From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
Inhibition
of the
Pentose
Mechanism
for
Phosphate
Shunt
Hemolysis
in Lead
By Neil A. Lachant,
Recent
investigations
have
shunt
activity
phosphate
5’-nucleotidase
disclosed
deficiency.
ciated
with
an
tidase
activity.
taken
(1 )
compare
The
current
determine
in
erythrocytes
the
hereditary
if
pentose
increased
reduced
glutathione.
a reversible
to
a positive
of
intact
erythrocyte.
lead
M
ANY
MECHANISMS
the
poisoning.1’2
development
Most
studies
acetate
(2)
seen
in vitro
formation.
ascorbate
pentose
acetate
of
and
that
cyanide
in clinical
ability
of lead
and
in the
added
to
red
have
been
indicted
for
anemia
in
have
centered
clinical
around
lead
the
have
nisms.”#{176} 2
Paglia
et
5’-nucleotidase
al.
first
activity
demonstrated
was decreased
cyte after
exposure
Recent
investigations
gested
that
pynimidine
suppression
caused
idine
to
the shortened
5’-nucleotidase
of pentose
5’-nucleotidase
activity
The
and
in lead
presence
impaired
to ( I ) determine
phosphate
shunt
present
investigations
red cell
pentose
pynimdehydro-
of an unexpynimidine
poisoning
prompted
the status
and
was similar
pynimidine
Division
Harbor-UCLA
rance,
Medical
the
Hematology.
Center,
tutes
reagents
of the
UCLA
School
of
Inc.,
Plainview,
Medicine,
by Grant
AM-14898from
the
National
made
fresh
in part
Submitted
Address
UCLA
at the national
April
reprint
Medical
1, /983:
accepted
requests
Center,
meeting
Washington.
to Dr. Neil
I I 24
West
90502.
(ci)
1984
by Grune
& Stratton,
0006-497//84/6303-0004$0/.00/0
Inc.
ofthe
D.C.,
American
May
September
TorInsti-
After
A. Lachant,
Carson
Street,
(15
U/mI
seen
in
of
shunt
hereditary
CA
to detershunt
in vitro
erythrocyte
METHODS
Co. (St.
(ICN
‘4C-glucose
Louis,
Pharmaceuticals,
(New
acetate
consent,
volunteers
blood)
kept
England
and
venous
by
was
routine
used
in ice water
cell
and
as
until
platelet-free
a-cellulose
Beutler.’7
3.2
Chemical
lead
Nuclear,
acetate
were
blood
samples
were
venipuncture.
the
Heparin
anticoagulant.
Blood
used.
Methods
White
using
Sigma
of sodium
informed
whole
General
and
Final
red
red
cell
suspensions
microcrystalline
cell
were
cellulose
counts
were
by
adjusted
the
prepared
method
of
2.8
and
to between
x l06/zI.
otherwise
normal
50 cM
a 20%
of 0.9%
sodium
prior
methods
G6PD,
reductase,
activities
formation
chloride,
used
30
was
determined
2-hr
incubation
was
calculated
(GSH)
in this
have
the
laboratory
mg/dI
of
Reduced
by
except
the
the
ratio
of
Blood, Vol.
the
The
63,
glycolytic
and
40%
that
assay
percent
Heinz
red
1984:
cell
buffer
stability
after
a
stability
reduced
The ascorbate
No. 3 (March),
glutatranske-
the
(GSH)
postincubation
GSH.
in
below.
(6PGD),
body
Heinz
acetylphenylhydrazine.
to preincubation
listed
Incubated
glutathione
with
washed
determining
published.’8
was
glucose,
or 50 pcM
twice
an approximately
Beutler,’t
cells
acetate,
then
transaldolase,
using
in conjunction
with
were
for
previously
glucose.
I MM
dehydrogenase
determined
method
red
and
of the studies
peroxidase,
been
intact
zM sodium
50
Red cells
6-phosphogluconate
glutathione
of
chloride
to the performance
was
by
suspension
sodium
at 37#{176}C
for 30 mm.
saline
enzyme,
thione
stated,
in a solution
either
contained
2, Harbor-
in hereditary
deficiency.
from
and
normal
were
suspension
Torrance,
of
Methods
from
samples
body
/983.
C-/-/
to
daily.
obtaining
obtained
tolase
Society
10, /982.
/3,
that
AND
purchased
NY)
Preparation
thione
Investigation.
mechanism
tert-butyl-hydroperoxide
Solutions
lead acetate
of Health.
Clinical
for
MA).
Unless
Medicine,
of
were
except
The
in part
Presented
518
Department
in part,
inhibition
deficiency.
to that
seen
5’-nucleotidase
MO)
antioxidant
CA.
Supported
for
of
to
to
the
erythro-
Materials
with
the
similar
MATERIALS
incubated
From
enhanced
shortened
to be due.
The
is
concentrations
secondary
shunt.
part.
the
for
which
Magnesium
Thus.
appears
5’-nucleotidase
Boston,
red cell survival
in hereditary
deficiency
is due, in part, to
phosphate
shunt
(PPS)
activity
(G6PD)
activity.’5”6
hemolytic
anemia
respectively.
defense
system
after
lead exposure,
and (2)
mine
if the mechanism
of pentose
phosphate
inhibition
in red cells exposed
to lead acetate
All
in vitro.’3”4
have sug-
effects
of the increased
glucose-6-phosphate
in
(NADP)
lead
poisoning.
pentose
is,
pyrimidine
mecha-
that
pynimidine
in the erythro-
lead
in vivo and
from this laboratory
by the inhibitory
5’-nucleotides
on
genase
plained
as possible
inhibition
M.
poisoning
and
phosphate
2.1
G6PD.
glucose-
glucose-6-phosphate
sensitivity
the
of
phosphofructoki-
cell
fragility
suggested
lead
in lead
and
for
intraerythrocytic
oxidant
G6PD
lead
and
of
activities
and
dinucleotide
to inhibit
survival
the
(G6PD)
of
MM
range
found
of heme3
and globin6’7
synthesis.
Despite
with
the 5tCr labeling
technique,
red cell
has been shown
to be shortened.8’9
Altered
of the Na7K
adenosine
tniphosphatase
dependent
pump
and increased
membrane
been
the
increased
Ks
1 .5
in
impairment
problems
survival
function
(ATPase)
also
within
cyte
inhibited
adenine
were
Normal
test,
mean
G6PD
demon-
activity
normal
markedly
dehydrogenase
to
decreased
shunt
hemolysates
nicotinamide
was
deficiency.
lead
body
suppression
lead,
R. Tanaka
6-phosphate
The
A Potential
Poisoning
and Kouichi
nase.
under-
activity
to
5’-nucleotidase
Heinz
were
inhibition
with
is asso5’-nucleo-
shunt
exposed
incubated
strated
poisoning
in pyrimidine
of
pyrmidine
pyrimidine
investigations
mechanism
erythrocytes
lead
decrease
Tomoda,
in pentose
hereditary
Clinical
acquired
to
decreased
a decrease
in
Akio
by Lead:
cyanide
gluta-
test
pp. 5 18-524
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
INHIBITION
was
OF THE
performed
PENTOSE
by
intermediates,2’
the
SHUNT
method
adenosine
UV),
and
sured
on perchloric
of
iacob
of washed
red cells
5’-nucleotide
were
phoresis
was
for
on
35-UV)
methods.
spectra.22
cellulose
acetate
377-
were
mea-
The
8.6
electroby
standard
studies.
that
Phosphate
Pentose
phosphate
14C- I -glucose
or
electrometer
B-463
was
10 sI of
I ). ‘4CO2
‘4C02
release
of ‘4C02
Final
was
activity
determined
hemolysate
before
2-mercaptoethanol
was
tested
as outlined
incubated
or GSH
or immediately
to
was
for
after
above,
with
various
15 mm
the
in
at 37#{176}C,
I 5-mm
incuba-
acetate.
method
After
new
reed
in
was continuously
deviation.
mined
a closed
blue
monitored
and
paired
methods.26
The
from
kinetics
All
Student’s
data
effect
are
of lead
on red
Eadie-Hofstee
was calculated
t tests
plots,
from
were
expressed
cell
and
performed
as
mean
G6PD
the
activity
effect
Lineweaver-Burk
by stan1 standard
±
was
of lead
deter-
on G6PD
plots.27
in I ml of
I hr, the system
methylene
dard
has been
was suspended
Analysis
Two-tailed
from
in a vibrating
is measured
buffer.
M
by a modification
original
red cells
l0
production
The
of
of packed
bicarbonate
with
The
measured
chamber.
build-up
7.4 Krebs-Ringer
stimulated
2
the
microliters
lead
Statistical
in the preincu-
section)
and Tanaka.24
ionization
so that
Fifty
was determined
Methods
‘4C-2-glucose
and
modified
activity
of Davidson
cell
and
in G6PD
of 2-mercaptoethanol
immediately
with
glutathione
changes
activity
red
substrate.
Activity
(see General
of the method
pH
shunt
erythrocytes
system.
Shunt
reduced
G6PD
the
starter
I ml.
lead-induced
except
either
of the appropriate
was
of
the
other
tion
bated
ability
concentrations
methods,23
Pentose
the addition
of all assays
prevent
of pyrimidine
pH
after
volume
extracts
Hemoglobin
at
tometer
Kit
Acid
the determination
absorption
done
(Sigma
Kit
by standard
used
ultraviolet
iandl.2#{176}Glycolytic
(AlP)
(Sigma
extracts
519
and
triphosphate
2,3-diphosphoglycerate
acid
BY LEAD
was
RESULTS
(Sigma
for a total
of
Effect
ofLead
on Red
effects
of lead
Cell
Metabolism
hr.
Pentose
made
phosphate
from
an
modification
buffer
shunt
aliquot
of the
with
distilled
blue
was not added
Enzyme
water)
direct
of Smith.25
I mM
to this
cells
by
a
bicarbonate
and 2 mM
dilution
The
in hemolysates
red
Krebs-Ringer
ATP
( 1 :5
hemolysate
was added
NADP.
of packed
to 400 zl of buffer.
Fifty
red
New
cells
6PG D, glutathione
on the activity
hemolysates.
reductase,
methods)8
the
5’-nucleotidase
deficiency.’6
found
thawed
pH
red
and
the
prepared
cells
was
pH
Hexokinase
at
necessary
incubated
then
performed
at 37#{176}C
for
water,
g
chosen
15 mm.
Table
because
20
is
determined
Fresh
of
in
activity
red
cell
leukocyte
was
then
mm
at
and
freeze-
4#{176}C.The
substrates
were
and brought
to volume
added and the solution
enzyme
2400-S
that
pyrimidine
was
glycolytic
The
kinase
EDTA,
hexokinase
which
for
and
at 37#{176}C
in a Guilford
pyruvate
specimens.
added in sequence
to the buffer
in a cuvette
with distilled
water.
Lead acetate
was then
was
for
30 mm.
activity
assay
recording
1 . Metabolic
spectropho-
Changes
Heinz
body formation
Reduced
(%)
was
exposure
to lead,
hemolysis
occurred
when
red
preincubated
the red cell reduced
by 1 1% (p
glutathione
the red
incubated
with
acetate
cells incubated
with sodium
with
lead
Heinz
body formation
with
acetylphenylhydrazine.
content
was decreased
the content
of reduced
<
0.05).
was
(p
(g/10’#{176}RBC)
5
599
glutathione
Although
decreased
in
lead compared
after
exposure
to those
to ace-
those
incubated
with sodium
these metabolic
changes
were
presence
of acetate.
Aft er Incubating
Intac
t
Erythrocytes
with
acetate,
indicating
that
due to lead and not to the
lead
I versus II
Value of p
III
Sodium
Acetate
NS
13
±
150
NS
601
±
15
NS
53
II versus III
Value of p
±
3
<0.005
±
147
<0.05
±
19
Lead
Acetate
82
±
11
5
54
535
±
159
±
22
glutathione
stability(%)
Ascorbate
cyanide
Isopropanol
t
test
test.
NS, not significant.
test
<
tylphenylhydrazine
(341
±
164 versus
278
±
171
jsg/10’#{176} RBC,
p < 0.08),
there
was no significant
difference
in the percent
glutathione
stability.
The
ascorbate
cyanide
test was positive.
There
were
no
significant
differences
in these variables
when red cells
incubated
with
sodium
chloride
were
compared
to
6
±
variables
hemolysis
occurred
with
50 .tM lead
II
Sodium
Chloride
13
metabolic
glutathione
Reduced
‘Paired
3
No
erythrocytes
I
n
cell
1. Minimal
preincubated
had significantly
increased
0.005)
after
incubation
After
isomerase,
in hereditary
a I :5 dilution
10,000
cofactors,
transaldolase,
7.1, without
activity
control
from
in distilled
centrifuged
hemolysate,
of 7.1
pH
pH 8.0, as no measurable
7. 1 in
were
platelet-free
pH
intraerythrocytic
buffer,
at
The
and
buffer,
was
of G6PD,
phosphoglucose
dehydrogenase,
by standard
hemolysates
peroxidase,
nase,
in 0.1 M Tris-HCI
approximately
enzymes
for the activity
glutathione
glyceraldehyde-3-phosphate
0. 1 M Tris-HCI
of red cell
Assays
phosphofructoki
determined
acetate
on red
in Table
cells were
cells were incubated
in the sodium
chloride
or sodium
acetate
containing
solutions.
Although
Heinz
bodies
were not present
prior to the incubation
with acetyl-
methylene
system.
of lead
in red cell
transketolase,
are shown
when
red
phenylhydrazine,
effect
determined
was
determined
preincubated
Studies
The
were
was
same
with
red cell
of
the
method
was supplemented
microliters
activity
of
NS
45
Neg
Neg
Pos
Neg
Neg
Neg
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
LACHANT,
520
Effects
ofLead
Intermediates,
on Enzyme
Pyrimidine
Hemoglobin
in Intact
Intact
red
cells
lead,
sodium
washed
with
enzymes
of the
and
were
nase
which
exposed
30 mm
pathway
Our
was decreased
to sodium
activities
were
98%, respectively,
cells; nor were
intraerythrocytic
phoglycerate,
with
twice
of the
Stimulated
sodium
those
(hexoki-
were
to 85%
in
of that
seen
Hexokinase
unchanged,
seen in the
and
being
unleaded
(data
did
for pynimidine
not shown).
Red
not show a shift
5’-nucleotides
with
(data
bin instability
in the isopropanol
trophoresis
of hemolysate
from
tose
red
not show
the presence
(data
not shown).28
of
ofLead
on Pentose
Phosphate
Eleceryth-
washed
ty.
saline,
Table
with
2.
Pentose
before
Phosphate
the
Activity
(Mmole
Shunt
made
sodium
Intact
sodium
(p
when
chloride
0.005)
<
acetate
acetate.
0.001)
<
in
compared
(Table
to
2).
of a decrease
acetate.
was
in the
Stimulated
decreased
(p
pen0.05)
<
due
in
from
acetate,
red
cells
or sodium
exposed
chloride
to
lead
2).
The
enzymes,
PPS
Glucose
activities
glutathione
of
Activity
the
pentose
reductase
and
idase,
and key glycolytic
enzymes
fresh
hemolysates
made
from
Enzyme
activities
were determined
phosphate
glutathione
shunt
perox-
were determined
in
normal
red
cells.
after incubation
of
the hemolysate
with
various
concentrations
of lead
acetate
for 15 mm (Fig.
I ). There
was a significant
decrease
in the activities
of G6PD
and phosphofructokinase
at concentrations
of lead achieved
in the eryth-
activi-
Oxidized
.
1 O’#{176}
RBC
.
hr
‘) After
Incubating
Intact
Erythrocytes
With
II
Sodium
Chloride
I versus
Value
of
II
p
III
Sodium
II versus
Acetate
Value
Ill
Lead
of p
Acetate
red cell
Unstimulated
8
0. 1 2
±
0.07
NS
0. 1 1
±
0.05
<0.005
0.06
Stimulatedt
8
2.12
±
0.56
<0.001
1.64
±
0.44
<0.005
1.08
Unstimulated
3
0.08
±
0.04
NS
0.04
±
0.01
NS
Stimulatedt
3
0.97
±
0.20
<0.05
0.66
± 0.15
NS
3
2.42
±
1.26
NS
1.84
0.92
NS
±
0.03
±
0.42
0.03
±
0.01
0.72
±
0.06
2.34
±
0.67
Red cell hemolysate
t
test.
10 ‘M new methylene
NS. not significant.
blue.
the
(Table
‘4C-2-glucose
‘Paired
to
in red cell
a revers(denatu-
“C- 1-glucose
‘4C-1-glucose
in
of the shunt.
There
were
no
in the PPS activities
in red cell
Effect
ofLead
on Enzyme
in Red Cell Hemolysates
I
n
decreased
red cells
sodium
by
methy-
Activity
with the sodium
chloacetate
solutions
and then
measuring
new
(p
acetate
exposed
red cells compared
to those
with sodium
chloride
(Table
2), and again,
hemolysates
activity
and
pentose
phosphate
recycling
through
the shunt
were
measured
in intact
erythrocytes
by the release
of ‘4C02
from
‘4C-l-glucose
and
‘4C-2-glucose,
respectively,
before
and after
stimulation with new methylene
blue. Pentose
shunt
activity
was also
measured
in hemolysates
made
from
an
aliquot
of the same
batch
of red cells.
In all studies,
were preincubated
acetate,
or lead
with
recycling
ration)
of the enzymes
significant
differences
PPS
erythrocytes
ride, sodium
decreased
with
after
intact
red cells, PPS activity
was measured
hemolysates
to determine
if lead had caused
ible inhibition
or an irreversible
inactivation
a fast-moving
Shunt
was
is a reflection
incubated
phosphate
acetate,
Effect
was
this most likely
represents
a pH-related
effect
the presence
of intraerythrocytic
acetate.
Because
lead
decreased
the PPS
activity
hemoglo1 ).
activity
exposed
with
likely
the sodium
incubated
not shown).
test (Table
the lead-exposed
most
those
95%
cells incubated
in the spectral
lead did not show
activity
stimulation
decreased
by 34%
TANAKA
intraerythrocytic
pH due to the presence
of acetate.
Conversely,
there was no measured
difference
in the
rate of pentose
phosphate
recycling
through
the PPS
before
or after
new methylene
blue stimulation
when
red cells incubated
with lead acetate
were compared
to
In the lead-exposed
in phosphofructoki-
acetate.
basically
of that
Red cells incubated
rocytes
did
hemoglobin
findings
shunt
0.005)
PPS
acetate
preincubated
This
there
any significant
differences
in the
concentrations
of ATP,
2,3-diphosglucose-6-phosphate,
fructose-6-phos-
phate,
or lactate
with lead acetate
scan
for
chloride
and
for the activity
Embden-Meyerhof
peroxidase.
activity,
and
preincubated
with those of Paglia.’3
the largest
decrease
was
in red cells
G6PD
lene
phosphate
before
(p <
blue and was
AND
stimulation,
when
red cells exposed
to lead
were compared
to red cells exposed
to sodium
lactate
dehydrogenase),
G6PD,
6PGD,
transketolase,
glutathione
reductase,
glutathione
agreement
red cells,
45%
RBC
acetate,
or sodium
saline prior to assay
nase through
transaldolase,
Pentose
Activity,
Glycolytic
5’-Nucleotides,
and
TOMODA,
±
lead
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
INHIBITION
OF THE
PENTOSE
SHUNT
BY LEAD
521
Glutathione
Transaldolase
100
Peroxidase
Transketolase
Pvruvate
Kinase
AOC
G)yceraldehyde-3-phosphate
Dehydrogenase
a
U)
-I
75
0
I
C-
C-
Fig. 1 .
The effect of lead on enzyme
activity
in
red cell hemolysate.
The reaction
mixtures
contamed 20 ILl of normal
red cell hemolysate.
0.1 M
Tris-HCI
buffer.
pH 7.1 . and appropriate
substrates/cofactors.
lead
acetate
(0-200
MM final
concentration)
was then
added
to the reaction
mixture.
After
incubation
at 37’C for 1 5 mm, the
reaction
was started.
The residual
enzyme
activity
after lead exposure
is expressed
as the percent
of
the enzyme’s
activity
determined
in the absence
of
lead. The shaded area represents
the intraerythrocytic lead concentration
attained
in clinical leading
poisoning.
>
C- 50
0
CHexokinase
N
z
U)
25
Glutathione
LEAD
rocyte
in clinical
lead poisoning
(3-6 jsM).
Phosphofructokinase
activity
was decreased
by 50% with 0.9
.tM
lead
and
was
completely
activity
G6PD
was
inhibited
glutathione
completely
6PGD,
less affected,
by 8-9 j.tM
inhibited
zM
lead.
inhibited
50% by 1 .8 M
lead, and
by 10 .zM lead. The activities
of
reductase,
and hexokinase
were
with 6PGD
activity
being inhibited
50%
lead. The activities
of glutathione
peroxi-
dase,
transaldolase,
tra nsketolase,
isomerase,
glyceraldehyde-3-phosphate
nase, and pyruvate
kinase
were not
200
by 8 .zM
phosphoglucose
dehydrogeaffected
by up to
lead.
CONCENTRATION
(tiM)
incubated
at
conditions:
or without
( I ) in 0. 1 M Tnis-HC1
37#{176}Cfor
2 .tM
buffer,
pH
NADP
and
7.1,
the assay.
3 as
Eadie-Hofstee
lead had
of G6PD.
and
lead,
with
mm
and
under
(2)
10 mM
These
data
NADP
for
G6PD
calculated
K1 of lead
for G6P
in 0.1
M
,
and
were
In the
in the
from
was
following
pH 7. 1 with
Tris-HC1
2 M
then
are expressed
plots.
When
the
buffer,
MgCl2
in Figs.
effect
presence
Effect
of Lead
G6PD
activity
on G6PD
was
Kinetics
measured
lead
or absence
in the
in hemolysates
and
from
normal
red cells in 0.1 M Tnis-HC1
buffer,
pH 7.1,
with 10 mM MgCI2.
Prior to measuring
G6PD
activity, hemolysates
were preincubated
for I 5 mm with or
without
lead.
G6PD
activity
was measured
in the
presence
of varying
glucose-6-phosphate
in Figs.
2 and
3 as Eadie-Hofstee
noncompetitive
G6PD.
the
concentrations
(G6P).
These
inhibitor
When
calculated
with
from
of
data
G6P
Lead
and
was
NADP
Lineweaver-Burk
for
mean
Km for G6P
was 41.3
sM, with a mean
lead. The mean
Km for NADP
was 34.5
a mean K of 2. 1 .tM lead.
with
The Effect
of Thiol
ofG6PD
by Lead
Red
cell
presence
hemolysates
K of
M,
were
on the Inhibition
incubated
of 2-mercaptoethanol
Iz
of
The K1 of
of MgC12
of MgC12.
Reagents
with
various
or reduced
gluta-
co
Mg(-)Pb(-)
200
a
plots,
1.5 tM
in the absence
concentrations
NADP
and
are expressed
plots.
4.6 zM
of
plots,
in the absence
of MgC12.
was 2.1 jM in the presence
for NADP
activity
of G6P
Lineweaver-Burk
I .5 .tM
to
2 and
of MgCI2,
on the
inhibitor
MgC12 and 4.9 iM
The
lead.
added
presence
an increased
inhibitory
Lead was a noncompetitive
MgCl2.
the
15
glucose-6-phosphate
start
Reductase
Mg(-)Pb(+)
‘-.----
Mg(+)Pb(+)
0
,; 100
ci
>
The
Effect
ofMagnesium
on the Inhibition
of G6PD
v/s (LO.D.
by Lead
Zinc,
aluminum,
and
to a lesser
extent,
selenium,
have been shown
to antagonize
the inhibitory
effect of
lead on #{244}-ALAdehydratase
activity.29
As the concentration
of magnesium
in the red cell is approximately
3
mM,
we
inhibition
examined
of G6PD
the effects
of magnesium
by lead. Red cell hemolysates
on
the
were
x104MIN1/nMOL
G6P
.
Ml))
Fig. 2.
The inhibitory
effect
of lead on the activity
of G6PD
expressed
as an Eadie-Hofstee
plot for substrate
G6P. The reaction mixture
contained
20 isl of normal red cell hemolysate
and 0.1
M Tris-HCI
buffer at pH 7.1, with or without
10 mM MgCI2 (final
concentration).
After the addition
of 2.0 MM lead (final concentration) or water,
the mixture
was incubated
at 37’C for 1 5 mm. The
reaction
was started
by adding
13.125-210
MM G6P and 200 MM
NADP (final concentrations).
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
522
LACHANT.
_
Mg(-)Pb(-)
200
Table
Mg(-)Pb(+)
Iz
-.---,
Mg(+)Pb(+)
-
3.
When
Effect
Added
of 2-Mercaptoethanol
After
Incubating
(2ME)
AND
on G6PD
Red Cell Hemolysate
TANAKA
Activity’
With
lead
Lead Concentration
‘0
,;
TOMODA,
100
2ME (mMI
TTTT
0
0
‘1
v/s
(L0.D.
xld’4MIN1/nMOL
10
G6PD
with
the inhibition
0.4-1.7
mM
activity
reduced
In a similar
incubated
for
tion for I 5
glutathione.
could
not be
or 10 or 100
activity
by lead
was completely
(Fig.
4).
prevented
by
glutathione.
experiment,
I 5 mm
red cell
with
7.48’
5.44
7.56
5.44
7.48
5.57
lead
hemolysates
acetate
prior
gHgb’
.
.
suggested
that the hemolytic
anemia
seen in hereditary
pynimidine
5’-nucleotidase
deficiency
is due, in part, to
an increased
sensitivity
of the erythrocyte
to oxidant
stress,
of the
phate
pression
dine
which
results
from a reversible
inhibitory
effect
pynimidine
5’-nucleotides
on the pentose
phosshunt.
The primary
mechanisms
of shunt
supare: ( 1) competitive
inhibition
of the pynimi5’-nucleotides
with
glucose-6-phosphate
for
and (2) noncompetitive
inhibition
of the pynimidine
5’-nucleotides
with
NADP
for G6PD.
The
increased
concentrations
of the nucleotide
phosphates
decrease
the intraerythrocytic
pH, which
further
suppresses
G6PD
activity.’5”6
Although
the various
components
of the pentose
were
to incuba-
mm with
2-mercaptoethanol
or reduced
The lead-induced
loss of G6PD
activity
reversed
by 0.4 mM reduced
glutathione
mM 2-mercaptoethanol
(Table
3).
phosphate
shunt
and glycolytic
examined
in looking
for evidence
in the erythrocytes
of individuals
pathway
have
been
of oxidant
sensitivity
with
clinical
lead
exposure
or toxicity,
ings have
not been
confirmatory
findobserved
pattern
of
glycolytic
DISCUSSION
that
Paglia
the
et al.’3”4
activity
and others30’3’
of pynimidine
decreased
in erythrocytes
vitro.
Recent
investigations
have demonstrated
5’-nucleotidase
exposed
from
to lead in vivo
this laboratory
is
or in
have
C-
0
a
-I
living
in other
content
able,
suggests
that
60
2
40
20
reported
tase532’33
not been
d......o....................:1:
GSH
0
-
industrial
areas.34
2-MEOo
.2
GSH
.4
.
AND
.
2-ME
1
2
4
CONCENTRATION
10
(mM)
Fig. 4.
The effect
of reduced
glutathione
(GSH) and 2-mercaptoethanol
(2ME)
on G6PD activity
when added prior to lead. The
reaction
mixtures.
containing
20 isl of normal
red cell hemolysate.
0.1 M Tris-HCI
buffer,
pH 7.1 . and 200 MM NADP (final concentration). were incubated
for 1 5 mm at 37’C with GSH (-)
or 2ME
(0-O)
before
being incubated
for 1 5 mm with 2 pM lead acetate
(final concentration).
The reaction
was started
with 200 .tM G6P
(final
concentration).
The enzyme
activity
is expressed
as the
percent
of the original
G6PD activity
prior to lead exposure.
The
shaded area is the normal
intraerythrocytic
GSH concentration.
to be increased,38
Abnormalities
and glutathione
reported.
In the
Heinz
-.
present
body
study,
formation
the
activity
of
pathway
is normal.32’33
In the
Heinz
body formation,
Ghelberg
living near a lead smelter
had
formation
compared
to children
of reduced
glutathione
being
increased,32
332334,42
80
<
0
intermediates
the Embden-Meyerhof
only study evaluating
observed
that children
increased
Heinz
body
been
<100
z
reproducible
found.
The
The
of
highly
or
vanun-
examined
withcount
and has
decreased,39’4#{176} or norglutathione
reduc-
peroxidase32’33
we observed
was
intraerythrocytic
has been
decreased,5’35’36
changed.’4’33’37
G6PD
activity
has been
out respect
to cell age or reticulocyte
C>
0.
min’.
G6PD,
of red cell hemolysates
2-mercaptoethanol
pre-
ofG6PD
The loss ofG6PD
0
10
100
‘IU
at 37#{176}C.They were then incubated
15 mm with lead prior to assay
for
activity.
Preincubation
reduced
glutathione
and
vented
2 MM
NADPMC1)
Fig. 3.
The inhibitory
effect
of lead on the activity
of G6PD
expressed
as an Eadie-Hofstee
plot for substrate
NADP.
The
reaction
mixture
contained
20 p1 of normal red cell hemolysate
and
0.1 MTris-HCI
buffer at pH 7.1, with or without
10 mM MgCI3 (final
concentration).
After the addition
of 2.0 MM lead (final concentration) or water,
the mixture
was incubated
at 37’C for 1 5 mm. The
reaction
was started
by adding
1 2.5-200
MM NADP
and 200 MM
G6P (final concentrations).
thione
for I 5 mm
for an additional
0 M
increased,
activities
that
reduced
have
incubated
gluta-
thione
was decreased,
and the ascorbate
cyanide
test
was positive
in erythrocytes
that
had been
preincubated with lead acetate.
As the normal
isopropanol
test
and
hemoglobin
formation
of an
did not occur,
we
phosphate
shunt
electrophoresis
suggested
that
the
unstable
lead hemoglobin
complex28
evaluated
the activity
of the pentose
by the release
of ‘4CO2 in intact
red
cells before
and after
new methylene
blue stimulation
and in red cell hemolysates
in order to further
explain
our findings.
Pentose
phosphate
shunt
activity
was
significantly
decreased
(p < 0.005)
both before
and
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
INHIBITION
after
OF THE
new
PENTOSE
methylene
SHUNT
blue
523
BY LEAD
stimulation
in erythrocytes
the
inhibition
of
the
pentose
shunt
is the
most
likely
lead
for the inability
of erythrocytes
exposed
in vitro to handle
oxidant
stress.
the release
of ‘4C02
from
‘4C-l-glucose
was
in lead-exposed
red cells, the release
of
‘4C-2-glucose
was normal.
Although
the
controling
pentose
phosphate
recycling
are
poorly
understood,
phosphate
reflection
the
apparently
normal
to
pentose
recycling
that we observed
is most likely a
of the effects
of other
metabolic
defects
on
the flux of glycolytic
intermediates
and
phate
sugars.
The pattern
of decreased
pentose
‘4CO2
effect
in magnesium,
of lead
on G6PD.
with
intraerythrocytic
concen-
3 mM.45
Although
been evaluated
red cell
in clinical
an
tration
of approximately
magnesium
levels have
explanation
lead acetate
Whereas
decreased
‘4C02
from
mechanisms
inhibitory
rich
that
were
previously
incubated
with
lead
acetate.
Because
the pentose
shunt
activity
of the hemolysates
from
these
same
red cells
was normal,
a reversible
phosrelease
poisoning,
not
changes
in the
The
red
cell
concentration
of
cation
could
have significant
effects
on enzyme
tion and red cell survival
in the clinical
setting.
As the most likely mechanism
between
lead and G6PD is through
is
this
func-
for the interaction
the formation
of a
lead-sulfhydryl
complex,46’47
we evaluated
the ability
of thiol reagents
to protect
G6PD
from the effects
of
lead. Thiol
reagents
were able to prevent
the loss of
activity
if added
to red cell hemolysates
prior to
the addition
of lead. Having
a readily
available
source
of reducing
equivalents
may be an important
in vivo
protective
mechanism
for
the
intact
erythrocyte
G6PD
from
‘4C-l-glucose
and normal
‘4C02
release
from
‘4C-2-glucose
is rather
unusual,
as a relative
increase
in pentose
phosphate
recycling
is usually
seen in conjunction
with increased
pentose
phosphate
shunt activity.43 Ribose-5-phosphate
generated
by the pentose
against
trations
lead,
of
against
the
Although
activity
thiol reagents
can prevent
the loss of G6PD
due to lead,
they are unable
to reverse
the
phosphate
shunt
may be used as a substrate
for the
generation
of fructose-6-phosphate
a nd glyceraldehyde-3-phosphate
or it may be converted
to phosphoni-
G6PD
bosyl
Paglia
decreased
should
pyrophosphate
has
shown
(PRPP)
by PRPP
that
PRPP
kinase
kinase.
activity
As
is
in red cells exposed
to lead in vitro,’3
there
be an increased
conversion
of ‘4C-nibulose-
5-phosphate
and ‘4C-nibose-5-phosphate
to ‘4C-fructose-6-phosphate
and ‘4C-glucose-6-phosphate
due to
the metabolic
block
of the salvage
pathway.
The
i ncreased
‘ 4C-glucose-6-phosphate/glucose-6-phosphate
ratio
molecules
‘4CO.,,
will lead to an increase
of ‘4C-glucose-6-phosphate
albeit
at a decreased
activity
of the
instantaneously
pentose
phosphate
rate.
in the
Because
‘4C-glucose-6-phosphate
determined,
a spuriously
recycling
will
number
oxidized
the
of
to
specific
pool cannot
high rate
be
of
be calculated.
Lead
appears
to decrease
pentose
phosphate
shunt
activity
by inhibiting
the activity
of G6PD.
When
lead
was added
to normal
red cell hemolysates,
it acted as a
noncompetitive
G6PD.
The
inhibitor
calculated
of both
K1 of lead
G6P and NADP
for
for G6P was I .5 1uM
and that for NADP
was 2. 1 M.
The
concentration
of G6P is about
27 tM.
intraerythrocytic
Its Km for G6PD
is 41 .3 j.tM in the current
study,
and 44 ± 4 j.M at pH
7.3 in the literature.21’44
Similarly,
the estimated
red
cell NADP
concentration
is about
I .zM with Km5 of
34.5 jM
in the current
study and I 2.5 ± 2. 1 .tM at pH
7.3 in the literature.44
Because
the intraerythrocytic
lead concentration
is approximately
3-6 j.M in clinical
lead poisoning,4#{176} these data strongly
suggest
bition of G6PD
by lead should
occur
in the
vivo.
It is of interest
that
magnesium
is able
that inhired cell in
to potentiate
as subphysiologic
(0.4-2.0
mM)
reduced
glutathione
protected
effects
inhibition
It is quite
lead.
inhibitor
would,
of
2 j.tM
if added
possible
of G6PD
in addition,
lead
concenG6PD
in vitro
(Fig.
4).
to red cell hemolysates
after
that lead acts as a reversible
activity
inactivate
in the red cell cytosol,
(denature)
G6PD
but
in an
assay
system
where
enzyme
and reduced
glutathione
concentrations
had been diluted
250-fold.
In summary,
a reversible
suppression
of pentose
phosphate
shunt
function
due to a decrease
in the
activity
of G6PD
with lead.
Kinetic
competitive
occurs
in erythrocytes
studies
suggest
that
inhibitor
manner
G6PD.
The
activity
is similar
of
in
to that
both
which
preincubated
lead is a non-
G6P
and
NADP
for
lead
inhibits
G6PD
of the
pynimidine
5’-nucleo-
tides for NADP,
but is different
for G6P. Thus,
both
lead
itself
and the increased
concentrations
of the
pynimidine
5’-nucleotides
inhibit
pentose
phosphate
shunt
function,
which
should,
in turn,
render
the
lead-intoxicated
erythrocyte
more
susceptible
to oxidant damage
and shortened
survival
in vivo.
ACKNOWLEDGMENT
The
authors
ionization
wish
to thank
chamber
Dr.
Kawaguchi
Purcell
and
and
Larson
for secretarial
Evelyn
Warren
and vibrating
Eichin
Davidson
for the use of his
reed electrometer
Chang
Lim
for
apparati:
technical
Eileen
assistance;
assistance.
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From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
1984 63: 518-524
Inhibition of the pentose phosphate shunt by lead: a potential mechanism
for hemolysis in lead poisoning
NA Lachant, A Tomoda and KR Tanaka
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