J Mol Cell Cardiol22,935-938
(1990)
RAPID
Glutatbione Transferase
Peroxidase, Activities
COMMUNI
CATION
Glutathione Reductase in the Human Artery,
amd Glutathione Vein and Heart
(Received 6 December 1989, and accepted 17 April 1990)
The contiunous exposure to blood components, including prooxidants, makes the blood vessel wall susceptible to oaidative stress and ikee radical mediated reactions (Henning and Chow, 1988; Stamm et al., 1989; Halliwell and Gutteridge, 1984). Free radicals can be produced eatracellularly via the respiratory bursts of activated neutrophils, or intracellulariy, via oxidation of hypoxanthine by xanthine osidase (Henning and Chow, 1988; Stamm et al., 1989, Rubanyi, 19gg). Microsomial epspaes such as lipoaygenase and cyclooxygenase may also be a source of reactive species of oxygen (Hendng and Chow, l!M& Stamm et al., 1989; Rubanyi, 1988; Mason et al., 19S9). It has been proposed that free radicals are involved in the initiation and progression of various cardiovascular diseases including arteriosclerosis (Henning and chow, 1988; stamm et al., 1989; Yagi, 19g& Jiirgens et al., 1987). Thus the adequacy of the defence systems against free radicals is critical for the susceptibility of blood vessel wall to oxidative damage. Among the enzymatic systems capable of protecting the cell against oxidative iujury, selenium dependent glutathione peroxidase (Se-GSH-pa), glutathione reductase (GSSG-ra) and glutathione transferase (GST) play a crucial role (Flohe’ et al., 197%Mannervik and Danielson, 1WS). Using glutathione (GSH) as cofirctor, Se-GSHpx reduces Hz02 to water and organic hydroperoxides to the corresponding alcohols (Flohe’ et al., 1976). This reaction leads to conversion of GSH into its oxidized form (GSSG). In the presence of NADPH, GSSG-rx is able to reduce the oaidised glutathione. GST consists of a family of enzymes that contributes to the cellular detoxiiication by catalyzing GSH conjugation to electrophilic substrates, including carbonyl compounds like 4hydrosyalkenals, which are toxic products of lipid peroxidation (Mannervik and Danielson, 19@ Danielson et al., 1981). Since there is not much information about the mechanisms devoted to protect the human blood vessels against free radical aggression, we have characterized the SeGSH-pa, GSSG-rx and the GST activities of the internal m =-=Y--Y~ saphenous vein obtained from patients undergoing coronary revascnkisation. For comparison, we also studied the activities of the GSH-dependent enzymes of the human heart. Our data show that signi6cantIy Werent levels of Se-GSH-px, GSSGrx and GST are present in the human artery, vein and heart thus suggesting a diiferent antioxidative capacity of the three tissues to counteract the osidative stress.
Please address all correspondence Valignani, 6610-Chieti, Italy 0022-2828/90/090935
+ 04 S03.00/0
to: Andrea
Mezzetti,
1st. di Patalogia
Medica,
c/o Osp. SS. Annunziata, 0
1990 Academic
via PA.
Press Limited
936
A. Mezzetti
Samplesof internal mammary artery (IMA), saphenous vein (SV) and right atria1 myocardium (RA), were obtained intraoperativeIy from 23 male patients undergoing coronary by-passsurgery. The mean (+ S.D.) age of the patients was 54 + 8.7 years (range 39 to 68). Informed written consent was obtained from each patient before surgery, for coronary revascularization and researchpurposes. The extra length of artery and vein not used for coronary grafting was used in this study. The sampleswere immediately transferred to isotonic saline solution, washed thoroughly and stored at -70°C until used. No loss of enzymatic activity was noted after storage for at least 2 months. Each sample was weighed, minced and homogenized with two 20-sbursts of a Ultraturrax homogenizer, allowing 30-s restsbetween bursts in 4 vol of 10 mM phosphate buffer, pH 7, containing 1 mM EDTA and 1 mM dithiotreitol. The homogenate was centrifuged at 50000 x g for 60 min. and the resulting supernatant immediately used for determination of enzymatic activities. SeGSH-px activities were measured according to the method of Paglia and Valentine ( 1967). The final concentration of peroxides was 0.25 mMfor HzOs. The assaysolution contained 50 mM potassiumphosphate buffer, pH 7.0, 1 mM EDTA, 1.5 mM NaNs, 1 mM GSH, 0.16 mM NADPH, 4 pg of glutathione reductase and a suitable sample of enzyme solution. After 5 min of preincubation the reaction was started with the addition of peroxide. The value for a blank reaction with the enzyme source replaced by water wassubtracted for each assay. The rate of reaction was recorded at 25°C by following the decrease in absorbance at 340 nm. Specific activity was expressedas nanomoles of GSH oxidized per minute per mg of protein, GST activity was measured by the method of Habig et al. (1974). The assay mixture contained 0.1 M potassium phosphate buffer pH 6.5, 1 mM EDTA, 2 mM GSH, 1 mM 1-chloro-2,4-dinitrobenzene (CDNB) and a suitable amount of enzyme source. The reference cuvette contained the complete assay mixture with the enzyme replaced by water. Enzymatic assay was carried out at 25°C. Specific activity was expressedas nanomolesof GSH conjugated per minute per mg of protein, GSSG-rx activity was determined as described previously (Di Ilio et al., 1983).
et al.
The assaymixture contained 0.1 M potassium phosphate buffer, pH 7.4, 1 mM EDTA, 1 mM GSSG, 0.16 mM NADPH and an appropriate amount of enzyme source. The blank did not contain GSSG. Enzyme activity was determined at 25°C by measuring the disappearance of NADPH at 340 nm and expressed as nanomolesof NADPH oxidized/min/mg of protein. All measurements,in a final volume of 1 ml, were performed in duplicate and at least two different protein concentrations. The method of Bradford ( 1976) was employed for the determinations of protein concentrations. Gamma-globulin was used as protein standard. The results are expressed as mean f S.E.M. The comparisons between the three tissueswere perfomed using the one-way analysis of variance (ANOVA) followed by the Bonferroni t test (Wallenstein et al., 1980). Values of P < 0.05 were considered significant. The activity values of Se-GSH-px, GST and GSSG-rx assessed in IMAs, SVs and RA are reported in Table 1. The present study shows,apparently for the first time, that human blood vesselwall contains detectable levels of Se-GSH-px, GST and GSSG-rx activities. Moreover, these enzymatic activities are significantly lower than those present in the right human atrium. We also found the existence of statistically significant differencesin the activities of Se-GSH-px and GST between veins and arteries. These data suggestthat the three tissuesmay have a different ability to counteract the oxidative stress. Se-GSH-px activity was significantly higher in IMAs than in SVs (12.0 & 0.50 nmol/min/mg vs 8.7 + 0.62 nmol/min/mg, P < 0.05). Since Se-GSH-px has been shown to be crucial in stabilizing the polyunsaturated membrane lipids (Christophersen, 1969; Flohe’ et al., 1976)) this difference may suggest that human arteries are more subjected than veins to lipid peroxidation, probably because of constant exposure to higher oxygen tension. Schuman et al. (1988) recently demonstrated that a jugular vein grafted into the carotid artery of rabbits showed lessintimal hyperplasia if pretreated with deferoxamine, an oxygen free radical scavenger. The authors concluded that veins could have a lower free radical scavenger capacity than arteries, and
Glutathione
Depeudeat Enzyme
TABLE 1. Glutathione-dependent internal mammary artery (IMA), right atria1 myocardium (RA) Enzyme Se-GSH-px GST GSSG-rx
IMA(23) 12.0 + 0.50 18.8 + 1.43 2.9 + 0.32
Activities
937
enzyme activities in the saphenous vein (SV), and
SV(23) 8.7 &- 0.62* 33.8 f 2.04* 3.8 f 0.34
RA(23) 21.8 f 0.83§ 45.7 f 3.41g 17.1 If: 1.1%
Se-GSH-px activity is expressed as nmol of GSH oxidized per min. per mg.; GST activity is expressed as nmol of GSH conjugated with CDNB per min. per mg.; GSSG-rx activity is expressed as nmol of NADPH oxidized per min. per mg.; IMA = internal mammary artery; SV = saphenous vein; RA = right atrium. The numberof samplesinvestigatedare givenin brackets.Data areexpressed asmean+ s.E.M.; SV vs IMA: *P
(1990)
RAPID
Glutatbione Transferase
Peroxidase, Activities
COMMUNI
CATION
Glutathione Reductase in the Human Artery,
amd Glutathione Vein and Heart
(Received 6 December 1989, and accepted 17 April 1990)
The contiunous exposure to blood components, including prooxidants, makes the blood vessel wall susceptible to oaidative stress and ikee radical mediated reactions (Henning and Chow, 1988; Stamm et al., 1989; Halliwell and Gutteridge, 1984). Free radicals can be produced eatracellularly via the respiratory bursts of activated neutrophils, or intracellulariy, via oxidation of hypoxanthine by xanthine osidase (Henning and Chow, 1988; Stamm et al., 1989, Rubanyi, 19gg). Microsomial epspaes such as lipoaygenase and cyclooxygenase may also be a source of reactive species of oxygen (Hendng and Chow, l!M& Stamm et al., 1989; Rubanyi, 1988; Mason et al., 19S9). It has been proposed that free radicals are involved in the initiation and progression of various cardiovascular diseases including arteriosclerosis (Henning and chow, 1988; stamm et al., 1989; Yagi, 19g& Jiirgens et al., 1987). Thus the adequacy of the defence systems against free radicals is critical for the susceptibility of blood vessel wall to oxidative damage. Among the enzymatic systems capable of protecting the cell against oxidative iujury, selenium dependent glutathione peroxidase (Se-GSH-pa), glutathione reductase (GSSG-ra) and glutathione transferase (GST) play a crucial role (Flohe’ et al., 197%Mannervik and Danielson, 1WS). Using glutathione (GSH) as cofirctor, Se-GSHpx reduces Hz02 to water and organic hydroperoxides to the corresponding alcohols (Flohe’ et al., 1976). This reaction leads to conversion of GSH into its oxidized form (GSSG). In the presence of NADPH, GSSG-rx is able to reduce the oaidised glutathione. GST consists of a family of enzymes that contributes to the cellular detoxiiication by catalyzing GSH conjugation to electrophilic substrates, including carbonyl compounds like 4hydrosyalkenals, which are toxic products of lipid peroxidation (Mannervik and Danielson, 19@ Danielson et al., 1981). Since there is not much information about the mechanisms devoted to protect the human blood vessels against free radical aggression, we have characterized the SeGSH-pa, GSSG-rx and the GST activities of the internal m =-=Y--Y~ saphenous vein obtained from patients undergoing coronary revascnkisation. For comparison, we also studied the activities of the GSH-dependent enzymes of the human heart. Our data show that signi6cantIy Werent levels of Se-GSH-px, GSSGrx and GST are present in the human artery, vein and heart thus suggesting a diiferent antioxidative capacity of the three tissues to counteract the osidative stress.
Please address all correspondence Valignani, 6610-Chieti, Italy 0022-2828/90/090935
+ 04 S03.00/0
to: Andrea
Mezzetti,
1st. di Patalogia
Medica,
c/o Osp. SS. Annunziata, 0
1990 Academic
via PA.
Press Limited
936
A. Mezzetti
Samplesof internal mammary artery (IMA), saphenous vein (SV) and right atria1 myocardium (RA), were obtained intraoperativeIy from 23 male patients undergoing coronary by-passsurgery. The mean (+ S.D.) age of the patients was 54 + 8.7 years (range 39 to 68). Informed written consent was obtained from each patient before surgery, for coronary revascularization and researchpurposes. The extra length of artery and vein not used for coronary grafting was used in this study. The sampleswere immediately transferred to isotonic saline solution, washed thoroughly and stored at -70°C until used. No loss of enzymatic activity was noted after storage for at least 2 months. Each sample was weighed, minced and homogenized with two 20-sbursts of a Ultraturrax homogenizer, allowing 30-s restsbetween bursts in 4 vol of 10 mM phosphate buffer, pH 7, containing 1 mM EDTA and 1 mM dithiotreitol. The homogenate was centrifuged at 50000 x g for 60 min. and the resulting supernatant immediately used for determination of enzymatic activities. SeGSH-px activities were measured according to the method of Paglia and Valentine ( 1967). The final concentration of peroxides was 0.25 mMfor HzOs. The assaysolution contained 50 mM potassiumphosphate buffer, pH 7.0, 1 mM EDTA, 1.5 mM NaNs, 1 mM GSH, 0.16 mM NADPH, 4 pg of glutathione reductase and a suitable sample of enzyme solution. After 5 min of preincubation the reaction was started with the addition of peroxide. The value for a blank reaction with the enzyme source replaced by water wassubtracted for each assay. The rate of reaction was recorded at 25°C by following the decrease in absorbance at 340 nm. Specific activity was expressedas nanomoles of GSH oxidized per minute per mg of protein, GST activity was measured by the method of Habig et al. (1974). The assay mixture contained 0.1 M potassium phosphate buffer pH 6.5, 1 mM EDTA, 2 mM GSH, 1 mM 1-chloro-2,4-dinitrobenzene (CDNB) and a suitable amount of enzyme source. The reference cuvette contained the complete assay mixture with the enzyme replaced by water. Enzymatic assay was carried out at 25°C. Specific activity was expressedas nanomolesof GSH conjugated per minute per mg of protein, GSSG-rx activity was determined as described previously (Di Ilio et al., 1983).
et al.
The assaymixture contained 0.1 M potassium phosphate buffer, pH 7.4, 1 mM EDTA, 1 mM GSSG, 0.16 mM NADPH and an appropriate amount of enzyme source. The blank did not contain GSSG. Enzyme activity was determined at 25°C by measuring the disappearance of NADPH at 340 nm and expressed as nanomolesof NADPH oxidized/min/mg of protein. All measurements,in a final volume of 1 ml, were performed in duplicate and at least two different protein concentrations. The method of Bradford ( 1976) was employed for the determinations of protein concentrations. Gamma-globulin was used as protein standard. The results are expressed as mean f S.E.M. The comparisons between the three tissueswere perfomed using the one-way analysis of variance (ANOVA) followed by the Bonferroni t test (Wallenstein et al., 1980). Values of P < 0.05 were considered significant. The activity values of Se-GSH-px, GST and GSSG-rx assessed in IMAs, SVs and RA are reported in Table 1. The present study shows,apparently for the first time, that human blood vesselwall contains detectable levels of Se-GSH-px, GST and GSSG-rx activities. Moreover, these enzymatic activities are significantly lower than those present in the right human atrium. We also found the existence of statistically significant differencesin the activities of Se-GSH-px and GST between veins and arteries. These data suggestthat the three tissuesmay have a different ability to counteract the oxidative stress. Se-GSH-px activity was significantly higher in IMAs than in SVs (12.0 & 0.50 nmol/min/mg vs 8.7 + 0.62 nmol/min/mg, P < 0.05). Since Se-GSH-px has been shown to be crucial in stabilizing the polyunsaturated membrane lipids (Christophersen, 1969; Flohe’ et al., 1976)) this difference may suggest that human arteries are more subjected than veins to lipid peroxidation, probably because of constant exposure to higher oxygen tension. Schuman et al. (1988) recently demonstrated that a jugular vein grafted into the carotid artery of rabbits showed lessintimal hyperplasia if pretreated with deferoxamine, an oxygen free radical scavenger. The authors concluded that veins could have a lower free radical scavenger capacity than arteries, and
Glutathione
Depeudeat Enzyme
TABLE 1. Glutathione-dependent internal mammary artery (IMA), right atria1 myocardium (RA) Enzyme Se-GSH-px GST GSSG-rx
IMA(23) 12.0 + 0.50 18.8 + 1.43 2.9 + 0.32
Activities
937
enzyme activities in the saphenous vein (SV), and
SV(23) 8.7 &- 0.62* 33.8 f 2.04* 3.8 f 0.34
RA(23) 21.8 f 0.83§ 45.7 f 3.41g 17.1 If: 1.1%
Se-GSH-px activity is expressed as nmol of GSH oxidized per min. per mg.; GST activity is expressed as nmol of GSH conjugated with CDNB per min. per mg.; GSSG-rx activity is expressed as nmol of NADPH oxidized per min. per mg.; IMA = internal mammary artery; SV = saphenous vein; RA = right atrium. The numberof samplesinvestigatedare givenin brackets.Data areexpressed asmean+ s.E.M.; SV vs IMA: *P
