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Enzymes of oxygen metabolism and lipid peroxidation in erythrocytes from copper-deficient rats
ENZYMES OF OXYGEN METABOLISM AND LIPID PEROXIDATION IN ERYTHROCYTES FROM COPPER-DEFICIENT RATS ELEVTER M. RUSSANOV and TOLXJRKA A. KASSABOVA Institute of Physiology. Bulgarian Academy of Sciences, 1113Sofia, Bulgaria (Recriwd
13 Augusf 1981)
Abstract--t. Copper deficiency in the rat results in a high decrease of erythrocyte superoxide dismutase activity (by ?O?/,), an increase of glutathione peroxidase activity (by 17”;) and glucose (i-phosphate dehydrogenase activity (by 4Ou/,) and no change in catalase activity. 2. Ascorbate (30 nM) and copper (10 and 50 nmol/mg protein) enhance about 2-fold the lipid peroxidation of erythrocyte membranes from copper-deficient rats. 3. The osmotic stability of copper-deficient rat erythrocytes is higher compared with that of the controls.
The present study was undertaken to investigate some enzymes scavenging toxic products of oxygen metabolism as well as the spontaneous and induced hemolysis and lipid peroxidation of erythrocytes from copper-deficient rats.
INTRODUCTION
The intermediates of oxygen metabolism, superoxide radical (0;) and hydrogen peroxide (H,O,), are precursors of more reactive species which damage a variety of cellular components. Thus the enzymes which catalytically scavenge them, superoxide dismutase (SOD), catalase and glutathione peroxidase (GSHperoxidase), have a protective action against this damage (Fridovich, 1976). Recently, the mechanism of these processes has been extensively studied on invitro models. Data about the action of in-&o formed 0; and H20, are scarce and our knowledge of this problem is still in a primitive state (Lynch & Fridovich, 1978). Erythrocytes, being structurally simple and easily obtainable, are an excellent object for studies of oxygen metabolism and lipid peroxidation. It is known that 0; is generated upon in-vitro autooxidation of hemoglobin (Brunori et al., 1975) and about 2-3”/, of human hemoglobin is oxidized to methemoglobin daily (Winter~urn et ai., 1976). The regular production of 0; in blood could account for the high and relatively constant (with respect to the hemoglobin content) amount of SOD in many vertebrates including man (Maral er al., 1977). In cirro 0; induces lysis of erythrocytes and erythrocyte vesicles and regardless of the way of 0; generation the process is in-
MATERIALS AND METHODS Animais
and diets
Male Wistar albino rats weighing 150-160 g Control animals received a standard laboratory taining 15 ppm copper. Copper deficiency was feeding the rats a diet based on powder milk less than 1 ppm copper and glass-redistilled 45-50 days.
were used, diet coninduced by containing water for
Red blood cells
Blood was taken by a silicon syringe from the left ventricle under light ether anesthesia. Erythrocytes were obtained at 600 g for 10 min at 4-C and the “huffy” coat at the top was removed by aspiration. Packed cells were twice washed with a double volume of isoosmotic solution (IS) containing (in mM): NaCi, 136.8; KCI, 26.9; KH,PO,. 1.4: MgC12, 0.5: CaC12, 0.5 and NaHCO, to pH 7.4. Washed cells were suspended in an equal volume IS. Spontarwous
and induced
hemolysis
Hypoosmotic stability was determined as follows: 20/tl of erythrocyte suspension were added to plastic tubes containing 2 ml increasing concentrations of IS (from 0 to 100”;). After 3 hr-incubation at 4 C the tubes were centrifuged and the hemolysis was determined by absorbance (540nm) of the supernatant. Hemolysis in water was considered to be IOO’?,; and in IS OO.,. Maximal A,,, was hemolysis was measured 0.800-0.920. The C; 2+-induced after 2Ohr-incubation at room temperature (2&22 C). Erythrocyte suspensions. equivalent to 3.5 mg protein. were added to 2 ml IS containing the corresponding agents fcopper sulfate and bathocuproine sulfonate (BCS)). The extent of hemolysis was determined as described above. All assays were run in triplicate,
hibited by SOD and/or catalase (Fee & Teitelbaum, 1972; Brunori ef ul., 1975; Goldberg & Stern, 1975, 1977). Copper deficiency is generally associated with mic-
rocytic anemia, similar to that resulting from iron deficiency. Furthermore, copper-deficient erythrocytes have a decreased SOD activity (Bohnenkamp & Weser. 1976; Williams et al.. 1975) and a shorter lifetime (Lynch ef al., 1972), while in patients with Menkes’ syndrom the H,O,-induced hemolysis is enhanced (French er al., 1972). It is not not clear as yet whether anemia appears as a result of destroyed hemesynthesis and/or of oxidative disruption of hemoglobin and membrane lipids.
Erythrocyte
lysates and enzyme assays
Red cells were lysed in 10 vol of cold 5 mM phosphate buffer, pH 7.4. In this lysate glucose 6-phosphate dehydro321
ELEVTEK M. RVSSANOV and TODORKA A. KASSABOVA
322 Table
1. Hemoglobin
content,
cyte enzymes
hematocrite
from control
and activities
and copper-deficient Control rats
Hemoglobin (g/l00 ml blood) (g/ml packed red cells) Hematocrite (in “,) Superoxide dismutase (U/g hemoglobin) Catalase (AE240/mg hemoglobm) Glutathione peroxidase (pmol NADPH oxid./min/g hemoglobin Glucose 6-phosphate dehydrogenase (pmol NADP reducedjminjg hemoglobin)
of some
erythro-
rats C&deficient rats
12.1 f 0.1 0.23 i: 0.01 50.9 2 0.4
7.9 + 0.2* 0.20 * 0.01** 37.3 + 0.6”
4737 zf: 421
1501 * 139*
2.65 k 0.22
2.99 k 0.85
25.3 I): I.2
29.8 + IS**
13.4 + 0.8
18.6 -r_0.Y
GSH-peroxidase and glucose &phosphate dehydrogenase activities were measured in soluble, hemoglobin-free erythrocyte protein and were recalculated per g hemoglobin. Superoxide dismutase and catalase activities were determined in erythrocyte lysates. One SOD unit is the amount of protein that causes 50”” inhibition of the reaction with Nitro Blue Tetrazolium. Catalase activity was determined by the disappearance of HZO, followed at 240 nm. The data are the means of 8 experiments + SEM. *P c 0.01: **p < 0.05.
genase activity was measured by the method of Cartier er al. (1967) and GSH-peroxidase activity was determined according to Maral et al. (1917) with tert-butyl hydroperoxide as substrate. The spontaneous and ascorbateand Cu”-induced lipid peroxidation were followed by the formation of malonyl dialdehyde (MDA) by the method of Hunter er a. (1963). Molar extinction coefficient, I.56 x lo5 M-i cm-’ was used for calculation. Soluble. hemoglobin-free erythrocyte protein was obtained according to Maral et al. (1977) and was used for determining SOD activity (Beauchamp & Fridovich. 1971) and cataiase activity (Aebi. 1970). The hemoglobin content in both blood and erythrocyte lysate was determined by Merckotest (Cat. No. 3317). Protein was measured by the method of Lowry er 01. (1951).
Glutathione (reduced and oxidized). glutathione reductase. NADP and NADPH were purchased from Boehringer-Mannheim GmbH. tert-butyl hydroperoxide was synthetized and purified in the Institute of Organic Chemistry. Bulgarian Academy of Sciences. All other chemicals were of reagent grade purity obtained from Merck-Schuchardt (Darmstadt) or BDH Chemicals Ltd. Student’s r-test was used for statistical analysis. The means + SEM are presented.
REWLTS Rats fed on a copper-deficient diet for 6-7 weeks had a decreased content of hemoglobin in the blood (by 359, compared with the controls) and in milliliters of packed red cells (by 13”b); the values of hematocrit decreased by 27’?;,(Table 1). Copper deficiency exerted a different effect on the activities of the enzymes studied. SOD. which in erythrocytes is only a Cu, Zncontaining enzyme, sharply decreased (by 70”/,) and catalase did not change. GSH-peroxidase. utilizing
lipid peroxides or H,Ot as substrates, slightly increased and glucose &phosphate dehydrogenase was activated by 404, compared with the control level (Table 1). Copper-deficient erythrocytes were more stable in hypo~smotic medium at low temperature (4’C) and relatively short time of incubation (3 hr) as compared with the control erythrocytes. Fifty percent hemolysis occurred at 54u/, IS for the controls and at 44”:, IS for the copper-deficient erythrocytes (Fig. 1). At room temperature and prolonged incubation (20 hr) in isoosmotic medium the copper-deficient erythrocytes also had a higher stability than the controls (Table 2, without additions). Low copper concentrations (5 and 1Onmol~mg protein) caused hemolysis which was expressed to an equal degree in both control and copper-deficient red cells. The Cu’ ‘.-induced hemolytic effect was abolished by BCS, a Cu+-chelating agent. (50 nmol/mg protein) The higher Cu2+ concentration induced hemoiysis but to a smaller degree which was not affected by BCS (Table 2). Twenty-hour storage of erythrocyte lysates from control rats at room temperature increased the spontaneous formation of MDA and the latter was not changed in the presence of 10nmof. Cu’+/mg protein or 30 nM ascorbate but was inhibited by 50nmol Cu”/mg protein (Fig. 2). In the copper-deficient erythrocytes the spontaneous and ascorbateand 0.1 +-induced formation of MDA was increased and the high Cu” concentration was also effective (Fig. 2).
Anemia caused by copper deficiency has been known since 1928 and it is one of the earliest and the
Lipid peroxidation
IS, % Fig. 1. Hypoosmotic stability of erythrocytes from control (G-O) and copper-deficient (M) rats. 20~1 of erythrocyte suspensions were incubated for 3 hr at 4°C in tubes containing 2 ml increasing concentrations of IS (from 0 to 100%). The tubes were then centrifuged at 5OOOg for IOmin and the optical density of supernatantes were measured at 540 nm. The results are expressed as percentage of samples completely lysed in distilled water. Hemolysis in 100% IS was considered to be OT/,. The data are means of 10 experiments It: SEM. All assays were run in triplicate.
most pronounced symptoms of this state. The disturbed iron transport and the destroyed heme synthesis by copper deficiency (Williams ef al., 1976) cannot fully explain the pathogenesis of this anemia because the activity of catalase, which is also a hemeenzyme, is not decreased in this stage of copper depletion (Bohnenkamp & Weser, 1976). Furthermore, the life-span of erythrocytes and the turnover of hemoglobin are more prolonged, about 120 days, compared with the period during which anemia deTable
2. Copper-induced
in erythrocytes
velops. It has been found that the life-span of copperdeficient erythrocytes in the circulation of both normal and copper-deficient pigs is decreased (Lynch et al., 1972). The cause of the diminished survival is not clarified but in the erythrocytes of copper-deficient pigs the osmotic fragility is increased which suggests an enhanced pliability to oxidative attack. Copper deficiency induces an increase of the membrane permeability in both old and young red cells (Lynch ef al., 1972). The higher hypoosmotic stability of erthrocytes from copper-deficient rats we have found is in accordance with that observed by Bettger et al. (1978) in experiments on rats, too. This phenomenon could partly be due to the lower concentration of the main erythrocyte matrix proteins upon copper deficiency, hemoglobin and SOD, whereby the osmotic pressure in these erthrocytes in hypoosmotic medium is lower compared with normal erythrocytes. The present results show that copper-deficient erythrocytes have an increased spontaneous. and ascorbateand copper-induced peroxidation of the membrane lipids which is evidenced from the MDA formation. Lipid peroxides are metabolized by GSHperoxidase. The latter is functionally connected with GSH-reductase (supplying reduced glutathione) and with glucose 6-phosphate dehydrogenase (supplying NADPH). The higher activities of GSH-peroxidase and glucose 6-phosphate dehydrogenase are probably the response to the increased amounts of hydroperoxides formed in the copper-deficient erythrocytes. In other experiments of ours on copper-deficient rat liver, where lipid hydroperoxide level was twice increased, we found enhanced activity of GSH-reductase in both cytosol and mitochondria and activated mitochondria NADPH-generating system in (Balevska et al., 1981). Similar results were obtained by Nishiki et ul. (1976) on tocopherol-deficient rat liver while Bohnenkamp & Weser (1976) failed to find any changes in GSH-peroxidase activity in copperdeficient erythrocytes. The spontaneous lipid peroxidation of copper-deficient erythrocytes is slightly increased (compared with the ascorbate induced) and thus cannot cause hemo-
hemolysis of erythrocytes per-deficient rats
Additions
5 nmol Cu* + img protein - BCS + BCS (25 nmol/mg protein) 10 nmol Cu’+/mg protein - BCS + BCS (50 nmol/mg protein) 50 nmoles Cu” img protein - BCS + BCS (250 nmol/mg protein)
323
from control
Control rats
and cop-
Cu-deficient rats
39.0 + 4.3
29.5 k 2.2
84.0 + 4.2 37.5 + 2.0
83.1 f 4.9 29.7 f 1.0
88.6 + 1.1 31.5 f 1.4
87.9 + 2.0 28.3 + 2.6
44.3 + 1.8 49.2 + 7.8
37.6 + 2.8 43.8 f 8.2
Incubation conditions: 2 ml IS were placed in plastic centrifugal tubes and indicated amounts of CuS04 and BCS (bathocuproine sulfonate) dissolved in microvolumes IS were added. Erythrocyte suspensions equivalent to 3.5 mg protein were also added. After 20 hr at 20-22’C the tubes were centrifuged at 5C00 g for 10 min. The extent of hemolysis was measured and expressed as shown in the legend to Fig. 1. n = 7.
324
ELEVTER
M. Rcss~uov
and TODORKA A.
KASSAROVA
30 nk ascorbate
IO nmol
Cu/mg
* :
800 50
nmol
Cu/mg
; o
600
E -
400
Z E
200
0
1
2 20 Time
Fig. 2. Ascordate-
and Cu’ * -Induced ’
formation
I
I 2 20 of incubation, of malonyl
2 20
I 2
hr
dialdehyde
in erythrocytes
from control ( in 5 mM phosphate buffer. pH 7.0 and the corresponding additions at 20-22 C. At given time intervals a sample was taken and the amount of MDA formed was measured using the difference in the extinctions at 532 and 600 nm against the corresponding controls. n = 10.
and copper-deficient (Cl) rats. Erythrocyte lysates (IO mg protein~ml)
lysis as it is known that hemolysis begins to bccur when lipid peroxidation approaches 70”” of its maximum value (Fee rt ul., 1975). Cu2’-induced hemolysis being expressed to an equal degree in both control and copper-deficient erythrocytes could hardly be due to lipid peroxidation of the membranes. Probably it results from the Cu*‘-catalized oxidation of hemoglobin (Winterbourn et al.. 1976; Winterbourn & Carrel!. 1977). The hemolyzing effect of Cu2’ is removed by BCS suggesting the role of Cu’ whose reoxidation leads to generation of 0;. 0; in turn causes decomposition of hemoglobin to an unidentified green pigment which leads to hemoiysis regardless of the lipid ~roxidation (Goldberg & Stern, 1977). Kobayashi er lrl. (1979) have found that upon paraquate-induced 100”~ hemolysis where the lytic agent is IO2 there occurs no lipid peroxidation which also suggests that the mechanisms and the time-course of the two processes. hemolysis and lipid peroxidation, do not closely correlate as was stated by Bettger er al. (1978). Kellog & Fridovich (1975) have shown in inritro experiments that ‘02 (and OH 1 formation is mediated by H20z. This finding add the present result led us to believe that in the case of Cu-deficient erythrocytes the normal or even slightly increased activities of enzymes catalyzed decomposition of H,02 and lipid peroxides compensate the after-effects of the decreased SOD activity on the erythrocyte stability.
were incubated
BETTGER W. J.? FISH T. J. & O’DELL R. I_. (1978) Effects of copper and zinc status of rats on erythrocyte stability and superoxide dismutase activity. P,oc. Sot. esp. Biol. Med. 158, 279--282. BOHKENKAMP W. & ,WFSFR U. (1976) Copper defictency and erythrocuprein. ZCu.ZZn-superoxide dismutase. Biochini. hiophj~.s. Ada 444, 396406. BRCNOR~ M.. FALC~ONI G.. FIORETTI E.. GIARDINA B. 81 ROTILIO G. (1975) Formation of superoxide in the autoxidation of the isolated Y and /I chains of human hemoglobin and its involvement in hemichrome precipitation. Eur. J. Biochrn1. 53, 99-- 104. CARTIER P.. LEROUX J-P. & MARCHAND J.CL. (1967) Techniques de dosage des enzymes glycolytiques tissulaires. Atznls Biol. flirt. 25, 109-I 36. FEE J. A, & TEITELBAUM H. D. (1972) Evidence that superoxide dismutase plays a role in protecting red blood cells against peroxidative hemolysis. Biocl~rnl. hiophp. Rr.5. Conlntrrn. 49, 150-158. FEE J. A.. BERCA~~VI R. & BRIGGS R. G. (1975) Observations on the mechanism of the oxygen:dialuric acidinduced hemolysis of vitamin E-deficient rat red blood cells and the protective roles of catalase and superoxide dismutase. Arclts Bioci~crn. Biophx~. 169, 160-167. FRENCH J. H.. SHERARU S. E.. LC’BELL H.. BROTX M. & MOORI C. L. (19721 Trichopoiiodystrophy~ 1. Report of a case and biochemical studies. Arc,hs .%‘(,tirol. 26, 229-244. FRIDO~I~H 1. (1976) Oxygen radicals. hydrogen peroxlde and oxygen toxicity. In free rc&cols it? Biolo!~~. Vol. I (Edited by PRYOR W. A.). pp. 239-277. Academic Press. New York. GOLDBERG B. & STERN A. (1975) The generation of 0; by REFERENCES the interaction of the hemolytic agent. phenylhydrazine. J. hiol. C/wn. 250, 2401.-2403. AEBI H. (1970) Kataiase. fn Mefhc$e!r der ~~~l~~~)iur;s~~l~~~ with human hemoptobm. GOLDBERG B. & STERN A. (1977) The role of superoxide At&!su (Edited by BERCMEYERH. U.). pp. 637-641. Akaanion as a toxic species rn erythrocyte. Arch.\ Biochm. demie Verlag. Berlin. Biophys. 178, 218-225. BALEVSKA P. S.. RUSSANOV E. M. & KASSABOVA T. A. HUNTER E. JR. GEBICK~ J. M.. HOFFSTEI~~~ P. E., WEIMSTE~~ (1981) Studies on lipid peroxidation in rat liver by copJ. & SCOTT A. (1963) Swelling and lysis of rat liver mitoper deficiency. Itzt. J. Biockem. 13, 489-493. chondria induced by ferrous ions. J. hid. Chrm. 238, BEAUCHAMP C. & FR~DOV~CH 1. (1971) Superoxide dismu828- 835. tase: improved assays and an assay applicable to acryl KELLOGG E. & FR~DOVICH 1. (1975) Superoxide. hydrogen amide gels. Anulrt. Biochrm. 270. 276-287.
Lipid peroxidation peroxide and synglet oxygen in lipid peroxidation by a xanthine oxidase system. J. hiol. Chem. 250. 8812-8817. KOBAYASHI Y., OKAHATA S. & USUI T. (1979) Hemolysis of human erythrocytes by paraquat in relation to superoxide dismutase activity. Biochem. hiophps. Res. Commun. 91, 1288-1294. LOWRY 0.. R~~EBROUGH N., FARR A. & RANDALL R. J. (1951) Protein measurement with the Folin phenol reagent. J. hiol. Chem. 193, 265-278. LYNCH R. E.. LEE G. R. & CARTWRIGHT G. E. (1972) Copper and the red cell membrane. Blood 40, 9633971. LYNCH R. E. & FRIWVICH I. (1978) Effects of superoxide on the erythrocyte membrane. 1. hiol. Chem. 253. 1838-1845. MARAL J., PUGET K. & MICHELSON A. M. (1977) Comparative study of superoxide dismutase. catalase and glutathione peroxidase levels in erythrocytes of different animals. Biochem. biophys. Res. Commun. 77, 152551535.
in erythrocytes
325
NISHIKI K., JAMIESON D., OSHINO N. & CHANCE B. (1976) Oxygen toxicity in the perfused rat liver and lung under hyperbaric conditions. Biochem. J. 160, 343-355. WILLIAMS D. M., LYNCH R. E., LEE G. R. & CARTWRIGHT G. E. (1975) Superoxide dismutase activity in copperdeficient swine. Proc. Sot. exp. Biol. Med. 149, 534 536. WILLIAMS M. D., LOUKOPOULOS D., LEE G. R. & CARTWRIGHT G. E. (1976) Role of copper in mitochondrial iron metabolism. Hood 48, 77-85: _ WINTERBOURN C. C.. MCGRATH B. M. & CARRELL R. W. (1976) Reactions involving superoxide and normal and unstable hemoglobins. Biochem. J. 155, 493-502.
WINTERBOURN C. C. & CARRELL R. W. (1977) Oxidation of human hemoglobin by copper. (Mechanism and suggested role of the thiol group of residue b-93). Biochem. J. 165, 141-148.
13 Augusf 1981)
Abstract--t. Copper deficiency in the rat results in a high decrease of erythrocyte superoxide dismutase activity (by ?O?/,), an increase of glutathione peroxidase activity (by 17”;) and glucose (i-phosphate dehydrogenase activity (by 4Ou/,) and no change in catalase activity. 2. Ascorbate (30 nM) and copper (10 and 50 nmol/mg protein) enhance about 2-fold the lipid peroxidation of erythrocyte membranes from copper-deficient rats. 3. The osmotic stability of copper-deficient rat erythrocytes is higher compared with that of the controls.
The present study was undertaken to investigate some enzymes scavenging toxic products of oxygen metabolism as well as the spontaneous and induced hemolysis and lipid peroxidation of erythrocytes from copper-deficient rats.
INTRODUCTION
The intermediates of oxygen metabolism, superoxide radical (0;) and hydrogen peroxide (H,O,), are precursors of more reactive species which damage a variety of cellular components. Thus the enzymes which catalytically scavenge them, superoxide dismutase (SOD), catalase and glutathione peroxidase (GSHperoxidase), have a protective action against this damage (Fridovich, 1976). Recently, the mechanism of these processes has been extensively studied on invitro models. Data about the action of in-&o formed 0; and H20, are scarce and our knowledge of this problem is still in a primitive state (Lynch & Fridovich, 1978). Erythrocytes, being structurally simple and easily obtainable, are an excellent object for studies of oxygen metabolism and lipid peroxidation. It is known that 0; is generated upon in-vitro autooxidation of hemoglobin (Brunori et al., 1975) and about 2-3”/, of human hemoglobin is oxidized to methemoglobin daily (Winter~urn et ai., 1976). The regular production of 0; in blood could account for the high and relatively constant (with respect to the hemoglobin content) amount of SOD in many vertebrates including man (Maral er al., 1977). In cirro 0; induces lysis of erythrocytes and erythrocyte vesicles and regardless of the way of 0; generation the process is in-
MATERIALS AND METHODS Animais
and diets
Male Wistar albino rats weighing 150-160 g Control animals received a standard laboratory taining 15 ppm copper. Copper deficiency was feeding the rats a diet based on powder milk less than 1 ppm copper and glass-redistilled 45-50 days.
were used, diet coninduced by containing water for
Red blood cells
Blood was taken by a silicon syringe from the left ventricle under light ether anesthesia. Erythrocytes were obtained at 600 g for 10 min at 4-C and the “huffy” coat at the top was removed by aspiration. Packed cells were twice washed with a double volume of isoosmotic solution (IS) containing (in mM): NaCi, 136.8; KCI, 26.9; KH,PO,. 1.4: MgC12, 0.5: CaC12, 0.5 and NaHCO, to pH 7.4. Washed cells were suspended in an equal volume IS. Spontarwous
and induced
hemolysis
Hypoosmotic stability was determined as follows: 20/tl of erythrocyte suspension were added to plastic tubes containing 2 ml increasing concentrations of IS (from 0 to 100”;). After 3 hr-incubation at 4 C the tubes were centrifuged and the hemolysis was determined by absorbance (540nm) of the supernatant. Hemolysis in water was considered to be IOO’?,; and in IS OO.,. Maximal A,,, was hemolysis was measured 0.800-0.920. The C; 2+-induced after 2Ohr-incubation at room temperature (2&22 C). Erythrocyte suspensions. equivalent to 3.5 mg protein. were added to 2 ml IS containing the corresponding agents fcopper sulfate and bathocuproine sulfonate (BCS)). The extent of hemolysis was determined as described above. All assays were run in triplicate,
hibited by SOD and/or catalase (Fee & Teitelbaum, 1972; Brunori ef ul., 1975; Goldberg & Stern, 1975, 1977). Copper deficiency is generally associated with mic-
rocytic anemia, similar to that resulting from iron deficiency. Furthermore, copper-deficient erythrocytes have a decreased SOD activity (Bohnenkamp & Weser. 1976; Williams et al.. 1975) and a shorter lifetime (Lynch ef al., 1972), while in patients with Menkes’ syndrom the H,O,-induced hemolysis is enhanced (French er al., 1972). It is not not clear as yet whether anemia appears as a result of destroyed hemesynthesis and/or of oxidative disruption of hemoglobin and membrane lipids.
Erythrocyte
lysates and enzyme assays
Red cells were lysed in 10 vol of cold 5 mM phosphate buffer, pH 7.4. In this lysate glucose 6-phosphate dehydro321
ELEVTEK M. RVSSANOV and TODORKA A. KASSABOVA
322 Table
1. Hemoglobin
content,
cyte enzymes
hematocrite
from control
and activities
and copper-deficient Control rats
Hemoglobin (g/l00 ml blood) (g/ml packed red cells) Hematocrite (in “,) Superoxide dismutase (U/g hemoglobin) Catalase (AE240/mg hemoglobm) Glutathione peroxidase (pmol NADPH oxid./min/g hemoglobin Glucose 6-phosphate dehydrogenase (pmol NADP reducedjminjg hemoglobin)
of some
erythro-
rats C&deficient rats
12.1 f 0.1 0.23 i: 0.01 50.9 2 0.4
7.9 + 0.2* 0.20 * 0.01** 37.3 + 0.6”
4737 zf: 421
1501 * 139*
2.65 k 0.22
2.99 k 0.85
25.3 I): I.2
29.8 + IS**
13.4 + 0.8
18.6 -r_0.Y
GSH-peroxidase and glucose &phosphate dehydrogenase activities were measured in soluble, hemoglobin-free erythrocyte protein and were recalculated per g hemoglobin. Superoxide dismutase and catalase activities were determined in erythrocyte lysates. One SOD unit is the amount of protein that causes 50”” inhibition of the reaction with Nitro Blue Tetrazolium. Catalase activity was determined by the disappearance of HZO, followed at 240 nm. The data are the means of 8 experiments + SEM. *P c 0.01: **p < 0.05.
genase activity was measured by the method of Cartier er al. (1967) and GSH-peroxidase activity was determined according to Maral et al. (1917) with tert-butyl hydroperoxide as substrate. The spontaneous and ascorbateand Cu”-induced lipid peroxidation were followed by the formation of malonyl dialdehyde (MDA) by the method of Hunter er a. (1963). Molar extinction coefficient, I.56 x lo5 M-i cm-’ was used for calculation. Soluble. hemoglobin-free erythrocyte protein was obtained according to Maral et al. (1977) and was used for determining SOD activity (Beauchamp & Fridovich. 1971) and cataiase activity (Aebi. 1970). The hemoglobin content in both blood and erythrocyte lysate was determined by Merckotest (Cat. No. 3317). Protein was measured by the method of Lowry er 01. (1951).
Glutathione (reduced and oxidized). glutathione reductase. NADP and NADPH were purchased from Boehringer-Mannheim GmbH. tert-butyl hydroperoxide was synthetized and purified in the Institute of Organic Chemistry. Bulgarian Academy of Sciences. All other chemicals were of reagent grade purity obtained from Merck-Schuchardt (Darmstadt) or BDH Chemicals Ltd. Student’s r-test was used for statistical analysis. The means + SEM are presented.
REWLTS Rats fed on a copper-deficient diet for 6-7 weeks had a decreased content of hemoglobin in the blood (by 359, compared with the controls) and in milliliters of packed red cells (by 13”b); the values of hematocrit decreased by 27’?;,(Table 1). Copper deficiency exerted a different effect on the activities of the enzymes studied. SOD. which in erythrocytes is only a Cu, Zncontaining enzyme, sharply decreased (by 70”/,) and catalase did not change. GSH-peroxidase. utilizing
lipid peroxides or H,Ot as substrates, slightly increased and glucose &phosphate dehydrogenase was activated by 404, compared with the control level (Table 1). Copper-deficient erythrocytes were more stable in hypo~smotic medium at low temperature (4’C) and relatively short time of incubation (3 hr) as compared with the control erythrocytes. Fifty percent hemolysis occurred at 54u/, IS for the controls and at 44”:, IS for the copper-deficient erythrocytes (Fig. 1). At room temperature and prolonged incubation (20 hr) in isoosmotic medium the copper-deficient erythrocytes also had a higher stability than the controls (Table 2, without additions). Low copper concentrations (5 and 1Onmol~mg protein) caused hemolysis which was expressed to an equal degree in both control and copper-deficient red cells. The Cu’ ‘.-induced hemolytic effect was abolished by BCS, a Cu+-chelating agent. (50 nmol/mg protein) The higher Cu2+ concentration induced hemoiysis but to a smaller degree which was not affected by BCS (Table 2). Twenty-hour storage of erythrocyte lysates from control rats at room temperature increased the spontaneous formation of MDA and the latter was not changed in the presence of 10nmof. Cu’+/mg protein or 30 nM ascorbate but was inhibited by 50nmol Cu”/mg protein (Fig. 2). In the copper-deficient erythrocytes the spontaneous and ascorbateand 0.1 +-induced formation of MDA was increased and the high Cu” concentration was also effective (Fig. 2).
Anemia caused by copper deficiency has been known since 1928 and it is one of the earliest and the
Lipid peroxidation
IS, % Fig. 1. Hypoosmotic stability of erythrocytes from control (G-O) and copper-deficient (M) rats. 20~1 of erythrocyte suspensions were incubated for 3 hr at 4°C in tubes containing 2 ml increasing concentrations of IS (from 0 to 100%). The tubes were then centrifuged at 5OOOg for IOmin and the optical density of supernatantes were measured at 540 nm. The results are expressed as percentage of samples completely lysed in distilled water. Hemolysis in 100% IS was considered to be OT/,. The data are means of 10 experiments It: SEM. All assays were run in triplicate.
most pronounced symptoms of this state. The disturbed iron transport and the destroyed heme synthesis by copper deficiency (Williams ef al., 1976) cannot fully explain the pathogenesis of this anemia because the activity of catalase, which is also a hemeenzyme, is not decreased in this stage of copper depletion (Bohnenkamp & Weser, 1976). Furthermore, the life-span of erythrocytes and the turnover of hemoglobin are more prolonged, about 120 days, compared with the period during which anemia deTable
2. Copper-induced
in erythrocytes
velops. It has been found that the life-span of copperdeficient erythrocytes in the circulation of both normal and copper-deficient pigs is decreased (Lynch et al., 1972). The cause of the diminished survival is not clarified but in the erythrocytes of copper-deficient pigs the osmotic fragility is increased which suggests an enhanced pliability to oxidative attack. Copper deficiency induces an increase of the membrane permeability in both old and young red cells (Lynch ef al., 1972). The higher hypoosmotic stability of erthrocytes from copper-deficient rats we have found is in accordance with that observed by Bettger et al. (1978) in experiments on rats, too. This phenomenon could partly be due to the lower concentration of the main erythrocyte matrix proteins upon copper deficiency, hemoglobin and SOD, whereby the osmotic pressure in these erthrocytes in hypoosmotic medium is lower compared with normal erythrocytes. The present results show that copper-deficient erythrocytes have an increased spontaneous. and ascorbateand copper-induced peroxidation of the membrane lipids which is evidenced from the MDA formation. Lipid peroxides are metabolized by GSHperoxidase. The latter is functionally connected with GSH-reductase (supplying reduced glutathione) and with glucose 6-phosphate dehydrogenase (supplying NADPH). The higher activities of GSH-peroxidase and glucose 6-phosphate dehydrogenase are probably the response to the increased amounts of hydroperoxides formed in the copper-deficient erythrocytes. In other experiments of ours on copper-deficient rat liver, where lipid hydroperoxide level was twice increased, we found enhanced activity of GSH-reductase in both cytosol and mitochondria and activated mitochondria NADPH-generating system in (Balevska et al., 1981). Similar results were obtained by Nishiki et ul. (1976) on tocopherol-deficient rat liver while Bohnenkamp & Weser (1976) failed to find any changes in GSH-peroxidase activity in copperdeficient erythrocytes. The spontaneous lipid peroxidation of copper-deficient erythrocytes is slightly increased (compared with the ascorbate induced) and thus cannot cause hemo-
hemolysis of erythrocytes per-deficient rats
Additions
5 nmol Cu* + img protein - BCS + BCS (25 nmol/mg protein) 10 nmol Cu’+/mg protein - BCS + BCS (50 nmol/mg protein) 50 nmoles Cu” img protein - BCS + BCS (250 nmol/mg protein)
323
from control
Control rats
and cop-
Cu-deficient rats
39.0 + 4.3
29.5 k 2.2
84.0 + 4.2 37.5 + 2.0
83.1 f 4.9 29.7 f 1.0
88.6 + 1.1 31.5 f 1.4
87.9 + 2.0 28.3 + 2.6
44.3 + 1.8 49.2 + 7.8
37.6 + 2.8 43.8 f 8.2
Incubation conditions: 2 ml IS were placed in plastic centrifugal tubes and indicated amounts of CuS04 and BCS (bathocuproine sulfonate) dissolved in microvolumes IS were added. Erythrocyte suspensions equivalent to 3.5 mg protein were also added. After 20 hr at 20-22’C the tubes were centrifuged at 5C00 g for 10 min. The extent of hemolysis was measured and expressed as shown in the legend to Fig. 1. n = 7.
324
ELEVTER
M. Rcss~uov
and TODORKA A.
KASSAROVA
30 nk ascorbate
IO nmol
Cu/mg
* :
800 50
nmol
Cu/mg
; o
600
E -
400
Z E
200
0
1
2 20 Time
Fig. 2. Ascordate-
and Cu’ * -Induced ’
formation
I
I 2 20 of incubation, of malonyl
2 20
I 2
hr
dialdehyde
in erythrocytes
from control ( in 5 mM phosphate buffer. pH 7.0 and the corresponding additions at 20-22 C. At given time intervals a sample was taken and the amount of MDA formed was measured using the difference in the extinctions at 532 and 600 nm against the corresponding controls. n = 10.
and copper-deficient (Cl) rats. Erythrocyte lysates (IO mg protein~ml)
lysis as it is known that hemolysis begins to bccur when lipid peroxidation approaches 70”” of its maximum value (Fee rt ul., 1975). Cu2’-induced hemolysis being expressed to an equal degree in both control and copper-deficient erythrocytes could hardly be due to lipid peroxidation of the membranes. Probably it results from the Cu*‘-catalized oxidation of hemoglobin (Winterbourn et al.. 1976; Winterbourn & Carrel!. 1977). The hemolyzing effect of Cu2’ is removed by BCS suggesting the role of Cu’ whose reoxidation leads to generation of 0;. 0; in turn causes decomposition of hemoglobin to an unidentified green pigment which leads to hemoiysis regardless of the lipid ~roxidation (Goldberg & Stern, 1977). Kobayashi er lrl. (1979) have found that upon paraquate-induced 100”~ hemolysis where the lytic agent is IO2 there occurs no lipid peroxidation which also suggests that the mechanisms and the time-course of the two processes. hemolysis and lipid peroxidation, do not closely correlate as was stated by Bettger er al. (1978). Kellog & Fridovich (1975) have shown in inritro experiments that ‘02 (and OH 1 formation is mediated by H20z. This finding add the present result led us to believe that in the case of Cu-deficient erythrocytes the normal or even slightly increased activities of enzymes catalyzed decomposition of H,02 and lipid peroxides compensate the after-effects of the decreased SOD activity on the erythrocyte stability.
were incubated
BETTGER W. J.? FISH T. J. & O’DELL R. I_. (1978) Effects of copper and zinc status of rats on erythrocyte stability and superoxide dismutase activity. P,oc. Sot. esp. Biol. Med. 158, 279--282. BOHKENKAMP W. & ,WFSFR U. (1976) Copper defictency and erythrocuprein. ZCu.ZZn-superoxide dismutase. Biochini. hiophj~.s. Ada 444, 396406. BRCNOR~ M.. FALC~ONI G.. FIORETTI E.. GIARDINA B. 81 ROTILIO G. (1975) Formation of superoxide in the autoxidation of the isolated Y and /I chains of human hemoglobin and its involvement in hemichrome precipitation. Eur. J. Biochrn1. 53, 99-- 104. CARTIER P.. LEROUX J-P. & MARCHAND J.CL. (1967) Techniques de dosage des enzymes glycolytiques tissulaires. Atznls Biol. flirt. 25, 109-I 36. FEE J. A, & TEITELBAUM H. D. (1972) Evidence that superoxide dismutase plays a role in protecting red blood cells against peroxidative hemolysis. Biocl~rnl. hiophp. Rr.5. Conlntrrn. 49, 150-158. FEE J. A.. BERCA~~VI R. & BRIGGS R. G. (1975) Observations on the mechanism of the oxygen:dialuric acidinduced hemolysis of vitamin E-deficient rat red blood cells and the protective roles of catalase and superoxide dismutase. Arclts Bioci~crn. Biophx~. 169, 160-167. FRENCH J. H.. SHERARU S. E.. LC’BELL H.. BROTX M. & MOORI C. L. (19721 Trichopoiiodystrophy~ 1. Report of a case and biochemical studies. Arc,hs .%‘(,tirol. 26, 229-244. FRIDO~I~H 1. (1976) Oxygen radicals. hydrogen peroxlde and oxygen toxicity. In free rc&cols it? Biolo!~~. Vol. I (Edited by PRYOR W. A.). pp. 239-277. Academic Press. New York. GOLDBERG B. & STERN A. (1975) The generation of 0; by REFERENCES the interaction of the hemolytic agent. phenylhydrazine. J. hiol. C/wn. 250, 2401.-2403. AEBI H. (1970) Kataiase. fn Mefhc$e!r der ~~~l~~~)iur;s~~l~~~ with human hemoptobm. GOLDBERG B. & STERN A. (1977) The role of superoxide At&!su (Edited by BERCMEYERH. U.). pp. 637-641. Akaanion as a toxic species rn erythrocyte. Arch.\ Biochm. demie Verlag. Berlin. Biophys. 178, 218-225. BALEVSKA P. S.. RUSSANOV E. M. & KASSABOVA T. A. HUNTER E. JR. GEBICK~ J. M.. HOFFSTEI~~~ P. E., WEIMSTE~~ (1981) Studies on lipid peroxidation in rat liver by copJ. & SCOTT A. (1963) Swelling and lysis of rat liver mitoper deficiency. Itzt. J. Biockem. 13, 489-493. chondria induced by ferrous ions. J. hid. Chrm. 238, BEAUCHAMP C. & FR~DOV~CH 1. (1971) Superoxide dismu828- 835. tase: improved assays and an assay applicable to acryl KELLOGG E. & FR~DOVICH 1. (1975) Superoxide. hydrogen amide gels. Anulrt. Biochrm. 270. 276-287.
Lipid peroxidation peroxide and synglet oxygen in lipid peroxidation by a xanthine oxidase system. J. hiol. Chem. 250. 8812-8817. KOBAYASHI Y., OKAHATA S. & USUI T. (1979) Hemolysis of human erythrocytes by paraquat in relation to superoxide dismutase activity. Biochem. hiophps. Res. Commun. 91, 1288-1294. LOWRY 0.. R~~EBROUGH N., FARR A. & RANDALL R. J. (1951) Protein measurement with the Folin phenol reagent. J. hiol. Chem. 193, 265-278. LYNCH R. E.. LEE G. R. & CARTWRIGHT G. E. (1972) Copper and the red cell membrane. Blood 40, 9633971. LYNCH R. E. & FRIWVICH I. (1978) Effects of superoxide on the erythrocyte membrane. 1. hiol. Chem. 253. 1838-1845. MARAL J., PUGET K. & MICHELSON A. M. (1977) Comparative study of superoxide dismutase. catalase and glutathione peroxidase levels in erythrocytes of different animals. Biochem. biophys. Res. Commun. 77, 152551535.
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325
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WINTERBOURN C. C. & CARRELL R. W. (1977) Oxidation of human hemoglobin by copper. (Mechanism and suggested role of the thiol group of residue b-93). Biochem. J. 165, 141-148.