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Active Involvement of Catalase During Hemolytic Crises of Favism
By Gian Franco Gaetani, Michela Rolfo, Sara Arena, Rosa Mangerini, Gian Franco Meloni, and Anna Maria Ferraris
The endemic occurrence of favism in certain Mediterranean
regions provided an investigative opportunity for testing in
vivo the validity of claims as to the role of catalase in protecting human erythrocytes against peroxidative injury. Reduced activity of catalase was found in the erythrocytes of
six boys who were deficient in erythrocytic glucose-6-phosphate dehydrogenase (GGPD) and who were studied while
suffering hemolysis after ingesting fava beans. Activity of
catalase was further reduced when their red blood cells were
incubated with aminotriazole. In contrast, minimal reduction
of catalase activity was found, both with and without incubation with aminotriazole, in erythrocytes of a GGPD-deficient boy who had ingested fava beans 7 days earlier and
in erythrocytes of seven GGPD-deficient men with a past
history of favism. These results confirmed earlier studies in
vitro indicating that catalase is a major disposer of hydrogen
peroxide in human erythrocytes and, like the glutathione
peroxidaselreductase pathway, is dependent on the availability of reduced nicotinamide adenine dinucleotide phosphate (NADPH). The effect of divicine on purified catalase
and on the catalase of intact GGPD-deficient erythrocytes
was similar to the previously demonstrated effect on catalase of a known system for generating hydrogen peroxide.
This effect of divicine strengthens earlier arguments that
divicine is the toxic peroxidative component of fava beans.
0 1996 by The American Society of Hematology.
P
largely, if not entirely, from experiments in vitro. In the
early years of research on G6PD deficiency, much was
learned from studying volunteers who took primaquine.’
With the present knowledge of the predictable and harmful
consequences of primaquine exposure, such studies are no
longer ethically justifiable.
Favism in many ways resembles drug-induced hemolysis,
but differs from it in that only some G6PD-deficient subjects
appear to be sensitive to fava beans. It is confined mainly
to Mediterranean countries with a prevalence of the GdMed
gene,’.*although sporadic cases carrying other G6PD mutations (GdA-; Gd Aures) have been recently reported.’.lo Efforts to educate people about the danger of eating fava beans
have reduced the incidence of favism in these Mediterranean
areas.’’ Nevertheless, sufficient cases occur to test in vivo
the validity of claims as to the role of catalase in protecting
human erythrocytes against peroxidative injury. In the present report, we offer such evidence. Moreover, we have observed an active participation of catalase under conditions
of oxidative stress in vitro caused by the addition of divicine,
a compound previously suspected of being one of the components of fava beans leading to AHA in G6PD-deficient people.
EOPLE WHO HAVE a deficiency of glucose-6-phosphate dehydrogenase (G6PD) are prone to acute hemolytic anemia (AHA) on exposure to certain substances, such
as fava beans, primaquine, and nitrofurantoin.’.’ The traditional explanation for the AHA triggered by these substances
is that G6PD-deficient cells are unable to generate enough
nicotinamide adenine dinucleotide phosphate (NADPH) to
maintain high levels of reduced glutathione (GSH). GSH
is used by the glutathione peroxidase (GSH-Px)/reductase
system to detoxify H202and organic peroxides within the
cell provided that sufficient GSH is made available by the
hexose monophosphate shunt (HMS), of which G6PD is the
first and rate-limiting
In recent years, however,
catalase has gained new consideration in cellular defense,’
based on the discovery that each tetrameric molecule of
mammalian catalase has four molecules of tightly bound
NADPH that prevents and reverses the accumulation of
Compound 11, the inactive form of ~ a t a l a s e . These
~ , ~ and
other finding^^.^ showed that both mechanisms are dependent
on NADPH generation and that a failure in the generation
of NADPH, as in G6PD deficiency, impairs both systems
for H202detoxification. These findings brought a unity to
the concept of two different mechanisms for disposing of
H202.Evidence for the role of catalase in protecting human
erythrocytes from peroxidative injury, however, has come
From the Division of Hematological Oncology, lstituto Nazionale
per la Ricerca sul Cancro and lstituto di Oncologia Clinica e Sperimentale, University of Genoa, Genoa; and the Department of Pediatrics, University of Sassari, Sassari, Italy.
Submitted January 11, 1996; accepted March 26, 1996.
Supported by grants from the Consiglio Nazionale delle Ricerche,
Special Project “lngegneria Genetica ” (contract no. 93.00042.PF
99), and by funds from Progetto Finaliuato Minister0 Sanith 1993
(105 34.2/RF 93.57).
Address reprint requests to Gian Franco Gaetani, MD, Division
of Hematological Oncology, lstituto Nazionale per la Ricerca sul
Cancro and Istituto di Oncologia Clinica e Sperimentale, University
of Genoa, Viale Benedetto XV, 10. 16132 Genoa, Italy.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1996 by The American Society of Hematology.
0006-4971/96/8803-00$3.00/0
1084
MATERIALS AND METHODS
In vivo studies. Favism was diagnosed as an acute hemolytic
episode occurring after ingestion of fava beans, with anemia, hemoglobinuria, and jaundice. Blood was drawn before transfusion from
seven boys having AHA of various degrees after fava beans ingestion
(see Table 1) and from seven healthy G6PD-deficient (GdMed)adult
men with a past history of favism. Leukocytes and platelets were
removed by the method of Beutler et al.’’ Plasma was removed after
each sample was centrifuged, and the erythrocytes were divided into
two portions: one was washed twice by suspension in 5 volumes of
0.15 moUL NaC1, and the other in 5 volumes of 0.15 molL NaCl
containing 20 mmom 3-amino-1:2:4-triazole (AT), a catalase inhibiforms an irreversible complex
tor, which in the presence of HZOZ,
with the enzyme. The first portion was promptly assayed for concentration of GSH and for activity of G6PD, 6-phosphogluconate dehydrogenase, and GSH-Px by the methods of Beutler.” Catalase activity was measured by the method of AebiI4 and expressed as Wg Hb.
The other portion of packed erythrocytes was mixed with 3 volumes
of Krebs-Ringer solution/20 mmolL Tes (N-tris[hydroxymethyl]methyl-2-aminoethanesulfonicacid) buffer (pH 7.4)/5 mmol/L glucose (KRTG) as previously described,15 and AT was added to a
final concentration of 20 mmoVL. After a 60-minute incubation in
Blood, Vol 88, No 3 (August 1). 1996: pp 1084-1088
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CATALASE AND FAVISM
1085
Table 1. Hematological Values of Seven GdW Subjects (males) During Acute Hemolytic Crisis Following Ingestion of Fava Beans
Patient
No.
1
2
3
4
5
6
7
Age
(yr)
6
3
8
7
2
3
3
Hemoglobin
WdL)
Bilirubin
(mg%l
Reticulocyte
6.9
7.2
7.4
11.9
7.1
4.8
7.6
7.7
4.3
8.1
4.9
6.1
0.7
6.0
5.8
20.0
25.0
2.5
7.2
(%I
2.3
4.2
Blood
Transfusion*
Days From Fava
Beans Ingestion
Yes
Yes
Yes
No
Yes
Yes
No
* Given after blood samples were drawn for the present study.
a metabolic shaker at 37°C at 120 oscillations per minute, the erythrocytes were collected, washed in 5 volumes of 0.15 m o m NaCl,
and assayed as described above. AT and all other reagents were
from Sigma (St Louis, MO). All incubations and determinations
were in duplicate.
In vitro studies with intact erythrocytes and divicine. The effect
of one of the metabolites present in fava beans, 2,6-diamino-4,5dihydroxypyrimidine (divicine), was evaluated in vitro on intact
G6PD-deficient red blood cells (GdMsd)from four healthy male donors. DNA analysis of the Gd gene of some of these subjects indicated that they were carriers of the GdMd mutation. Divicine was
obtained from vicine (Serva, Germany), a glycoside which, through
the action of a 0-glycosidase (Boheringer, Mannheim, Germany) in
Tris-HCI, 10 mmoUL, pH 6.0, splits the glycosidic bond of vicine
with formation of one molecule of glucose and one of divicine.16
After completion of this reaction, the sample was filtered with a CF25 ultrafiltration cone (Amicon, Beverly, MA); the amount of divicine in the ultrafiltrate was measured by the amount of glucose
generated from an enzymatic reaction using NADP, adenosine triphosphate (ATP), and the enzymes hexokinase and G6PD.l’ Erythrocytes were incubated in a metabolic shaker at 37°C in KRTG buffer,
in the presence of divicine (200 pmom), with or without AT (20
mmoYL). Erythrocytes without divicine, but with AT, served as
controls. Catalase activity and GSH concentration were measured
every 30 minutes for 90 minutes.
In vitro studies with pur$ed catalase and divicine. Human purified catalase was prepared as previously r e p ~ r t e dThe
. ~ interaction
between divicine and catalase was followed in a spectrophotometer
at 435 nm and at 37°C for 120 minutes. Each cuvette contained, in
the following final concentration: KRTes buffer, pH 7.4, 2 nmoV
mL, of human purified catalase, 0-glycosidase (2U/mL), vicine (200
nmoVmL), and a NADPH regenerating system formed by the addition of glucose-6-phosphate (600 nmoUmL), NADP+ (2 nmoVmL)
and G6PD (5 pg/mL). The G6PD was added at different times (see
Fig 3, curves a and b).
RESULTS
Admission to the hospital and blood sampling of the seven
boys occurred within 24 to 48 hours after ingestion of the
fava beans, except for patient no. 7, who had ingested the
beans 7 days earlier (Table 1). All subjects showed signs of
hemolysis, such as reduced hemoglobin (Hb) level, high
reticulocyte count, and increased level of total bilirubin (Table l). Erythrocytic enzyme activities and GSH were measured during AHA and before transfusion. Increased G6PD
and lower concentration of GSH were consistently observed
(Table 2), as reported earlier,” whereas mean catalase activity was significantly decreased (P < .0002) compared with
the values of G6PD-deficient subjects without AHA (Fig
1 and Table 2). In two cases, catalase activity and GSH
concentration, when measured in older erythrocytes fractionated by gravity, were barely detectable. Values of GSH-Px
were slightly increased. When erythrocytes collected from
patients during acute hemolytic crisis were preincubated with
AT for 60 minutes, catalase activity was significantly lower
(Fig 1 and Table 2, P < .0003). Patient no. 7, who had
ingested fava beans 7 days earlier, had an unchanged catalase
value (Fig 1).
Incubation of G6PD-deficient erythrocytes in the presence
of divicine, obtained enzymatically from vicine, caused a
decrease of catalase activity, which was more pronounced
in the presence of AT (Fig 2A). A similar behavior was
observed for intracellular GSH (Fig 2B). Higher concentrations of divicine caused complete inactivation of catalase
and oxidation of GSH in a shorter period of time.
The effect of divicine on purified human catalase was also
evaluated. Catalase was exposed to divicine, progressively
generated in the native form by the action of @glycosidase
on vicine. As observed in Fig 3 (curve a), generation of
divicine caused the formation of Compound 11, the inactive
form of catalase, whereas the addition of a NADPH generating system (G6PD) reversed the reaction (Fig 2, second part
of curve a). The simultaneous addition of divicine and the
NADPH generating system protected catalase from inactivation (Fig 3 curve b).
DISCUSSION
The catalase reaction consists of the conversion of two
molecules of H202to one molecule of oxygen and two molecules of water. Catalase becomes an enzyme-substrate complex called Compound I on reacting with the first molecule
of H2O2. Reaction with the second molecule of H202brings
catalase back to its initial state. In the absence of bound
NADPH, however, Compound I is capable of becoming
Compound 11, an inactive form of catalase. Compound I1
reverts to active catalase spontaneously, but a steady-state
ratio of Compound I and Compound I1 exists when catalase
is constantly exposed to H202in the absence of NADPH.I8*l9
Moreover, existence of catalase in the Compound I1 state is
accompanied by an increased rate of irreversible inactivation
of the catalase. As a consequence of the ability of catalase
to regain activity spontaneously, the extent of catalase
involvement during oxidative stress in vivo must have been
even greater than is reflected in the reduced activity of the
erythrocytic catalase of subjects with AHA from favism (Ta-
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1086
GAETANI ET AL
Table 2. Mean Values (+SDI of Catalase and GGPD Activities and GSH Level of Erythrocytes From Six GGPD-Deficient Subjects With Acute
Hemolysis After Fava Beans Ingestion Compared With Seven GGPD-Deficient Controls Without Hemolysis
Controls
Catalase (k/g Hb)
G6PD CU/g Hb)
GSH (pmol/g Hb)
Subjects With AHA
Plain
After Incubation With AT*
Plain
After Incubation With AT*
328 i- 35
0.04 i- 0.01
3.96 ? 0.61
312 i- 28
0.05 It 0.02
3.79 5 0.57
221 c 20
1.81 2 0.73
2.96 5 0.83
149 ? 34
1.75 i- 0.45
2.29 ? 1.04
* Erythrocytes were incubated with 20 mmol/L AT for 60 minutes at 37°C in KRTG, pH 7.4, before determination of enzyme activities and GSH
concentrations.
ble 2 ) . The most severely affected erythrocytes had undergone hemolysis during the 1 to 2 days after ingestion of fava
beans. There was a high percentage of reticulocytes (Table
l), which have higher G6PD levels (Table 2 ) and generate
more NADPH that should prevent catalase inactivation (Fig
3, curve b). Although it was done rapidly, the erythrocytes
had been washed twice with 5 volumes of saline before
catalase activity was measured. In this study, erythrocytes,
which had been subjected to in vivo oxidative stress, were
also exposed for a period of time to AT before preparation
of the hemolysates. AT is an irreversible and specific inhibitor of catalase, once the enzyme reacts with the first molecule
of H202with formation of Compound I.I9 The inhibition was
observed in six of the seven patients studied (Fig 1). In the
one patient who had ingested fava beans 7 days earlier,
erythrocytes had normal catalase activity and were refractory
to AT inhibition. Even without exposure to AT, erythrocytes
from subjects with AHA had a 32% reduction of catalase
activity and 25% decrease of GSH, compared with G6PDdeficient subjects without AHA, and these values reached
55% and 40%, respectively, in the presence of AT (Table
2).
Two agents, among those present in fava beans, have been
suspected of being responsible for the hemolytic crises in
G6PD-deficient subjects. These are two glycosidic compounds, vicine and convicine, which upon splitting of the
P-glycosidic bond between glucose and the hydroxyl group
at the C5 level, generate the redox aglycones divicine and
isouramil. Both pyrimidine derivatives, although never identified in vivo for technical difficulties, cause oxidative stress
in vitro, as described by several investigator^.^'-^^ Toxicity
of divicine and isouramil is very similar: reduced divicine
is believed to be oxidized to the semiquinoid free radical
form by the one-electron reduction of dioxygen and subse-
1 .o
400
0
350
a
300
2
0
c
.-
0
0
0
8
0
0
? .z
0.6
.=
m
3 4 0.4
m a
0.2
.,X 250
0.0
1 .o
>
.
c
e
)
U
-3
200
S
.-0
E
m
3
0.8
;
.
150
0.8
2z
'3 0
2
2z
100
z
0.6
0.4
50
G6PD deficient RBC G6PD deficient RBC
(controls)
(during AHA)
Fig 1. Distribution of ,atalese activity of erythrocytes of GGPDdeficient controls without (0)and with (0)incubation for 60 minutes
at 37°C in the presence of 20 pmol/mL AT and of erythrocytes from
subjects with AHA after ingestion of fava beans, without (V) and
with incubation with AT (V). Asterisks denotes a patient (no. 7, Table
1). who ingested fava beans 7 days before hospitalization and blood
sampling.
0.2
1,
0
,
,
,
,
,
15
30
45
60
75
I - /
90
Time (min)
Fig 2. Mean relative activities ( 2 standard deviation SDI) of catalase (A) and GSH concentration (B) in GBPDdeficient red blood cells
from four different male subjects without AHA, incubated in various
conditions: (0)plain, (0)in the presence of divicine 200 nmollml,
and with (VI divicine plus aminotriazole (20 pmol/mL).
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CATALASE AND FAVISM
primaquine administration to GGPD-deficient subjects. Other
investigators during the same period reported, through in
vitro studies, an increased H M S activity of actalasemic red
blood cells (Swiss type) in resting condition and during oxidative stress.’6 Those findings did not receive large credit
until Eaton et a12’ reconsidered those data after showing that
catalase was protected by the addition of NADPH to crude
hemolysate challenged with some oxidizing agents. Through
in vitro studies with normal, G6PD-deficient and acatalasemic erythrocytes, we proposed that catalase and GSH-Px
are equally active in the erythrocyte^.'^*^^ More recently, by
the use of a cell-free system, we obtained evidence for an
even more preeminent involvement of catalase.” The main
function of GSH-Px, at least in human erythrocytes, probably
involves the reduction of organic hydroperoxides rather than
H202, and this demand can be easily met by this enzyme
because of its broad specificity with respect to hydroperoxide
0.50
G6PD
zc
0.45
cc,
b,
E
8
4
0.40
$
s
0.35
4
Divicine & C6PD
D
I
I
I
I
I
30
60
90
120
150
Time (min)
Fig 3. Formation of Compound II (curve a) during exposure of
human purifiedcatalaseto divicine generated by 2 U of /?-glycosidase
from vicine (200 nmollml) and kurve b) in the presence of a NADPH
generating system formed by the addition of glucosa-6-phosphate
(600 nmollmU, NADP+ (2 nmollml), and G6PD (5 pglml). Addition
of G6PD after 80 minutes to reaction (curve a) promptly reversedthe
formation of Compound II.
quent generation of H202.’4 Divicine and isouramil have
been shown in vitro to cause a fall of GSH and NADPH,
generation of Heinz bodies, membrane cross-bonding, and
Ca2+-ATPasei n h i b i t i ~ n . ’ ~ Their
. ~ ’ - ~potential
~
toxic effect on
catalase, however, had never been tested. The in vitro findings with one putative toxic metabolite present in fava beans
are quite similar to the in vivo observations. During exposure
of G6PD-deficient erythrocytes to divicine, used at concentration considered probably realistic after ingestion of a small
amount of beans,23catalase decreases its catalatic activity to
less than 10% after a 90-minute incubation and about 50%
of GSH is in the oxidized form (Fig 2). As with in vivo
studies, these effects are more evident in the presence of AT,
when inactivation of catalase seems to anticipate oxidation of
GSH (Fig 2A and B). If divicine is interacting with catalase,
the same phenomenon should be observed in a cell-free system containing purified human catalase. As previously reported, generation of HzOzcauses a progressive inactivation
of catalase4.‘*that can be reversed or prevented if adequate
generation of NADPH is provided! This phenomenon has
been observed in the presence of native divicine (Fig 3,
curve a) when the increased formation of Compound I1 is
promptly stopped by addition of the NADPH generating
system (Fig 3, second part of curve a). On the other hand,
when there is simultaneous generation of NADPH and divicine, as occurs in erythrocytes with normal G6PD levels,
the formation of Compound I1 is negligible (Fig 3, curve b).
The present in vivo data fully agree with the observations
made by early investigators of G6PD who reported a fall in
GSH accompanied by a decrease of catalase activity” during
substrate^.^'
ACKNOWLEDGMENT
The authors express appreciation to Dr H.N. Kirkman for his
advice in the preparation of this report.
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1996 88: 1084-1088
Active involvement of catalase during hemolytic crises of favism
GF Gaetani, M Rolfo, S Arena, R Mangerini, GF Meloni and AM Ferraris
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