From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
Catalase
and
Glutathione
Peroxidase
Hydrogen
Gian Franco
By
Genetic
deficiencies
nase
to
destruction
ciencies
predispose
do
served
to
principal
destruction.
These
the
assumption
cytes.
Recently,
to have
for
disposal
however,
tightly
erythro-
observations
that
peroxidase
means
bound
of
H202
and
require
A
LTHOUGH
the
of catalase
(H2O2: H2O2 oxidoreductase,
principal
means for disposal
of hydrogen
human
erythrocytes
is
erythro-
catalase
to
the
pathway
in human
mammalian
NADPH
defi-
affected
was
for
large
amounts
contain
stress
of cells
has
with
been
the
impaired
susceptibility
to
production
however,
revealed
that each tetrameric
and bovine
catalase
has four molecules
studies,
human
bound
NADPH.’
This
feature,
together
with
and
the
is that
product
One
by intracellular
proteins
other
explanation
for these observations
itself.23
malian
NADP,
catalase
releasing
peroxidative
cell
to
could
have
evolved
NADP
when
the
NADPH-dependent
tem.’ This suggestion
of
glutathione
is based
H2O2
occurs
reductase/peroxidase
mainly
Cohen
technique,
important
against
endogenous
glutathione
peroxidase
through
of the
the
and
have
Hochstein,
they
route
been
systhat
glutathione
offered
evidence
role in protecting
H2O2.4
Rather,
is the major
conclusions
the
using
that catalase
erythrocytes
concluded
for disposing
that
of
accepted
and
widely
A comparison
in
catalase
accounts
HO2 when
H202
and
Division
Department
Chapel
of
of
Pediatrics,
April
Supported
by Consiglio
85.0141
1.51
delle
and
29.
Malattie
reprint
ISMI,
16/32
Italy.
The
publication
payment.
indicate
© 1 989
requests
University
accepted
of
of
Genoa,
North
‘ingegneria
and
to
G.F.
of
ofthis
article
article
must
by
Grune
& Stratton,
Inc.
at
purified
of the
destruction
a rate
in G6PD-
& Stratton,
deficient
(Fig
and
finding
1). In this
has
report,
are actively
involved
a failure
in the
generation
binding
both
sites
bovine
catalase,
a unity
have
exposed
to
Italy;
Carolina,
12, 1988.
evidence
for
MATERIALS
AND
both
detoxification
a strict
order
of
of affinity
NADH
>
that
NADH
NADP,
thus
>
from
H202.
Chromatoto catalase
from erythroexcludes
however,
that
of H2O2 and that
as with
G6PD
this
possibility.
METHODS
Samples
of blood were obtained
from five healthy
men, one
acatalasemic
man, and four G6PD-deficient
men carrying
the
Mediterranean
variant.9
Collection
of CO2.
Leukocytes
and platelets
were removed
by
filtration
of the hepaninized
blood through
cellulose.’0
Plasma
was
removed, and the erythrocytes
were washed by dilution in 5 vol 0.15
mol/L
NaC1 and centnifugation
and then suspended
in KrebsRinger solution/N.Tris[hydroxymethyl]
methyl-2-aminoethanesulfonic
acid
(Tes)
mixed
buffer,
pH
7.4,
containing
glucose
at
a final
of 5 mmol/L.
For studies of hexose monophosphate
and glutathione,
suspensions of packed erythrocytes
with
an
equal
volume
of
Krebs-Ringer
solution/Tes
buffer
(pH 7.4)/S
mmol/L
glucose
(KRTG).
The packed
cell
volume ranged between 45% and 50% of total volume.
Incubation
mixtures
consisted of 1.0 mL erythrocyte
suspension,
1.0 mL KRTG
solution (with or without glucose oxidase),
and 0.3 mL (0.5 zCi) of
[I-’4Cj
glucose or [2-’4C] glucose
(specific
activity
3 MCi/smol;
Grants
No.
Moleco-
Amersham
10.
performed
in duplicate.
For each incubation,
the HMS activity
is
represented by the sum of the mean of the radioactivity
evolved from
the separate
incubations
with
[1-’4CJ
and [2-’4C]
glucoses.
For
assays of reduced glutathione
(GSH), labeled glucoses were omitted.
For studies of HMS, suspensions
of erythrocytes
were incubated
in
25-mL vials to which were attached rubber caps containing
disposable center wells (Kontes,
Vineland,
NJ). Throughout
the one-hour
incubation,
the center wells contained 0.2 mL I N NaOH. At the
end ofthe incubation,
0.7 mL 3.7 mol/L penchionic acid was injected
by needle
through
the rubber
cap into the incubation
mixture
Grant
HD-031
Gaetani,
MD,
Viale
defrayed
therefore
Division
Benedetto
be
section
in part
hereby
PF
H2O2,
to the
for disposing
of H2O2
are dependent
on
mechanisms
of catalase
how-
prevents
and
an inactive
brought
we offer
preventing
inactivation
of catalase
graphic
analysis
of NADH
bound
cytes
that
erythrocytes.
in the disposal
of NADPH,
systems
impairs
of
to
Inc.
human
This
that
comparable
that catalase-bound
NADPH
accumulation
of compound
of catalase.
acatala-
indicated
e Basi
with 18 U.S.C.
this fact.
half
and
study
Genetica
Genoa,
were
is generated
with
of normal
Ricerche
by NIH
costs
0006-4971/89/7301-0033$3.00/0
September
delle
This
in accordance
“advertisement”
334
University
Nazionale
Ereditarie”
Address
Genoa.
1988;
86.00083.51
Hematology,
charge
University
Hill.
Submitted
Ian
Hematology,
Neil Kirkman
present
than
to hemolysis
concentration
shunt (HMS)
the
the
for more
by Grune
ever, indicate
reverses
the
were
From
leads
The
reservoir
of
is under
ability
through
of
for the nicotinamide
dinucleotides
(NADPH
NADP
>
NAD).
Therefore,
it is possible
within
human
erythrocytes
might
displace
G6PD
mam-
reductase/peroxidase
largely
on the assumption
pathway.
the H2O2 diffusion
does not play an
H202.
Their
reported.5’6
into
a
erythrocyte
and thereby
enhancing
the
hydrogen
peroxide
(H202)
stress
destroy
disposal
than
erythrocytes.
erythrocytes
deficiency,
H2O2.
NADP-binding
the intracel(G6PD)
of substrate
and Henry
semic
NADPH
Recent
proteins,
represents
a system
by which
lular activity
of glucose-6-phosphate
dehydrogenase
is determined
largely
by the binding
and release
Ferraris,
human
form
molecule
of
of tightly
by other
Maria
Anna
concept
of two different
mechanisms
by revealing
that
both
mechanisms
peroxidative
of NADPH.
Canepa,
Studies
EC 1 .1 1.1.6),
peroxide
in
cells has been considered
to be the NADPH,
glutareductase/peroxidase
pathway.
Supporting
this tradi-
in Detoxification
Erythrocytes
. 1989
the
these
thione
tional
viewpoint
Active
in Human
which
found
NADPH
Equally
prevention
and reversal
of inactivation
by its toxic
substrate
(H202).
Since
both
catalase
and
the
glutathione
pathway
are dependent
on NADPH for function,
this finding
raises
the possibility
that both mechanisms
destroy
H202 in
erythrocytes
genetic
predispose
strengthen
Letizia
dehydroge-
affected
NADPH/glutathione/glutathione
the
Galiano,
Conversely,
not
to peroxide-induced
have
Silvana
peroxides.
catalase
Peroxide
glucose-6-phosphate
NADPH
from
of
cytes
of
and
(G6PD)
Gaetani,
Are
of
XV,
6.
by page
marked
1 734 solely
to
Int,
Buckinghamshire,
B/cod,
England).
Vol 73, No 1
All
(January),
incubations
1989:
were
pp 334-339
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
H2O2 DETOXIFICATION
IN HUMAN
ERVTHROCYTES
ii1-::.
H
OSH
.::III
335
‘ 1::
NAOP\
2
GLUTATHIONE
GLUTATHIONE
REDUCTASE
Mechanisms
Fig 1 .
containing
‘4C-glucose.
shaker
metabolic
vials
30 minutes.
and dropped
into
(Packard
Instrument)
removed
Instagel
normal
The
for
with
erythrocytes
Determination
described
of
by Beutler
an enzymatic
acid
and
using
GSH
glucose.
et al.’2 Glucose
method
(obtained
by exposure
and then ultrafiltration)
was determined
concentration
NADP,
ATP,
of
resulted
as
was measured
and
the enzymes
by
G6PD
and hexokinase.’3
The glucose concentration,
together with measurement of ‘4C radioactivity,
allowed calculation
of the specific activity
of labeled glucose
at zero time and the total amount
of glucose
consumed
over
60 minutes.
Measurement
ofrate
oxidase
to the incubation
to steady-state
ofgeneration
ofHlO2.
Addition of glucose
mixtures served to expose the erythrocytes
concentrations
of H202,
as described
by Chance’4
and
Aebi and Suter.”
Preliminary
assays of glucose
oxidase
permitted
a
choice
to be made of the concentration
of glucose oxidase that would
allow continuous
generation
of H202 at the desired rate. Assays for
glucose oxidase were carried out under conditions identical
to those
for incubation
of erythrocytes:
Variable
amounts
of glucose
oxidase
were added to 2.3 mL Krebs-Ringer/Tes
buffer, pH 7.4, to a final
enzyme concentration
of 2.2, 4.5, and 9.0 nmol/L.
The reaction was
followed at 37#{176}C
in a 3.0-mL
cuvette
with a light path of 1 cm. In
addition,
the assay mixture
contained
horseradish
penoxidase,
phenol, and 4-aminoantipynine
as specified
for the spectrophotometnic
determination
of H2O2 by the method of Green and Hill.’6 Under
these conditions,
the rate of generation
of H202 was constant for one
hour at concentrations
of glucose oxidase up to 9 nmol/L.
Catalase
and NADH.
Sephacryl
5-200 was a product of Pharmacia Fine Chemicals.
Ultrafiltration
was performed
with CF-25
ultrafiltration
Protein
cones
concentrations
lase activity
Amicon
were
with
a Cary
were
measured
recording
by
as described
as the first-order
a method
of
kinetic
and
The
Cata-
constant
of the
at 240 nm
and
extraction
alkaline
earlier.’
method.’7
NADP
alkaline
previously.’9
determined
described
by absorbance
spectrophotometer.’8
concentrations
were
manner
by the Folin
of H2O2 as measured
of dinucleotide
that represented
unbound
dinucleotide.
nation between bound
release
value
in the
measured
was expressed
rate of disappearance
cycling’3
from
and
extraction
NADPH
enzymic
led
to
from proteins and allowed determination
of a
the concentration
of both protein-bound
and
Preliminary
ultrafiltration
allowed discnimiunbound
dinucleotide.
by
use
of
NAD
the
same
and NADH
extraction
but with glutamate
dehydrogenase
and lactate dehydrogenase as cycling enzymes. The destruction ofNAD
in strong alkali
gave the final fluorescent
product for NAD determination,’3
A lO-mL
sample
of hepaninized
blood from a man with the
Mediterranean
variant
of glucose-6-phosphate
dehydrogenase
(n’
procedure,
I
,/
j
c
H,O
CN,CN
OH)
, NAOPN4
NADP’
GPO
SRP
,-
QOPO
were
incubated
again
in the
The disposable
center
wells were
‘4C-gluconic
GSH
GOP
scintillation
vials containing
10 mL
and I .0 ml of water.
Incubation
of
[1-’4C] glucose to glucose-oxidase
in no evolution of ‘4CO2.
-_
:
NAH
COMPOUND
HO
(.cH,cNO)
‘Y’
II
!
_7,//4.
GSSG
of H202
Ferricatalase
erythrocyte.
COMPOUND
PEROXIDASE
removal
and
compound
I represent
active forms
of
catalase.
Compound
II is an inactive
form
of the enzyme.
AH denotes
oneelectron
donors
(reducing
substances)
within
the catalase
molecule.
in the
L;
AH.--1
‘co,
SPOD
6-phosphate:
NADP
1-oxidoreductase,
E.C. 1.1.1 .49) was
filtered through
cellulose
for removal of leukocytes
and platelets.’0
One volume of washed, packed erythrocytes
was mixed with three
volumes of Krebs-Ringer/Tes
buffer that contained
glucose
at a
final concentration
of 5 mmol/L.
An identical
preparation
contained
glucose
oxidase at a final concentration
of 9 nmol/L.
At the end of
the incubation,
measurements
were made of the proportion
of the
volume
occupied
by packed
cells (hematocnit),
the activity
of
catalase,
and the concentrations
of nicotinamide
dinucleotides
(NAD and NADP).
The enythrocytes
that had been incubated
with glucose oxidase
were promptly
centrifuged
at 3,000 g for five minutes; the packed
cells were mixed with 6 vol distilled water at 0#{176}C.
After ten minutes,
the preparation
was centrifuged
for I S minutes
at 16,000
g. Four
volumes
of the supernatant,
stroma-free
hemolysate
were mixed
with one vol 0.75 mol/L
NaCI; and 7 mL solution,
containing
240
mg hemoglobin,
was applied to a Sephacryl 5-200 column that had
been equilibrated
with Krebs-Ringer/Tes
buffer. Fractions
of 7 mL
were collected at a flow rate of 7 mL/h.
glucose
RESULTS
Relationship
of
between
evolution
different
normal
of
amounts
of
and acatalasemic
chiometric
relationship
H2O2
the
and
rate
‘4C02.
ofgeneration
The
use
between
degree
of
the
stimulation
of glucose-6-phosphate
results
in two molecules
cient
to generate
glutathione.
Since
of H202,
of H202.
observed
and expected
normal
erythrocytes
of
of
the
of generation
GSH
flows
HMS.
of
Each
the
suffi-
from
oxidized
are necessary
in acatalasemic
and
of H2O2. In acatala-
semic
erythrocytes
-90%
of the
removed
by glutathione
peroxidase.
generated
However,
erythrocytes,
rates
of the
and
one molecule
of glucose
will
Table
I shows
the rates of
at different
at each
rate
presence
of
led to a stoi-
the
of GSH
CO2 evolution
‘
rate
and
glucose
(G6P)
passing
through
of NADPH,
which
are
four
molecules
only two molecules
to remove
one molecule
remove
two molecules
ofH2O2
labeled
glucose
oxidase
in
human
erythrocytes
molecule
shunt
of
different
H2O2
was
for normal
of generation
H2O2, the observed
of
‘4C02 evolution
corresponded
to -50%
the expected
values (Table
1); these
values
differed
by
SD from the rate observed
in acatalasemic
erythroyctes.
Relationship
ofdecrease
between
ofGSH
no stimulation
erythrocytes
are a model
of the
rate
HMS
the presence
for evaluation
in
ofgeneration
in G6PD-deficient
occurs
ofH2O2
and
erythrocytes.
in G6PD
of
>2
rate
Since
Mediterranean
of oxidizing
agents,
these
of the relationship
between
cells
the
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
336
GAETANI
Table
of Various
1 . Effect
Rates
of HO
of G eneration
Eryth
rocytes
From
H202 Rate
Monophosphate
Subject
61
465
266
882
454
of
‘4C02 Expected
(nmol/h)
(B)
A/B
-
121
0.89
205
233
0.88
393
441
0.89
erythrocytes
34 ± 8
93±11
-
-
59±9
-
124±6
0.47±0.06
0.47±0.06
478±31
145±23
112±20
239±16
891±76
237±37
205±35
445±38
the mean
H2O2 rate) both for
absolute
amounts
acatalasemic
of reduced
gluthathione
and normal erythrocytes,
decreased
(from 896
only slightly
0.46±0.08
nmol of baseline
values
to 870
nmol at the maximum
these values are not shown.
SD.
.±
of
decrease
regenerated
H202.
The
of
erythrocytic
after its oxidation,
results
in Table
GSH,
and the
2 indicate
which
cannot
cells of a
erythrocytes
an oxidizing
be
rate of generation
of
that
the observed
The
concentration
of human
catalase
in each
obtained
by dividing
the activity
of catalase
by the specific
activity
of highly
purified
(3.5 x i0
mol/L/s).
These
calculations
fraction
in each
human
indicate
shown
erythrocytes
or 5.3
in
zmol/L.
closely
to the concentration
erythrocytes
(5.4
zmol/L)
been incubated
Table
after
than
This
was
is available
incubation.
half
the
The
value
Catalase
obtained
Table 2. Content
the
decreased
of GS H Within
H202 Rate
(nmol/h)
Mediterranean
656
239±12
464±28
±
21
353
951±37
rates
of ‘4C02
H202 flow.
Similar
performed
3).
that
±
±
from
[1-’4CJ
Erythrocyte
steady-state
on
37
s During
are
in which
for H2O2
that GSH
At higher
becomes
derived
suspensions
concentration
the distance
Hochstein,
of intact
of H202
can
Exposure
to Differen
t
Rates
of HO3
-
49
-987
not shown
-
0.40±0.03
±
42
0.31
-1,901±74
since
the
differences
from
Production
GSH Found/
GSH Expected
-479±25
±
be expected
below
the meniscus.
As
-40%
to 60% of the H2O2
GSH Expected
(nmol/h)
-384±45
and [2-’4Cjglucose
to catacontribuconcluded
to the main compartment
is localized
in the vapor
the side walls.2’
Therefore,
for stoichiometric
-
-192±5
-303
ascribed
were exposed
to gaseous
H202. Although
the
is intrinsically
valid, the rate of entry of H202 into
depends
on many
variables,
such as tempera-
GSH Found
(nmol/h)
272±45
evolution
the
transferred
droplets
on the
24
earlier
the action
of catalase
These
conclusions
were
studies
to vary greatly
with
shown
by Cohen and
results
G 6PD-Deflcient
of the
ture, rate of shaking,
and the geometry
of the incubation
flask. Even with shallow
suspensions,
the rate of destruction
of H202 is so rapid, relative
to the rate of diffusion
of H2O2,
dinucleotides
GSH Found
(nmol)
0 (Baseline)
493
maximum
-30%.
was
experiment
diffusion
from
activity
is the major
route
conditions,
provided
operation
of the HMS.
the
mainly
a suspension
concentration
of NADPH
fell to less
for cells
incubated
without
glucose
activity
when
the protection
peroxidase
physiologic
of H2O2,
important.
corresponds
(Table
for the
through
erythrocytes
technique
oxidase
of the four
toward
Hochstein4
determined
the relative
and glutathione
peroxidase
and
concentrations
increasingly
nmol/mL
observed
in the
after
the cells had
of glucose
the concentrations
responsible
that
glutathione
breakdown
under
fraction
catalase
that
the
5.3
concentration
of NADPH
immediately
in the presence
3 shows
oxidase.
2 represented
Fig
of catalase
lase. Cohen and
tions of catalase
catalase
fractions
of Fig 2 contained
a total
of 1.7 nmol
catalase
or 6.8 nmol NADP-binding
sites. A total of 6.6 nmol
dinucleotide
was present
in the peak.
The NADPH
in the
peak
contribution
enzyme
was the same
in each of the four fractions
reprethe catalase
peak shown
in Fig 2. An estimate
of
catalase
agent,
with
G6PD
deficiency
whose
in the presence
of a-naphthol,
concentration
of 200 smol/L.
erythrocytes
has been a subject
of controversy
for many
years. Soon after the identification
of glutathione
peroxidase
by Mills,#{176}some investigators
suggested
that this was the
shown
in Fig 2, 76% was represented
by NADPH,
16% was
represented
by NADP
and only 8% was represented
by
NADH.
The ratio of the dinucleotide
content
to catalase
molar
subject
incubated
at a final
were
DISCUSSION
the results obtained
with normal
cells.
Evaluation
of the role of NADH
in intracellular
protection of catalase.
Of the dinucleotides
in the catalase
peak
activity
senting
second
was less than
that expected
at different
of H202. This finding
is in agreement
with
decrease
of GSH
rates of generation
The
Glucose)
Subjects
-
108
Because
Gluco se + [2-’4CJ
(I1-”Cj
Activity
Normal
-
169
0(Baseline)
248±12
were
Five
erythrocytes
242
Normal
Shunt
and
‘4C02 Found
Baseline
(nmol/h)
(A)
(nmol/h)
0(Baseline)
rate
Hexose
Acatalasemic
‘4C02 Found
(nmol/h)
Acatalasemic
on
One
ET AL
±
0.06
0.21±0.03
the
baseline
value were
neglibIe
even
at the
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
H2O2 DETOXIFICATION
IN HUMAN
337
ERVTHROCVTES
clearly
demonstrated
breakdown.
20
The
16
‘,
(3
a
119
133
volume
Elution
measurements
glucose,
this
147
(elution
80 to 120
volume
in
incubated
with
containing
activity
of
were concentrated
to a
of 0.4 mL with
concentrations
had been
ml)
(s).
cones.
Curves
of solid lines
of the dinucleotides
determined
after
ultrafiltration:
NADPH
NADP
(0). and NADH
(U). Curves
of
broken
lines represent
concentration
of protein
(0) and relative
activity
of catalase
(activity
at peak, 4.1 s’)
(A) before
ultrafiftration.
represent
the
involvement
between
ultrafiltration
constant
tion
H2O2
of diluted
addition
flow
of
the
catalase
and
H202.4
GSH,
This
of H2O2 and
relative
roles
in disposing
they
approach
is inadequate
of glutathione
of H2O2.
In support
used
the
dropwise
does
not
provide
the incubation
of the
in removing
H2O2.
strated
the
by
of the
preeminence
of the NADPH/glutathione
peroxidase
mechanism was the observation
that G6PD-deficient
subjects
are
prone
to develop
to oxidizing
agents.
particularly
exposure
acute
hemolytic
anemia
The
of
the
through
common
oxidase
expected
peroxide-generating
gible
effect
rate;
acid
therefore,
tative
study.
ascorbic
Table
3.
agents
on normal
The
Effect
observations
of HO2
H203
Catalase
(k/mg Hb)
0.262
0
400
0.180
mean
for
normal
when
acid)
after
Activity
of G6PD-Deficient
45.2
60.5
erythrocytes
and
undergo
to levels
that
have
agents
of
a neglisuch
as
These
inhibit
catalase
quantitative
Further
observed
16.8
5.4
52.8
54.9
from ten subjects
are
(Table
the
exposed
peroxi-
to the
of the HMS
1).
of Jacob
stimulation
with
agents,
Since
and
of
rates
NADPH
such
however,
either
other
or have
as cyanide
do
metabolic
50%
is the
perof the
with
reported
blocking
or sodium
not
completely
effects
that
prevent
active
role of catalase
is easily
G6PD-deficient
erythrocytes.
cells
to avoid overt chronic
hemolysis,
their
activity
being
almost
nil.a4
As reported
agents.
same
is almost
Jandl,23
who
the HMS
by
inhibitors,
evidence
of the
in Mediterranean
carry
a baseline
HMS
activity
that
no stimulation
of the HMS
of
erythrocytes
occurs
in the presence
Moreover,
reduction
gible.26
An estimate
glutathione
peroxidase
of oxidized
is barely
maximum
in several
such
G6PDof oxidizing
glutathione
is negli-
of the intracellular
involvement
of
is made possible by exposure
of these
at controlled
rates
in Table
2 suggest
of generation
that glutathione
disposes
of 20% to 40% of the generated
of 20%
being
obtained
at the
of H202. The
peroxidase
H2O2, the proportion
higher
rate
of generation
of
2).
present
results
on normal
clearly
indicate
of H2O2. Expected
evolution
NADH
an
and
G6PD-deficient
eryth-
active
role of catalase
in the
values
for glutathione
peroxidase
2.3
is unlikely,
owing
to the results
observed
from
study
semic erythrocytes.
The validity
of conclusions
2.9
2.0
was
is demon-
1 :1 between
involvement
are derived
from the rate of generation
of H202.
The possibility
of incomplete
diffusion
of H2O2 through
the
erythrocyte
membrane
or of an underestimation
of ‘4C02
Erythrocytes
NAD
erythrocytes)
of
sufficient
rocytes
removal
Intracellular
NADPH
(nmol/mI
ratio
These
The
et a122 nevertheless
and
system
studies.
H2O2 (Table
of H2O2 at an unknown
are inadequate
for quanti-
of Jacob
NADP
rate
exposed
However,
on Catalase
Concentration
(nmol/h/mL)
The
(ascorbic
erythrocytes.
lead to the generation
those experiments
Dinucleotide
0.5.
the normal
times
the
peroxidase
of this technique
the activity
values
function
azide.
G6PD
times
many
of establishing
hydrogen
donor for both systems,
glutathione
probably
accounts
for even less than 50%
rate (Table
1 ). These results
are in accordance
cells to H202
results shown
three
rate
is
the NADPH/glutathione
erythrocytes
expected
the HMS
in this
a means
constant
of H2O2,
of generation
articles,”5
deficient
at about
mechanisms
period. When coupled
with
of ‘4CO2 from ‘4C-labeled
reliability
almost
dase pathway.
When normal
Other investigators
reached different
conclusions.
Jacob et
al,22 studying
acatalasemic
subjects,
suggested
the possibility
that catalase
is a first, but not indispensable,
line of defense
against
H2O2 in erythrocytes.
They reported
for the first time
that acatalasemic
erythrocytes
metabolize
glucose
through
an increase
both
NADPH/glutathione
stoichiometrically
catalase
a
for precise
evaluaperoxidase
and
of the claim
of
of the generation
method
provides
earlier
observations
potentiation
of the
studies
of
A
observed
and the expected
HMS
rate of acatalasemic
erythrocytes
(Table
I). The result
indicates
that essentially
all of
the generated
H202
enters
the RBC
and
is inactivated
Fig 2.
Distribution
of dinucleotides
in catelase-containing
fractions
of a hemolysate
after
chromatography
in Sephacryl
5-200.
Results
are from
a lysate
of erythrocytes
deficient
catalase
volume
of catalase-
first time a unification
for disposing
of H202.
H2O2 throughout
, ml
glucose-S-phosphate
dehydrogenase
that
glucose
and glucose
oxidase.
Fractions
function
H2O2
0
of
involvement
105
and
in
.
4
91
catalase
appropriate
at this time. With the technique
of H202 generation used in the present
study,
the appropriate
use of glucose
and glucose
oxidase
gives a constant
rate of generation
of
C
77
of
reevaluation
8
a)
presence
allows
for the
mechanisms
role
E
V
0
of the
NADPH”7
two different
active
c;
12
Cl,
03
discovery
bound
these
the
±
accuracy
activity.
dase
were
of estimates
of rates
Phenol,
aminoantipyrine,
present
at
low
this
of H2O2 production
and horseradish
concentrations
in the
in acatalarests
on
the
and HMS
peroxicontinuous
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
338
assay
of
H2O2
solution,
of these
production
by
glucose
oxidase
but they were omitted
from
three reagents
were inhibitors
rate
of H2O2
We
found,
production
however,
produced
during
a
would
have
that
estimates
period
of
An alternative
method
peroxidase
yielded
of metabolism
study
been
same
which
greatly
stimulates
second
carbon
results
results.27
a
recycling
provided
small
through
(Table
oxidase
contrast
cells
the
to
very
that
little
and
of
in methe-
samples
of normal
of
1 . In
highest
concentration
shown
in Table
acatalasemic
cells,
As
G6PD-deficient
of cellular
that the
was depleted
the rate
or was
of H202
human
lowered
in concentration
production.
erythrocytes
The
rate
buffer,
pH
demonstrated
(through
II) of catalase
during
Therefore
erythrocytic
exposure
NADH
<20%
during
(Table
1)
the
than
of the glucose
that
NADH
formation
com-
and
to
a
functionally
system
erythrocytes).
bound
NADP
chromatography
may
of
of purified
catalase
might
have been
placed
by NADH
when intact
erythrocytes
H202 generation
at a rate of 400 nmol/mL/h.
demonstrated
the stability
of catalase-bound
Figure
2
was dis-
were subjected
to
Earlier
studies
nicotinamide
storage.’
The con-
centration
of NADH
in the erythrocyte
(Table
3) may be too
low to affect the displacement
of NADP
from the catalase
molecule,
and inactivation
of catalase
occurs
(Table
3).
of
produces
decrease
days.#{176}
primaquine
of -35%
to G6PD-deficient
in catalase
activity
In conclusion,
the present
investigation
two routes
for H2O2 breakdown,
namely
thione
peroxidase,
are equally
involved
subjects
within
several
indicates
that the
catalase
and glutain the removal
of
H2O2 in human
erythrocytes.
Failure
of only one of the two
mechanisms
for disposing
of H202 may not be deleteriousY’
Since both mechanisms
are dependent
on the generation
of
the NADPH
(Fig
1), however,
failure
of the NADPHsystem
erythrocytes
is
oxidase
keeping
catalase
NADPH-generating
(as in the case of G6PD-deficient
indicates,
however,
that very little
(as
with
particularly
G6PD
susceptible
deficiency)
makes
to oxidative
hemolysis.
the
ACKNOWLEDGMENT
of
7.4,
that
we
dinucleotides
shown
determinations
of glucose
under the conditions
of
inactivation
donor
for
an adequate
rates
by the 0.5 mL
can be calcuaddition
of the
and would
have caused
to be reduced
by less
assumption
study,
the
to slow
enough
Embden-Meyerhof
in Krebs-Ringer/Tes
the
a previous
prevent
generating
of G6PD-deficient
cells shown in Table
2 are less than
the ratios shown for normal
cells in Table
1 . Consideration
was also given to whether
the glucose
in the cell suspensions
ratios
HMS
smol/h.
This would
have
5% to 20% of the glucose
to 1 .85
of only
Actual
postincubation
of normal
erythrocytes
Administration
generated
no increase
when
at the
conditions
the
be expected
to undergo
oxidation
It is noteworthy
in this connection
would
was
in those
cells.
against
peroxidative
exhibited
incubated
under
the
normal
components.
of
H202
generated
in this
manner
excellent
defenses
erythrocytes
were
glucose
in
measurement
the one-hour
incubation
rate of H2O2 production
suitable
hydrogen
active even without
of the rates of H2O2 prohave also been in error
if
or in malonyldialdehyde29
erythrocytes
‘4C] glucose
-
accurate
of
1) indicates
normal
moglobin
Brin
the
by glucose
during
average
pound
H202.7
of the
with
consumption
the total
to 0.5
in consumption
In
CO2 arose from other
blue was present.28
[2
of glucose
brings
resulted
also
as well
blue,
the HMS.
rate
Table
1 confirmed
was consumed.
cellular
components,
such as hemoratio was close to I in the acatalasemic
H2O2
was consumed
expected
from their
injury,
Yone-
Coupled
in Table
1, the rate of glucose
consumption
erythrocytes
in the 2.3-mL
incubation
mixture
lated to have been 0.5 to 0.95 zmol/h.”
The
2% to 10%.
in suspensions
classic
and
Oxidation
and
an
fraction
consumed
in oxidizing
globin. That the A/B
subject
their
Brin
activity.
HMS
from comparisons
HMS
activity
would
than
In
erythrocytes,
- ‘4C] glucose
present
study
activity.
more
and
acid and horserad-
the second
carbon
of glucose
in the presence
of methylene
from
the use of [I
Conclusions
duction
and
glucose
these three reagents
end of the incubation.
and Yonemoto
showed
that s5% ofthe
carbons
of glucose,
even when methylene
HMS
of
that >90%
of evolved
CO2 comes
from
of glucose
when the cells are incubated
that
stimulate
the HMS.28
They
noted
substances
the
of H2O2
amount
homovanillic
the
that CO2 is evolved
from
when cells are incubated
Thus,
underestimated.
of the
incubation
in human
moto demonstrated
the first carbon
without
using
0.8 to 1 .0 mol/h/mL.
in cell-free
cell suspensions.
If any
of glucose oxidase,
the
glucose
oxidase
were the same whether
were added
at the beginning
or at the
ish
ET AL
GAETANI
We
dedicate
thank
A.B.
this
article
for
the
donation
to the memory
of
acatalasemic
of Professor
Hugo
blood.
We
Aebi.
REFERENCES
I.
with
USA
2.
Kirkman
HN, Gaetani GF: Catalase:
a tetrameric
enzyme
four tightly bound molecules of NADPH.
Proc Natl Acad Sci
81:4343, 1984
Kirkman
HN, Gaetani
GF: Regulation
of glucose-6-phosphate
dehydrogenase
3.
proteins
4.
HN,
causing
in human
erythrocytes.
in human
Kirkman
Gaetani
reduced
availability
erythrocytes.
Cohen
J Biol
G, Hochstein
for the elimination
Biochemistry
2: 1420, 1963
agent
5. Beutler
GF,
Chem
of
EH:
and sigmoid
261:4039,
hydrogen
E, Erslev
New
Chem
P: Glutathione
E: Glucose-6-phosphate
Williams
Wi, Beutler
(ed 2), McGraw-Hill,
J Biol
Clemons
York,
261:4033,
release
in
1977, p 466
RH
Hematology
in Piomelli
5, Yachnin
S (eds):
Current
Topics
in Hematology,
vol 1. Liss, New York, 1978, p 1
7. Kirkman
HN, Galiano 5, Gaetani GF: The function
lase-bound
NADPH.
J Biol Chem 262:660,
1987
8. Jouve
erythrocytes.
deficiency,
(eds):
jects,
L, Testa U: Human erythrocyte
glucose-6-phosphate
Structure
and function in normal and mutant sub-
HM,
Pelmont
J, Gaillard
J: Interaction
between
of catapyri-
and bovine liver catalase: A chromatoArch Biochem Biophys 248:71, 1986
Group: Standardization
of procedures
for the
dine adenine dinucleotides
graphic and.spectral
study.
The primary
peroxidase:
peroxide
Rundles
of NADP
1986
dehydrogenase
AJ,
1986
NADP-binding
6. Luzzatto
dehydrogenase:
9. WHO
in
Scientific
study of glucose-6-phosphate
dehydrogenase.
WHO
Tech Rep Ser
366:1, 1967
10. Beutler E, West C, Blume KG: The removal ofleukocytes
and
platelets
from whole blood. J Lab Clin Med 88:328, 1976
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
H2O2 DETOXIFICATION
1 1 . Gaetani
restraint:
stress
in
GF,
Parker
A new
basis
human
erythrocytes
glucose-6-phosphate
71:3584,
IN HUMAN
for
ERVTHROCVTES
JC,
the
Kirkman
limitation
HN:
Intracellular
in response
containing
dehydrogenase.
339
to oxidative
low-activity
Proc
NatI
Acad
variants of
Sci USA
1974
12. Beutler
E, Duron
0, Kelly
BM: Improved
method
for the
determination
of blood glutathione.
J Lab Clin Med 61:882,
1963
13. Lowry
OH, Passonneau
JV: A flexible
system of enzymatic
analysis.
San Diego, Academic
Press, 1972
14. Chance
B: The reactions of catalase in the presence of the
notatin system. Biochem J 46:387, 1950
15. Aebi H, Suter H: Protective
function of reduced glutathione
(GSH) against the effect of peroxidative
substances and of irradiation in the erythrocyte,
in Floh#{233}L, Benohr
H Ch, Sies H, Waller
HD, Wendell
A (eds): Glutathione,
Stuttgart,
Thieme,
1974, p 192
16. Green Mi, Hill HAO:
Chemistry
of dioxygen.
Methods
Enzymol 105:3, 1984
17. Lowry
OH, Rosebrough
NJ, Farr AL, Randall
Ri: Protein
measurement
with the Folin phenol
reagent.
J Biol Chem
193:265,
1951
18. Chance
B, Maehly
AC: Assay of catalases and peroxidases.
Methods
Enzymol
2:764,
1955
19. Kirkman
HN, Gaetani GF, Clemons EH, Mareni C: Red cell
NADP
and NADPH
in glucose-6-phosphate
dehydrogenase
deficiency. J Clin Invest 55:875, 1975
20. Mills
GC: The purification
and properties
of glutathione
peroxidase of erythrocytes.
J Biol Chem 234:502,
1959
21.
Cohen
P, Hochstein
and detoxification
Science
134:1756,
P: Glucose-6-phosphate
of hydrogen
1961
peroxide
in human
dehydrogenase
erythrocytes.
22.
Jacobs
erythrocyte
HS
Ingbar
SH,
metabolism
44:1187,
JH:
Jandl
in hereditary
Oxidative
acatalasia.
hemolysis and
J Clin Invest
1965
HS, Jandl JH: Effects of sulfhydryl
inhibition
on red
blood cells. J Biol Chem 241 :4243, 1966
24. Morelli A, Benatti U, Lenzerini
L, Sparatore
B, Salamino
F,
Melloni E, Michetti
M, Pontremoli
5, Dc Flora A: The interference
of leukocytes
and platelets
with
measurement
of glucose-6phosphate
dehydrogenase
activity of erythrocytes
with low activity
variants of the enzyme. Blood 58:642, 1981
25. Szeinberg
A, Marks
PA: Substances
stimulating
glucose
23.
Jacob
catabolism
by
pathway
26.
the
in human
Rieber
EE,
oxidative
reactions
erythrocytes.
J Clin
Jaff#{233}ER:
Reduction
of
Invest
the
pentose
40:914,
of oxidized
normal and glucose-6-phosphate
dehydrogenase
cytes and their hemolysates.
Blood 35:166, 1970
27.
tion
Ruch
by
W, Cooper
macrophages
horseradish
28.
pathway
peroxidase.
J, Dormandy
by hydrogen
30. Tarlov
rocytes.
J Immunol
M: Assay
with
Methods
Stimulation
by
glutathione
deficient
of H2O2
homovanillic
63:347,
producacid
and
1983
of the glucose
methylene
in
erythro-
blue.
oxidative
J Biol
Chem
1958
29. Stocks
quine.
Baggiolini
neutrophils
Brin M, Yonemoto
RH:
in human erythrocytes
230:307,
induced
PH,
and
phosphate
1961
XI.
AR,
Decreased
J Lab Clin
TL:
peroxide.
Kellermeyer
catalase
The autoxidation
Br J Haematol
RW:
activity
Med 58:204,
of red cells lipids
20:95,
The hemolytic
1971
effect
in primaquine-sensitive
of primaeryth-
1961
3 1 . Beutler E, Matsumoto
F: Ethnic
variation
thione peroxidase activity. Blood 46:103, 1975
in red cell gluta-
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
1989 73: 334-339
Catalase and glutathione peroxidase are equally active in detoxification of
hydrogen peroxide in human erythrocytes
GF Gaetani, S Galiano, L Canepa, AM Ferraris and HN Kirkman
Updated information and services can be found at:
http://www.bloodjournal.org/content/73/1/334.full.html
Articles on similar topics can be found in the following Blood collections
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of
Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.