Biochimica et Biophysica Acta, 1036(1990) 88 94
88
Elsevier BBAGEN 23390
Phospholipid hydroperoxide glutathione peroxidase in various mouse organs during selenium deficiency and repletion Frank Weitzel 1, Fulvio Ursini 2 and Albrecht Wendel 1 I Biochemical Pharmacology, Faculty of Biology, University of Konstanz, Konstanz (F.R. G) and -"Department of Biological Chemistry, University of Padova, Padova (Italy)
(Received 2 April 1990)
Key words: Seleniumdeficiency;Phospholipidhydroperoxideglutathione peroxidase; (Mouse)
An assay for the determination of the newly discovered selenoenzyme, phospholipid hydroperoxide glutathione peroxidase (PH-GPx) in biological material is described. Dietary selenium deficiency and repletion was used as a tool in order to modify this enzyme activity in various mouse organs and to compare it to the activity of the 'classical' selenium-dependent glutathione peroxidase (GPx) (EC 1.11.1.9). A semipurified diet containing less than 12 ppb Se was used for depletion. Controls received this diet supplemented with 500 ppb Se in the form of Na2SeO 3. The results showed that a rapid loss of GPx activity occurred in liver, kidney and lungs of selenium-deficient mice which reached undetectable levels within 130 days. In the heart, about 24% of control GPx activity was still present. In contrast, PH-GPx activity was more slowly depleted by Se deficiency and resulted in residual activities ranging from 30 to 70% in the different organs even after 250 days of depletion. In repletion experiments with a single application of I0 or 500 /Lg/kg Se, only the high dose restored either enzyme activity. The data demonstrate that the need for selenium of the two glutathione peroxidases is different. A markedly distinct organ distribution of both enzymes suggests that the heart may be the organ most sensitive to oxidative stress.
Introduction Since the discovery of selenium (Se) as an essential micro nutritient, the molecular role of the element in the mammalian organism has attracted much interest. One of the striking aspects of Se deficiency is the extraordinarily wide variety of organs which are affected in different species by this abnormal nutritional state [1]. Moreover, a discrete organotropy exists, e.g., the pancreatic degeneration and edema in poultry, the skeletal muscle dystrophy of cattle, cardiac muscle degeneration in pigs, and geochemically determined cardiomyopathy in adolescent man [2]. Se deficiency causes general symptoms in mammals such as growth retardation, infertility, loss of hair and nails. It is hard to conceive that only few discrete metabolic functions
Abbreviations: GSH, glutathione; GPx, glutathione peroxidase, EC 1.11.1.9; PH-GPx, phospholipid hydroperoxide glutathione peroxidase; PC-OOH, phosphatidylcholinehydroperoxide;CDNB, chlorodinitrobenzene; Se-, selenium deficient; Se+, selenium supplemented; ppb, parts per billion; EDs0, effectivedose, median. Correspondence: A. Wendel, Biochemical Pharmacology, University of Konstanz, POB 5560, D-7750 Konstanz, F.R.G.
of selenium account for this wide variety of pathophysiological effects. The first selenoenzyme recognized, glutathione peroxidase, is a tetramer containing 4 gatom Se and plays a major role in the cell's antioxidant defense [3]. It utilizes its highly hydrophilic electron donor substrate glutathione (GSH) in order to reduce chemically very different hydroperoxides to alcohols. Among them, fatty acid hydroperoxides appear to be biologically most relevant. Unless reduced to hydroxy fatty acids, they may lead to uncontrolled radical chain reactions that are deleterious for the integrity of membranes. Recently, two further selenium-containing glutathione peroxidases (GPx) were described. A protein with different properties has been detected in the extracellular space and may represent an enzyme species with complementary localization and similar properties to the intracellular enzyme [4]. The third GPx described differs from the other two by its substrate specifity, as well as by its localization. It acts preferentially upon esterified membrane phospholipid hydroperoxides [5]. This enzyme is an interfacial monomeric protein with 1 gatom Se [6] and seems to be an individual enzyme species. Another novel GPx with similar properties to PH-GPx has been recently purified to homogeneity [7].
0304-4165/90/$03.50 © 1990 Elsevier SciencePublishers B.V. (BiomedicalDivision)
89 However, it is not clear whether this enzyme contains Se. The complementary subcellular localization of the known peroxidases might provide a rationale for the perfect antioxidant defense system of mammalian cells. The importance of the individual enzyme species, however, requires quantitative knowledge on their activities in different organs during different phases of dietary Se supply. Recently, manifold enzyme modulations in mouse liver were observed in severe Se deficiency [8-10] which did not seem directly related to the lack of GPx. Subsequent work showed that a general increase of protein turnover or protein processing in the Golgi stack might explain this phenomenon [11,12]. The restorage of these various metabolic disturbances needed a low single dose of Se (EDs0 = 8/~g/kg) whereas GPx required a much higher one of 250/~g/kg. Until now, as many as 13 different selenoproteins were identified in different organs of the rat [13]. Studies on their replenishment by Se showed that the organs primarily affected in their function by Se deficiency are supplied as the last ones with the element. On the other hand, tissues such as testes and other endocrine organs contain very high amounts of Se [14] and take up most Se upon repletion. However, only little is incorporated into GPx. These findings indicate that the distribution of Se is subject to an efficient control presumably by reproductive and endocrine organs. These results suggest also that various selenoproteins may be differently supplied with Se according to their importance for the different organs. These possible relations are unknown for the couple GPx and PH-GPx. The aim of the present study was to demonstrate the dependence of the novel PH-GPx on dietary selenium during depletion and repletion of the element. We were interested in estimating whether the two peroxidases are subject to a common Se need during the two experimental nutrition phases and whether any indication for their functional importance could be derived from these findings. In particular those organs of the mouse were examined which are candidates for failure induced by oxidative stress.
the animals were killed by cervical dislocation. The abdominal cavity was opened, and the circulatory system immediately perfused via the right cardiac ventricle with ice-cold isotonic saline. The organs were removed and homogenized in four parts (w/w) 0.1 mol/1 Tris, 0.3 mol/1 KC1 buffer (pH 7.6) containing 0.1% peroxide-free Triton X-100 (Boehringer, Mannheim) to yield a 20% homogenate. The homogenates of liver, heart, lung and kidney were centrifuged for 10 min at 10000 x g. Aliquots of the supernatant were immediately frozen at - 8 0 ° C and stored no longer than 3 weeks until analysis. For the repletion experiments, the animals received a single intraperitoneal injection of Na2SeO 3 (10 or 500 /~g/kg) in 0.9% NaC1. Controls received the same volume of saline. After 114 h, the organs were removed, homogenized as described and assayed for enzyme activities.
Synthesis of the substrate (PC-OOH) for the PH-GPx
Materials and Methods
Basically, the substrate for PH-GPx was synthesized as described previously [6]. Minor modifications were as follows. A 50 mmol/1 solution of phosphatidyl choline in 50 mmol/1 sodium desoxycholate was prepared. 6.3 mg was transfered into 14.5 ml of a 0.2 mol/l sodium borate buffer (pH 9.0) and 2.3 ml 3% desoxycholate (Fluka AG, Buchs) were added. This solution was thermostated to 15°C and oxygenated with 99.999% 02 under continuous stirring. In order to overcome the self-inactivation of the enzyme, 200/~1 of soybean lipoxygenase type IV (Sigma, specific activity 440 k U / m g protein) corresponding to 730 kU were added every 20 min in eight portions of 25 /~1. After 160 rain the aqueous solution was passed over a Sep-Pak C18 cartridge (Waters Ass., Milford, MA) previously washed with methanol (Merck, Darmstadt, pro analysis grade) and equilibrated with water. The cartridge was washed with a 10-fold vol. of water, and then eluted with 1.5 ml methanol. Aliquots of this eluate were used for the assay of PH-GPx. The phosphatidylcholine hydroperoxide (PC-OOH) was analysed as previously described [16] or, alternatively subjected to FAB/MS analysis on a Finnigan MAT 312 mass-spectrometer, modified with an AMD high energy LSIMS-source.
Animals
Biochemical analysis
Female NMRI mice from Thomae, Biberach, F.R.G. were fed, from weanling, a self-produced, extremely low-Se diet prepared according to Ref. 15, which contained less than 12 ppb selenium. Controls received an identical diet supplemented with 500 ppb Se in the form of Na2SeO 3. The animals were maintained in Macrolon cages on stainless steel grids at 55% humidity and 27 °C with an artificial 12 h day/night rhythm. At the times indicated,
PH-GPx was assayed as follows. 10 txl of biological sample were added to a total volume of 1.0 ml containing 0.1 mol/1 Tris buffer (pH 7.6), 5 mmol/1 EDTA, 1 mmol/1 azide, 3 mmol/1 GSH (Sigma), 0.1% peroxidefree Triton X-100 (Boehringer-Mannheim), 0.1 mmol/1 NADPH (Boehringer-Mannheim) and 1.2 U glutathione reductase (Boehringer-Mannheim, specific activity 120 U / m g protein). The reaction was started at 37 °C by the addition of 10/~1 phospholipid hydroperoxide (PC-
90 PC-OOH
O O H ) in m e t h a n o l (final c o n c e n t r a t i o n 57/~mol/1) a n d the change in a b s o r p t i o n at 340 n m was r e c o r d e d ( K o n t r o n U v i k o n 930 s p e c t r o p h o t o m e t e r ) . G P x was assayed with H 2 0 2 (Merck, D a r m s t a d t ) as d e s c r i b e d p r e v i o u s l y [17]. G l u t a t h i o n e S - t r a n s f e r a s e activity was m e a s u r e d with 1-chloro-2,4-dinitrobenzene ( C D N B , F l u k a A G , Buchs) as d e s c r i b e d in Ref. 18. P r o t e i n was m e a s u r e d a c c o r d i n g to L o w r y ' s procedure.
PC -OOH
"-
88
Elsevier BBAGEN 23390
Phospholipid hydroperoxide glutathione peroxidase in various mouse organs during selenium deficiency and repletion Frank Weitzel 1, Fulvio Ursini 2 and Albrecht Wendel 1 I Biochemical Pharmacology, Faculty of Biology, University of Konstanz, Konstanz (F.R. G) and -"Department of Biological Chemistry, University of Padova, Padova (Italy)
(Received 2 April 1990)
Key words: Seleniumdeficiency;Phospholipidhydroperoxideglutathione peroxidase; (Mouse)
An assay for the determination of the newly discovered selenoenzyme, phospholipid hydroperoxide glutathione peroxidase (PH-GPx) in biological material is described. Dietary selenium deficiency and repletion was used as a tool in order to modify this enzyme activity in various mouse organs and to compare it to the activity of the 'classical' selenium-dependent glutathione peroxidase (GPx) (EC 1.11.1.9). A semipurified diet containing less than 12 ppb Se was used for depletion. Controls received this diet supplemented with 500 ppb Se in the form of Na2SeO 3. The results showed that a rapid loss of GPx activity occurred in liver, kidney and lungs of selenium-deficient mice which reached undetectable levels within 130 days. In the heart, about 24% of control GPx activity was still present. In contrast, PH-GPx activity was more slowly depleted by Se deficiency and resulted in residual activities ranging from 30 to 70% in the different organs even after 250 days of depletion. In repletion experiments with a single application of I0 or 500 /Lg/kg Se, only the high dose restored either enzyme activity. The data demonstrate that the need for selenium of the two glutathione peroxidases is different. A markedly distinct organ distribution of both enzymes suggests that the heart may be the organ most sensitive to oxidative stress.
Introduction Since the discovery of selenium (Se) as an essential micro nutritient, the molecular role of the element in the mammalian organism has attracted much interest. One of the striking aspects of Se deficiency is the extraordinarily wide variety of organs which are affected in different species by this abnormal nutritional state [1]. Moreover, a discrete organotropy exists, e.g., the pancreatic degeneration and edema in poultry, the skeletal muscle dystrophy of cattle, cardiac muscle degeneration in pigs, and geochemically determined cardiomyopathy in adolescent man [2]. Se deficiency causes general symptoms in mammals such as growth retardation, infertility, loss of hair and nails. It is hard to conceive that only few discrete metabolic functions
Abbreviations: GSH, glutathione; GPx, glutathione peroxidase, EC 1.11.1.9; PH-GPx, phospholipid hydroperoxide glutathione peroxidase; PC-OOH, phosphatidylcholinehydroperoxide;CDNB, chlorodinitrobenzene; Se-, selenium deficient; Se+, selenium supplemented; ppb, parts per billion; EDs0, effectivedose, median. Correspondence: A. Wendel, Biochemical Pharmacology, University of Konstanz, POB 5560, D-7750 Konstanz, F.R.G.
of selenium account for this wide variety of pathophysiological effects. The first selenoenzyme recognized, glutathione peroxidase, is a tetramer containing 4 gatom Se and plays a major role in the cell's antioxidant defense [3]. It utilizes its highly hydrophilic electron donor substrate glutathione (GSH) in order to reduce chemically very different hydroperoxides to alcohols. Among them, fatty acid hydroperoxides appear to be biologically most relevant. Unless reduced to hydroxy fatty acids, they may lead to uncontrolled radical chain reactions that are deleterious for the integrity of membranes. Recently, two further selenium-containing glutathione peroxidases (GPx) were described. A protein with different properties has been detected in the extracellular space and may represent an enzyme species with complementary localization and similar properties to the intracellular enzyme [4]. The third GPx described differs from the other two by its substrate specifity, as well as by its localization. It acts preferentially upon esterified membrane phospholipid hydroperoxides [5]. This enzyme is an interfacial monomeric protein with 1 gatom Se [6] and seems to be an individual enzyme species. Another novel GPx with similar properties to PH-GPx has been recently purified to homogeneity [7].
0304-4165/90/$03.50 © 1990 Elsevier SciencePublishers B.V. (BiomedicalDivision)
89 However, it is not clear whether this enzyme contains Se. The complementary subcellular localization of the known peroxidases might provide a rationale for the perfect antioxidant defense system of mammalian cells. The importance of the individual enzyme species, however, requires quantitative knowledge on their activities in different organs during different phases of dietary Se supply. Recently, manifold enzyme modulations in mouse liver were observed in severe Se deficiency [8-10] which did not seem directly related to the lack of GPx. Subsequent work showed that a general increase of protein turnover or protein processing in the Golgi stack might explain this phenomenon [11,12]. The restorage of these various metabolic disturbances needed a low single dose of Se (EDs0 = 8/~g/kg) whereas GPx required a much higher one of 250/~g/kg. Until now, as many as 13 different selenoproteins were identified in different organs of the rat [13]. Studies on their replenishment by Se showed that the organs primarily affected in their function by Se deficiency are supplied as the last ones with the element. On the other hand, tissues such as testes and other endocrine organs contain very high amounts of Se [14] and take up most Se upon repletion. However, only little is incorporated into GPx. These findings indicate that the distribution of Se is subject to an efficient control presumably by reproductive and endocrine organs. These results suggest also that various selenoproteins may be differently supplied with Se according to their importance for the different organs. These possible relations are unknown for the couple GPx and PH-GPx. The aim of the present study was to demonstrate the dependence of the novel PH-GPx on dietary selenium during depletion and repletion of the element. We were interested in estimating whether the two peroxidases are subject to a common Se need during the two experimental nutrition phases and whether any indication for their functional importance could be derived from these findings. In particular those organs of the mouse were examined which are candidates for failure induced by oxidative stress.
the animals were killed by cervical dislocation. The abdominal cavity was opened, and the circulatory system immediately perfused via the right cardiac ventricle with ice-cold isotonic saline. The organs were removed and homogenized in four parts (w/w) 0.1 mol/1 Tris, 0.3 mol/1 KC1 buffer (pH 7.6) containing 0.1% peroxide-free Triton X-100 (Boehringer, Mannheim) to yield a 20% homogenate. The homogenates of liver, heart, lung and kidney were centrifuged for 10 min at 10000 x g. Aliquots of the supernatant were immediately frozen at - 8 0 ° C and stored no longer than 3 weeks until analysis. For the repletion experiments, the animals received a single intraperitoneal injection of Na2SeO 3 (10 or 500 /~g/kg) in 0.9% NaC1. Controls received the same volume of saline. After 114 h, the organs were removed, homogenized as described and assayed for enzyme activities.
Synthesis of the substrate (PC-OOH) for the PH-GPx
Materials and Methods
Basically, the substrate for PH-GPx was synthesized as described previously [6]. Minor modifications were as follows. A 50 mmol/1 solution of phosphatidyl choline in 50 mmol/1 sodium desoxycholate was prepared. 6.3 mg was transfered into 14.5 ml of a 0.2 mol/l sodium borate buffer (pH 9.0) and 2.3 ml 3% desoxycholate (Fluka AG, Buchs) were added. This solution was thermostated to 15°C and oxygenated with 99.999% 02 under continuous stirring. In order to overcome the self-inactivation of the enzyme, 200/~1 of soybean lipoxygenase type IV (Sigma, specific activity 440 k U / m g protein) corresponding to 730 kU were added every 20 min in eight portions of 25 /~1. After 160 rain the aqueous solution was passed over a Sep-Pak C18 cartridge (Waters Ass., Milford, MA) previously washed with methanol (Merck, Darmstadt, pro analysis grade) and equilibrated with water. The cartridge was washed with a 10-fold vol. of water, and then eluted with 1.5 ml methanol. Aliquots of this eluate were used for the assay of PH-GPx. The phosphatidylcholine hydroperoxide (PC-OOH) was analysed as previously described [16] or, alternatively subjected to FAB/MS analysis on a Finnigan MAT 312 mass-spectrometer, modified with an AMD high energy LSIMS-source.
Animals
Biochemical analysis
Female NMRI mice from Thomae, Biberach, F.R.G. were fed, from weanling, a self-produced, extremely low-Se diet prepared according to Ref. 15, which contained less than 12 ppb selenium. Controls received an identical diet supplemented with 500 ppb Se in the form of Na2SeO 3. The animals were maintained in Macrolon cages on stainless steel grids at 55% humidity and 27 °C with an artificial 12 h day/night rhythm. At the times indicated,
PH-GPx was assayed as follows. 10 txl of biological sample were added to a total volume of 1.0 ml containing 0.1 mol/1 Tris buffer (pH 7.6), 5 mmol/1 EDTA, 1 mmol/1 azide, 3 mmol/1 GSH (Sigma), 0.1% peroxidefree Triton X-100 (Boehringer-Mannheim), 0.1 mmol/1 NADPH (Boehringer-Mannheim) and 1.2 U glutathione reductase (Boehringer-Mannheim, specific activity 120 U / m g protein). The reaction was started at 37 °C by the addition of 10/~1 phospholipid hydroperoxide (PC-
90 PC-OOH
O O H ) in m e t h a n o l (final c o n c e n t r a t i o n 57/~mol/1) a n d the change in a b s o r p t i o n at 340 n m was r e c o r d e d ( K o n t r o n U v i k o n 930 s p e c t r o p h o t o m e t e r ) . G P x was assayed with H 2 0 2 (Merck, D a r m s t a d t ) as d e s c r i b e d p r e v i o u s l y [17]. G l u t a t h i o n e S - t r a n s f e r a s e activity was m e a s u r e d with 1-chloro-2,4-dinitrobenzene ( C D N B , F l u k a A G , Buchs) as d e s c r i b e d in Ref. 18. P r o t e i n was m e a s u r e d a c c o r d i n g to L o w r y ' s procedure.
PC -OOH
"-
