Lowman
haemolytic
Br J Haematol
Najman
Baranko
dine-5’-nucleotidase
Three
Konrad
pyrophosphokinase
5’-nucleotidase
E,
Quesdada
6.
in
Top
17.
95:83,
Blood
W.
nucleotides,
new kindreds.
Kaplan
pyrimidine
Krivit
Nonspherocytic
increased
Buc
defects
Curr
14.
JM,
philic stippling
4.
acquired
erythrocytes.
#{149}
WN,
DE,
and
human
ET AL.
of man.
transport
NatI
Acad
of
Eur
by
Sci
From www.bloodjournal.org by guest on March 31, 2015. For personal use only.
1982 60: 1212-1218
Hemolytic anemia in hereditary pyrimidine 5'-nucleotidase deficiency:
nucleotide inhibition of G6PD and the pentose phosphate shunt
A Tomoda, NA Noble, NA Lachant and KR Tanaka
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