From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
Impaired
Nicotinamide
Adenine
Dinucleotide
Synthesis
Kinase-Deficient
Human
Erythrocytes:
A Mechanism
Total
NAD
Content
and a Possible
Secondary
Cause
By Charles
Erythrocytes
from
deficiency
have
reduced)
mal
for
individuals
nicotinamide
In order
decrease
in intact
in total
phate
the
presence
istics
of PK deficiency
had
which
NAD
Fluoride
RYTHROCYTES
not
from
are
abnormalities,’
the decreased
simulates
pyruvate
half
the
lower
This
is limited
total
NAD
of normal
G3PD
reaction
is significant
at
than
should
tion, immediately
reaction,
which
(ATP)-generating
decreased
flux
G3PD
reaction
which
may
following
are
the
is
steady
state,
physioG3PD
enhance
the
in decreased
hemolytic
ATP
may
decreased
the
rate
of
NAD.
it
is
causes
enhance
further
hemolysis
in
Inc.
rate
PK
regeneration,
PK-deficient
concentration
in
acid
elucidated
NAD
biosynthesis
is
of ATP:
in addition
NAD
biosynthesis
1-pyrophosphate
from
(PRPP)
(niacin)
in
by Preiss
nicotinic
(Fig
highly
to the
and
synthesis
three-
dependent
on
ATP
required
the
for
acid and 5-phosphoribosylI), ATP
is also required
the synthesis
of PRPP
from ribose-5-phosphate.’2
presented
in this paper suggest
that the decrease
NAD
concentration
in PK-deficient
erythrocytes
NAD
a
Handler’#{176}”
caused
for
The data
in the total
is due to
by decreased
ATP
regener-
a
of flux
reacreac-
in
nicotinic
first
AND
MATERIALS
a decreased
process
(Fig
I).
availability
impaired
have
the G3PD
reaction,
and the
only
adenosine
5’-triphosphate
result
of
synthe-
ation.
reactions
in mature
erythrocytes.
Thus,
through
the glycolytic
reactions
following
the
will
from
pathway
erythrocytes.
of flux through
G3PD
will also cause a decreased
rate
through
the glycolytic
reactions
that follow the G3PD
tion.
This includes
both the phosphoglycerate
kinase
by
Since
synthesis
thereby
ATP
NAD
availability
& Stratton,
synthesis
reaction
approximately
normal
impaired
is caused
NAD
impairment
by decreased
deficiency.
the
not
erythrocytes.
by Grune
NAD
of NAD.
with
erythrocytes,2’3
rate
because,
under
of the
that
for
does
in a
unexplained
and reduced)
concentration
erythrocytes,
in vivo
a 1987
ATP
and
that
PK
by
and
fluoride
is mediated
impaired
depletion
PK-deficient
metabolic
(G3PD)
the rate
by the
PK-deficient
ATP
(NAD)
in these
cells.2’3
in normal
erythrocytes
is
Km for NAD
of glycer-
dehydrogenase
is 143 ,mol/L,5
in vivo
Therefore,
character-
synthesis
to the
limited
on glycolysis
that
erythrocytes
due
that
eryth-
of NAD
PK-deficient
suggested
dependent
suggest
it is concluded
formation
incubation
is not
data
by fluoride
Thus,
in
ATP
(PK)-deficient
several
One abnormality
that remains
concentration
of total (oxidized
aldehyde-3-phosphate
logical
conditions
sis
that
synthesis
kinase
by
formation.
is
decreased
NAD
enzymes
synthesis
glycolysis
and
characterized
nicotinamide
adenine
dinucleotide
Since the total NAD
concentration
approximately
40 imol/L4
and the
reaction
the
the
that
These
5’-triphos-
normal
inhibit
inhibit
synthe-
enolase.
synthesis
did
After
system
generation.
of NAD
to a degree
cells.
ATP
of nor-
NAD
A. Tanaka
hemolysate
synthesis
adenosine
by inhibiting
impaired
individuals
and
and
NAD
that
of these
of fluoride,
concentrations.
E
activity
(PK)
mechanism(s)
examined
erythrocytes
concentration
in the
rocytes
PK
kinase
(oxidized
(NAD)
the
It is demonstrated
on
(ATP)
total
elucidate
we
in PK-deficient
is dependent
the
dinucleotide
to
NAD,
erythrocytes.
sis is impaired
pyruvate
half
adenine
erythrocytes.
the
with
approximately
R. Zerez and Kouichi
in Pyruvate
for Decreased
of Hemolysis
METHODS
Materials
D-Glucose
was purchased
burg, NJ. All other reagents
Co. St Louis. The potassium
from J.T. Baker Chemical
Co. Phillipswere purchased
from Sigma Chemical
salt of fluoride was used throughout
this
investigation.
Methods
Isolation
blood
of
was
erythrocytes.
obtained
by
After
routine
obtaining
consent,
informed
venipuncture
from
PK-deficient
erythrocytes.
The
decrease
deficient
rate
in
erythrocytes
of flux
trations
decreased.
lactate
the
Thus,
total
NAD
cannot
through
of both
the
be
attributed
dehydrogenase
oxidized
and
a decreased
since
reduced
total
NAD
the
ter,
concentration
are
must
be due to other
metabolic
processes
such as changes
in the
rate of synthesis
or degradation
of this coenzyme.
It is not
clear
how intraerythrocytic
NAD
is degraded
in humans
since
NAD
located
access
shown
other
the only
known
glycohydrolase
on the exterior
enzyme
(NADase:
of NAD
EC
3.2.2.6),
of the erythrocyte
to intraerythrocytic
to be an extracellular
mammalian
capable
NAD.6’7
NADase
membrane-bound
erythrocytes.8’9
Therefore,
the mechanism
responsible
for the decrease
concentration
in PK-deficient
erythrocytes,
trated
our efforts
on an examination
of the
cells to synthesize
NAD.
Human
erythrocytes
Blood, Vol 69. No 4 (April), i987:
pp 999-1005
degradation,
is known
membrane
has
to be
with
the Department
University
ofMedicine,
of California
at
Harbor-UCLA
Los
Angeles
Medical
School
Cen-
of Medicine,
Torrance.
concen-
of NAD
forms
From
PK-
to a decreased
to determine
in the total NAD
we have concencapacity
of these
are capable
of
March
3!,
1986;
Supported
by a grant
from
Foundation
ofLos
the National
Angeles
Institutes
Presented
in
for
and
Congress
at the
Sydney.
of
Medicine,
Carson
St.
“advertisement”
c(
May
Disease
by Grant
AM
Research
14898
from
National
Meeting
Washington.
of
the
DC.
Society
American
May
5. 1986,
of Haematology.
12. 1986.
CA
to Kouichi
R. Tanaka.
Medical
MD,
Department
Center,
1000
West
90509.
costs
ofthis
article
This
article
must
in
22. 1986.
Cell
(K.R.T.).
Harbor-UCLA
400.
Torrance,
indicate
this fact.
1 987 by Grune
and
Research,
requests
Bin
payment.
October
Sickle
of the International
reprint
The publication
charge
the
(C.R.Z.)
the
at
Clinical
Australia.
Address
accepted
ofHealth
part
Federation
no
also been
enzyme
in
Submitted
accordance
& Stratton,
with
were defrayed
therefore
/8
U.S.C.
in part
be hereby
§
1734
by page
marked
solely
to
Inc.
0006-4971/87/6904-0002$3.00/0
999
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
1000
ZEREZ
PRPp
erythrocyte
maintained
hemolysate
Pp,
0
(-OH
Q-LOH
.o-P-o-cH,
o
suspension,
and the complete
at 37#{176}C.
Conditions
for the
were developed
by modifying
mixture.
nicolinic
HH
i4
acid
(niacin)
Glucose
b
NAD
due
5.0
equivalent
(s-OH
times
0
HH
H
nicolinomide
oden
nicolinic
adenine
ne
dinucleotide
OH
hemolytic
trols.
anticoagulated
from
with
normal
anemia
The
nicotinic
5.0
acid;
mol
in a total
10 mol
I S mol
MgCl2;
1-glutamine;
volume
mixture
Tris-HC1,
and
of 1 .00 mL.
pH
5.0 imol
hemolysate
The
assay
was
Thus,
at the
times
indicated,
a 0.20-mL
aliquot
the
before
determination
sensitive
cycling
of the
method
total
of Bernofsky
NAD
and
concenSwan.’5
of oxidized
NAD (NAD),
be converted
to its reduced
formation
from
PRPP.
Conditions
for ATP formation
PRPP and endogenous
AMP and inorganic
phosphate
were
identical
to those used to measure
NAD synthesis
in the hemolysate
dinucleolide
-1(.5
1--
--
I
---(
.4
.0’
and
Blood
ATP;
the incubation
was
from
pyrophosphate.
blood).
PRPP
NaCl
ATP
acid
(NAD)
individuals
Mmol
tenfold
with
Fig i .
NAD biosynthesis
in human erythrocytes.
The reactions
shown
are catalyzed
by nicotinic
acid phosphoribosyltransferase
(EC 2.4.2.1 1 ), reaction
a; nicotinic
acid adenine
dinucleotide
pyrophosphorylase
(EC 2.7.7.1 8). reaction
b; and NAD synthetase
(EC
6.3.5.1
). reaction
c. AMP.
adenosine
5’-monophosphate;
PP. inorganic
1 .0 .tmol
Thus,
contained
The total NAD concentration,
instead
was measured
since NAD
can readily
form (NADH)
following synthesis.
HH
OH
KCI;
of incubation.
tration
0
HH
in hemolysate
to 42 mg Hb
was diluted
(X)
0-P-O-CH,
with
was withdrawn,
maintained
in a boiling water bath for 60 seconds,
and then cooled to 0#{176}C.
The heat-treated
aliquot was centrifuged
to
remove cell debris and denatured
proteins. To minimize
interference
by compounds
present in the incubation
mixture,
the supernatant
HH
OH
AlP
replaced
ofglycolysis.
started
by adding the appropriate
volume of hemolysate,
and the
complete
incubation
mixture was maintained
at 37#{176}C.
NAD synthesis
in both the intact erythrocyte
and hemolysate
incubation
mixtures was determined
by measuring
the accumulation
of the total (oxidized and reduced)
NAD concentration
after various
PP’
)
of the cell contents.
synthesis
5.0 zmol
PRPP;
Mg’
synthesis
to dilution
for NAD
AlP
7.4;
PP,
were
eliminated
since the maintenance
of isotonic
conditions
was no
longer
necessary,
and MgCl2 was added
since the endogenous
concentration
of magnesium
is, presumably,
decreased
after cell
lysis
+
be independent
OH
,/
AMP
phosphate
would
incubation
mixture
was
NAD synthesis
assay in
the previous
incubation
so that
nicoliniC
acid
mononucleol
Ide
‘-0-CM,
inorganic
TANAKA
HH
OH
0-
and
AND
of unknown
separation
and
etiology
and
U/mL
whole
individuals
with
as appropriate
con-
heparin
subjects
(15
from
served
washing
of
erythrocytes
have
0.5
I
been
0
described
previously.’3
Erythrocytes
were
used
immediately
after
E
isolation.
of
Preparation
PK activity
lysates
were
used
osmotic
prepared
to
lysis
hemolysates.
Hemolysates
by using
measure
NAD
of intact
cells
a freeze-thaw
synthesis
without
method.’3
were
4
z
Hemo-
prepared
freeze-thawing
l.5
to determine
used
by
since
-A
using
the
4
I0
latter
I-
manipulation
markedly
the
NAD
a).
Suspensions
precursor
minutes.
pH
7.0, and
the
volumes
after
NAD
NAD
in intact
acid
0.70
revealed
this
mmol/L
and
mixed
that
more
pH
I .0 zmol
nicotinic
containing
gently.
0.5
EDTA,
added
under
a light
of the
erythrocytes
were
99.9%
was
used
-
to the
Examination
hemolysate
#{149}.__#{149}
g for ten
mmol/L
were
-
1, reaction
at 3,000
2.7
.0
to
immediately
zoo
I
0
in
Conditions
were
incubation
7.4;
75
mixture
zmol
acid;
the NAD
for
a modification
KCI;
5.0
equivalent
to 42 mg
mL.
The
was started
pmol
synthesis
of the system
contained
75 /Amol
hemoglobin
by adding
40 zmol
NaCl;
L-glutamine;
(Hb)
assay
of Preiss
and
potassium
30
zmol
D-glucose;
and
intact
erythro-
in a total
the appropriate
volume
of I .00
volume
of
1
8
4
TIME
cytes
assay
(Fig
centrifuged
than
This
assay.
erythrocytes
phosphate,
mononucleotide’4
acid
assay.
synthesis
Handler.’#{176}The
of nicotinic
2-mercaptoethanol
treatment.
synthesis
conversion
were
of a solution
erythrocytes
microscope
lysed
nicotinic
the
of erythrocytes
Two
packed
decreases
OF
I
12
INCUBATION
16
1
20
24
(hours)
Fig 2.
NAD synthesis
in intact
normal
erythrocytes.
(A) Optimal conditions
were used (see Materials
and Methods).
(B) Physiological
conditions
were
used (5.0 mmol/L
glucose.
1 .0 mmol/L
glutamine.
and 5.0 mmol/L
inorganic
phosphate;
other conditions
are the same as for the optimal
system).
The following
incubation
conditions
were used: complete
system
(closed
circles).
without
glutamine
(open
circles).
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
IMPAIAED
NAD
SYNTHESIS
IN PK-DEFICIENT
Table
1 . The
ABC
1001
Quantity
of NAD
Synthesized
and PK-Deficient
in Intact
Patients
After
Red Cells
20 Hours
From
Normal
PK
Activity
Subject
Normal
Splenectomy
(imol/min
.
Subjects
of Incubation
Total
ATP
NAD Accumulated
(zmol/g Hbl
mL ABC)
(imol/g
Hbl
8)
No
reticulocytes)
Yes
0.60
i.90
0.53
No
0.57
2.54
0.70
No
i .52
2.83
1.04
(n
=
5.53
±
0.82
3.6i
0.66
±
i.35
0.24
±
PK deficient
Patient
1
(4i%
Patient
2
(7.5%
reticulocytes)
Patient
3
(7.5%
system
reticulocytes)
(see
mixture.
presence
earlier)
tioned
reaction
and
20
times,
mmol/L
was
were
Following
ATP
was
omitted
supplemented
potassium
from
with
phosphate
withdrawn
(pH
and
centrifugation,
tate ATP as described
The rate of ATP
continuous
that
the
reaction
formation
from PRPP was measured
in the
AMP and inorganic
phosphate,
the aforemen-
mixture
aliquots
her.
except
When AlP
ofexogenous
the
7.4).
was
was
the
indicated
used
measured
that
AMP
as described
also
assay
mmol/L
At
heat-treated
supernatant
by Beutler.”
formation
spectrophotometric
1.0
to quanti-
10 zmol
a
and
hemolysate
equivalent
I .00 mL. The reaction
started
by adding
monitored
252
spectrophotometer
lin,
OH)
was
with
lar
a slit
width
of 340
nm
mm.
amount
The
by using
The
volume
of
absorbance
a Gilford
relatively
Ober-
wide
of hemolysate
slit
necessary
coefficient
of NADPH
remains
6.22
at a slit
width
of
1.0 mm.’6
methods.
PK
activity
was determined
as described
by
Tanaka.’7
The whole blood ATP content
was determined
spectrophotometrically’8
by using neutralized
perchloric
acid extracts.’6
This method
was also used to measure
the ATP
concentration
in
intact erythrocytes
during incubation
with fluoride.
The Hb concen-
Data
was
determined
by using
were expressed
the
as the mean
±
cyanmethemoglobin
phate,
glutamine,
and
nicotinic
NAD
was
accumulated
incubation
NAD
the
mixture
synthesis
more
NAD
significant.
in the
total
incubation
is due
NAD
also
mixture
to NAD
mature
from
suggests
synthesis.
accumulated
and
were
(Fig
2B).
RBC
that
NAD
latter
total
is essential
this
glutamine,
conditions
in normal
ofglutamine
glutamine
erythrocytes,”
of glucose,
physiological
synthesis
Since
accumulation
the
extent,
concentrations
phate
2A).
in human
NAD
total
lesser
in the absence
(Fig
found
their
original
total
incubation
with
the
2A).
Relatively
little
inorganic
reduced
This
size
NAD
is physiologically
five patients
(9.0%
0.I4mol
with
ATP.
had
to
NAD/g
I 5%
Hb.
activity
In
NAD/g
and
ATP
only
this patient
a greater
I).
1 , who
patient
accumulated
Hb (Table
1 ). Since
(Table
I ), presumably
the
PK-
Hb (Table
PK-deficient
content,
and
from
PK activity
total
from
ATP
PK
Erythrocytes
the highest
I .04 tmol
the
total
correlated
NAD
fluoride
Incubation
conditions
to
depletion
tion
0.53
zmol
had undergone
proportion
of
Hence,
phosthat
accumulation
ATP
content,
in normal
that
added
as little
using
erythrocytes
was
dependent
on the
incubation
mixture
after
fluoride
six hours
of incubation
(Fig
mixture
in normal
3).
resulted
intact
The
concentra(Fig
led to a 28%
of incubation
concentration
of I .0 mmol/L
was
decrease
in the ATP
concentration
incubation
synthesis
to synthewe examined
to the
as 0.1 mmol/L
ATP content
3).
decrease
(Fig
3). A
sufficient
to cause
after
only three
addition
of fluoride
in marked
erythrocytes.
inhibition
A fluoride
to the
of NAD
concen-
tration
ofonly
0.10 mmol/L
caused
a 17% inhibition
of total
NAD
accumulation
(Fig 4). Higher
concentrations
of fluoride were more effective
in impairing
NAD
synthesis,
with
1.0 mmol/L
fluoride
causing
full inhibition
of total
NAD
The
the
their
inhibit
enolase2#{176} and
reduce
ATP
formation.
of intact
normal
erythrocytes
with fluoride
under
that yield optimal
NAD synthesis
resulted
in ATP
fluoride
accumulation
To a
with
to a degree
of
for
to simulate
suggests
±
positively
erythrocytes
lowest
the
that
when
1.14
from
etiology
defective
erythrocytes
was in circulation.
Since
the capacity
of PK-deficient
erythrocytes
hours
acid.’#{176}”9We have
accumulated
correlated
the
fluoride
a 34%
erythrocytes
are capable
of NAD
synthesis
in the presence
of glucose,
inorganic
phos-
normal
erythrocytes
increased
content
five to I 5 times
after
compounds
for 20 hours
(Fig
I). RBC
of unknown
accumulated
in the
method.’6
I SD.
RESULTS
Intact
human
when
incubated
had
(Table
anemia
erythrocytes
from three PK-deficient
patients
accusubstantially
less total NAD than erythrocytes
from
subjects
(Table
I ). NAD
synthesis
in PK-deficient
In contrast,
the
Other
tration
contrast,
mulated
normal
Hb
accumulated
after 20 hours of
from eight
normal
subjects
was
more
width
for
imoL/g
total NAD/g
splenectomy
model
Diagnostics,
0.24
content,
at 37#{176}C
and then
of PRPP.
Ciba-Corning
of0.80
the
in a total
hemolytic
NAD
level
of intraerythrocytic
deficient
patient
3, who
resulted
in a relatively
high initial absorbance.
However,
the absorbance
band of NADPH
is fairly wide, the millimo-
extinction
upto
volume
(Gilford,
because
Hb
was preincubated
appropriate
at a wavelength
required
reaction
because
mixture
the
was
to I .7 mg
±
erythrocytes
Tris-
HC1, pH 7.4; 5.0 mol KCI; I .0 mol nicotinic
acid; 1 5 zmol MgCl2;
5.0 mol PRPP;
5.0 mol L-glutamine;
0.40 zmol NADP;
I .0 mol
D-glucose;
I .2 lU glucose-6-phosphate
dehydrogenase;
8.0 lU hexokinase;
1.35
with
reticulocytes)
ear-
by using
contained
The quantity
of total
incubation
in erythrocytes
(Fig
4).
likely
impairment
of
due to decreased
is not
known
synthesis,
what
we
effect
NAD
ATP
fluoride
determined
by measuring
system
that
total
is not
enolase).
Since
the present
experiment
of NAD
synthesis
has
the
enzymes
lysate
tion
synthesis
regeneration.
effect
on
by
fluoride
However,
the
enzymes
of
fluoride
is most
since it
of NAD
on
these
NAD
accumulation
in a hemodependent
on glycolysis
(and
is the first
in a human
known
hemolysate
demonstrasystem,
the
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
ZEREZ
i002
Table
2.
The
Effect
Incubation
of Omissions
Mixture
of Various
on NAD
AND
TANAKA
Components
Synthesis
of the
in Hemolysate
4
Total NAD Accumulated
Contents
of
Mixture
Incubation
Complete
3
0.36
Without
I
0
E
in 20 Hours
(tmoI/g
Hb)
2
0.
nicotinic
acid
1
0
Without
MgCI2
0
Without
glutamine
0
Without
PRPP
Without
ATP
0
Without
ATP and PRPP
0.247
0
4
U..
due to ATP regeneration
Previous
studies
have
I
0
-
-
I
4
8
TIME
Fig
3.
intact
The
effect
normal
synthesis.
plete
system
effect
of fluoride
with
was
in
PRPP
the
after
However,
mmol/L
concentration
conditions
were
fluoride
components
There
of
20 hours
of
for
NAD
used:
corn-
(triangles).
with
was
nicotinic
no total
acid,
of incubation
of the
incubation
NAD
accumula-
MgCI2,
(Table
glutamine,
no total
NAD
or
2). In contrast,
resulted
only in a 32% decrease
after
20 hours of incubation
was
the
in the total
(Table
2).
accumulation
when
both
ATP and PRPP
were excluded
from the incubation
mixture
(Table
2). This suggests
that the substantial
accumulation
of
total NAD
observed
in the absence
of added
ATP
may be
I.5
hemolysate
system.
does
AMP.
(EC
for PRPP
synthesis
catalyze
the reverse
from
reac-
E
0.5
4
I0
I-
interfere
that
ATP
NAD
the
with
reaction
to the
PRPP
PRPP
(Fig
SA).
was
suggests
mixture
mixture.
The
in a time-dependent
No
absent
ATP
from
was
the
that the NAD
do not interfere
the
absence
(Fig
SB).
of PRPP,
The
0
4
TIME
Fig 4.
erythrocytes
8
OF
conditions
were
glutarnine
gles), with
(open circles).
0.50 rnmol/L
(squares).
used:
complete
with
12
results
indicate
that
in the
presence
found
fluoride
20
6
mixture
present
formation
of
the
rate
ATP
ATP
ATP
accumulate
of
when
5A). This
(Fig
in the reaction
from PRPP.
content
and a concomitant
In this system,
ATP
was
formed
(4.6
only
from
system
PRPP
and
and inorganic
phosphate
was not rapid
the rate of ATP utilization.
To confirm
conditions,
with
rapidly
in this
formation
we devised
a continuous
the formation
of ATP
formation
decreased
accumulation
of ATP
by the detection
synthesis
and/or
(closed
circles).
fluoride
triangles).
with
NAD
formation
hemolysate
synthesis
in intact
normal
The following
incubation
mrnol/L
(open
to
reaction
“trapped”
for NAD
the
(hours)
system
0.10
PRPP
sup-
increase
in the absorbATP
that is formed
is
the
system
other
before
it can be utilized
processes.
Under
these
readily
tmol/h
spectrophotometric
results
in the produc-
from
endogenous
presence
that
g Hb
.
‘/25th the hemolysate
AMP
of PRPP
a significant
at steady
and
rate
state)
(1 .7 mg Hb)
was
used
for
I
INCUBATION
The effect of fluoride
on NAD
under
optimal
conditions.
NAD
synthe-
formation
from
were exogenously
precursors
with ATP
the
lack
that
obtained
I
of the
PRPP
Subsequently,
we examined
ATP
formation
from
PRPP
using
the hemolysate’s
endogenous
AMP
and
inorganic
phosphate.
Under
these conditions,
no accumulation
of ATP
was observed
in the presence
of PRPP
(Fig 5B). However,
in
of ATP
I
reverse
under
in our
manner
and
inorganic
phosphate
in the
hemolysate
(Fig SC). It is noteworthy
I
determine
under
our
formation
synthesis
presence
the
plied
accumulated
To
PRPP
from
we first examined
ATP
and inorganic
phosphate
tion of NADPH
ance at 340 nm.
z
reaction.2’
tase reaction,
when AMP
this hypothesis,
system
in which
l.0
0
fluoride
and endogenous
PRPP
synthetase
we measured
yield optimal
To ensure
not
endogenous
AMP
enough
to overcome
=
_1
experimental
conditions,
the same conditions
that
suggested
.
‘.-
the enzyme
responsible
ribose-5-phosphate,
can
and
precursors
of various
examined.
there
ATP
PRPP
that
tion nearly
as well as the forward
whether
ATP was being
regenerated
(squares).
absence
absence
of ATP
NAD
accumulation
the
conditions
O.iO
ATP
20
(hours)
optimal
incubation
(circles).
of omissions
tion
on
under
foIowing
fluoride
mixture
INCUBATION
erythrocytes
The
1 .0 mmol/L
OF
2.7.6.1),
I
6
2
from
shown
without
(closed
trian-
1 .0 mmol/L
nous
more
with
that
and
assay
was observed
(Fig SC).
AMP
and
rapid rate
the rapid
confirm
AMP
synthesis
(Fig
SC).
No
significant
in the absence
of either
Furthermore,
the addition
inorganic
phosphate
of ATP formation
ATP
accumulation
ATP
is formed
inorganic
phosphate
ATP
PRPP
or
of exoge-
to this system
yielded
a
(Fig SC) that is consistent
of Fig SA. These
data
from
under
PRPP
optimal
and
endogenous
conditions
for
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
IMPAIRED
NAD
SYNTHESIS
IN PK-DEFICIENT
RBC
1
I
1003
I
2C
I
I
‘
4
.4
.0
I
I
,,,.
-..
0
0
E
E2
10
Q.
.4
0
.4
5
.I
.
O-._.
I
0
2
TIME
OF
3
INCUBATION
20
I
I
I
I
0
1
2
3
(hours)
TIME
OF
INCUBATION
I
..
‘
20
‘
(hours)
C
Fig 5.
ATP formation
from PRPP in hemolysate
from
normal
erythrocytes.
Conditions
were identical
to those
used to measure
NAD synthesis
in hemolysate
except
that ATP was omitted.
(A) Reaction
mixtures
contained
exogenous
AMP and inorganic
phosphate
(see Materials
and Methods).
(B) Reaction
mixtures
contained
no exogenous AMP and inorganic
phosphate.
The following
incubation conditions
were used: complete
system
(closed cirdes).
without
PRPP (open
circles).
(C) ATP formation
measured
using a continuous
spectrophotometric
assay
system
(see Materials
and Methods).
The following
incubation conditions
were used: complete
system (tracing
1).
without
PRPP (tracing
2). without
hemolysate
(tracing
3).
without
PRPP and hemolysate
(tracing
4). The reactions
were started
(arrow a) by the addition
of PRPP (tracings
1
and 3) or water
(tracings
2 and 4). At the indicated
time
(arrow
b).
the reaction
mixtures
were supplemented
with
AMP and inorganic
phosphate
to a final concentration
of
1 .0 mmol/L
and 20 mmol/L,
respectively.
NAD
synthesis
ties
of total
the
presence
in hemolysate.
NAD
of PRPP
(Table
ATP from PRPP.
In the hemolysate-NAD
initial
first
decrease
two hours
NAD
fluoride
absence
2) is due
synthesis
at
(Fig
to the
the
in the
substantial
to the
6).
incubation
(Fig
6)
intraerythrocytic
bound
NADase6’7
may
NAD
after
,---
a
The
system,
was
NAD
concentration
within
(Fig 6). Subsequently,
in
of
I
I
0
30
in the
total
(Figs
2A
to
20
addition
of up to 1.0 mmol/L
did not affect
total NAD
hours
of
when
intact
is consistent
NAD
I
120
(minutes)
is
erythrocytes
with
not
this
exposed
are
hypothesis
to
used
since
NADase
under
conditions.
initial
in this
We
NAD
higher
red cell
tion
of a decrease
have
shown
that
intact
PK-deficient
impaired
NAD
synthesis
since they
NAD
than normal
erythrocytes
when
sors essential
for NAD
biosynthesis
cell
lack
I
90
DISCUSSION
be caused
by the exposure
of the
pool to the extracellular
membraneThe
content
2B)
I
60
an
rate
lysis.
NAD
and
intraerythrocytic
the
total
linear
mixture
up
-
TIME
quanti-
there
----- -
-.------.------
4--__--.---__,-_
of ATP and
regeneration
accumulation
in the hemolysate
system
(Fig 6). The
decrease
in total NAD
concentration
that is observed
system
3
these
in the total
of incubation
accumulated
incubation
Thus,
accumulated
- -- -
2...._
synthesis
proportion
samples
significantly
between
in PK-deficient
of reticulocytes
without
impaired
NAD
incubated
less
with
(Table
1 ).
PK
total
precur-
Impaired
red cells is not due
since high reticulocyte
PK deficiency
NAD
synthesis.
synthesis.
have
erythrocytes
accumulated
to the
red
did not demonstrate
The positive
correlaactivity,
and
the
level
of
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
ZEREZ
1004
phosphate.
Under
be dependent
these
for PRPP
formation.
of total NAD
in this
I
Since
nents
a
glutamine,
there was
essential
0
sate
-A
accumulation
z
4
I0
or PRPP
the
system
NAD
hence
TANAKA
synthesis
enolase
will
to provide
not
ATP
accumulation
2 and Fig 6).
no accumulation
of total NAD
when compofor NAD
synthesis
such as nicotinic
acid,
that
4
and
There
was a substantial
hemolysate
system
(Table
MgCI2,
suggested
conditions,
on glycolysis
AND
total
was
due
to
of total
were
NAD
excluded
accumulation
NAD
NAD
synthesis.
observed
(Table
2),
in the
hemoly-
The
it is
substantial
in the absence
of added
ATP is due to ATP regeneration
from PRPP
and endogenous
AMP since no total NAD
accumulation
is observed
in the
I-
absence
of both
formation
from
demonstrated
8
TIME
Fig
6.
The
effect
INCUBATION
of fluoride
on NAD
Ihoursl
synthesis
in hemolysate
and
in
thesis
PK-deficient
decreased
ATP
provide
presence
has
of fluoride.
been
used
for
This
synthesis
found
in
that
tions
intact
of NAD
the
(Fig
partial
depletion
synthesis
by decreased
ATP
itself.
Furthermore,
measured
the
even
of
to inhibition
may
trations
as high
activity
in
.
fluoride
To
PRPP
determine
PRPP
Tanaka
KR,
8:367,
the
of
be
have
that
of
is caused
to the PK deficiency
that
impaired
NAD
the
decreased
total
erythrocytes.
shown
that
impaired
erythrocytes
that
NAD
Previous
studies
PK-deficient
in these
erythrocytes.24
impaired
NAD
synthesis
Thus,
in vivo activity
NAD
because
is limited
erythro-
NAD
and
by
Thus,
we have sugmay also be mediated
of PRPP
synthetase.24
synthesis
in PK-deficient
erythrocytes
is
the rate of the G3PD
reaction
and hence
by the NAD
content
of the erythrocyte.
impaired
depletion
PK-deficient
fluoNAD
in
and
inorganic
may
synthesis
be
may
a secondary
cause
further
cause
of
ATP
hemolysis
in
erythrocytes.
ACKNOWLEDGMENT
We
thank
Jasmine
Whe-Yon
Hseih
for
performing
the
ATP
determinations.
twelve
Paglia
DE:
kinase
deficiency.
Semin
5.
A, Dacie
A study
JV: Hereditary
of red-cell
of pyruvate-kinase
non-spherocytic
carbohydrate
metabolism
Br J Haematol
deficiency.
in
10:403,
I 964
3.
Mills
GC,
erythrocytes.
anaemia.
cases
Pyruvate
1971
AJ, Meisler
Grimes
haemolytic
PB,
de Gruchy
11:21,
1965
4.
Beutler
Williams
E:
Wi,
(ed
congenital
York,
Red-cell
enzymes
haemolytic
Biochemistry
Beutler
3). New
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and
E, Erslev
Arch Biochem
6. Goodman
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McGraw-Hill,
of
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7.
Br J Haematol
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MA
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FL:
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Chapman
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1982
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Diphosphopyridine
Physiol
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phosphate
Biophys 146:306, 1971
SI, Wyatt
RJ, Trepel
Enzyme
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Comp
Kuchel
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41:53, 1985
in
Hema-
Hill
The
erythrocytes.
Loder
in non-spherocytic
tology
laboratory
hypothesis
due
suggest
cause
suggests
normal
erythrocytes
by decreased
ATP
synthetase
whether
for glucose
Impaired
significant
glycolysis
concen-
activity
of the enzymes
of
the total NAD
accumulation
by substituting
regeneration
our data
were
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2.
since
do not affect
hemolysate.23
with
the
we measured
hemolysate
synthetase
as 50 mmol/L
crude
ride interfered
biosynthesis,
I
of PRPP
this
measured
enolase
effect
on total
NAD
that fluoride
did not
in hemolysate.
biosynthetic
enzymes
PK-deficient
primary
was
in hemolysate
the
ATP
be readily
and
in intact
is caused
in PK-deficient
concentrations
gested
that
be
requirement
of several
ATP
molecules
for
synthesis
of one NAD
molecule
(Fig 1). The inhibition
NAD
synthesis
in intact
erythrocytes
by fluoride
cannot
to inhibition
a
by the decreased
to the
due
from
3). The
by fluoride
is
since
can
cytes have decreased
PRPP
synthetase
subunit
aggregation
and therefore
a less active enzyme
in vivo.24 This is caused
the decreased
ATP
and increased
2,3-diphosphoglycerate
concentra-
(Fig
synthesis
glycolysis
support
in intact
concentration
of NAD
at
of ATP
synthesis
synthesis
used.
inhibitor
2) and
AMP
NAD
and
synthesis
fluoride
data
To
conditions
4)
These
activity.
characteristics
(Table
by fluoride
of NAD
presence
of
formation.
of enolase2#{176}and
is a potent
synthetase
NAD
in
When
system
by
we
the
erythrocytes
SC).
caused
erythrocytes
under
fluoride
only
syn-
in the ATP concentration
with
used (Fig 3) suggests
that
(and glycolysis)
and, presum-
deficiency
that
cause
sensitivity
due
PK
have
hypothesis,
to simulate
The
simulating
PK
ion is an inhibitor
previously
NAD
be
low
normal
time
We
to
this
in
decrease
at all fluoride
concentrations
fluoride
was inhibiting
enolase
ably,
may
due
accumulation
PK deficiency.22
impaired
erythrocytes
evidence
NAD
that
regeneration
further
total
suggested
endogenous
(Fig
PRPP
impairment
in the
ATP
PRPP
and
bypassed,
fluoride
had no significant
accumulation
(Fig 6). This suggests
inhibit
the enzymes
of NAD
biosynthesis
The lack of inhibition
of the NAD
from
normal
erythrocytes.
The following
incubation
conditions
were
used: complete
system
(closed
circles).
with 0.10 mmol/L
fluoride
(closed triangles).
with 0.50 mmol/L
fluoride
(open triangles). with 1 .0 mmol/L
fluoride
(squares).
intraerythrocytic
and
in the hemolysate
2
OF
ATP
PRPP
of
the
NMR
NAD
spec-
metabolism
of erythro-
of
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
IMPAIRED
NAD
SYNTHESIS
IN PK-DEFICIENT
ABC
1005
9. Pekala PH, Anderson
BM: Studies of bovine erythrocyte
NAD
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LM:
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KR:
Pyruvate
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in Yunis
JJ (ed):
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From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
1987 69: 999-1005
Impaired nicotinamide adenine dinucleotide synthesis in pyruvate kinasedeficient human erythrocytes: a mechanism for decreased total NAD
content and a possible secondary cause of hemolysis
CR Zerez and KR Tanaka
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