Individual Differences in Activity of Glutathione Peroxidase and Catalase Studied in Monozygotic Twins Discordant for
Smoking Lars Björkman, 1,2 1 Magnus Svartengren Department 1
of Environmental
&
Monica
1 Nordberg
Department of Occupational Hygiene, Karolinska Institutet & 2
Health, Karolinska Hospital, S-104 01 Stockholm, Sweden 1 Cigarette smoke contains free radicals. The enzymes glutathione peroxidase (GSH-px) and catalase are important parts of the anti-oxidative protecting system. 2 Ten pairs of monozygotic twins, who were discordant for smoking, were analysed in order to determine their erythrocyte glutathione peroxidase and catalase activities and their plasma concentrations of selenium. 3 Analysis of variance (ANOVA) revealed that the difference in activities of catalase and glutathione peroxidase was much less within pairs than between pairs, indicating a large individual variation due to genetic expression or shared environment and no major effect from smoking. 4 The plasma selenium levels of the investigated twins revealed sufficient intake of selenium to maintain maximal activity of GSH-px in erythrocytes. The mean±s.d. -1 and for non-smokers selenium concentration in plasma for smokers was 98 ± 16 μg 1 111 ± 16 μg . -1 There was no correlation between plasma selenium and glutathione 1 peroxidase in erythrocytes.
Introduction
Cigarette smoke contains free radicals’ which may initiate lipid peroxidation and biological damage. The enzymes glutathione peroxidase (GSH-px) and catalase are important parts of the antioxidative protecting system, in addition to vitamin E and superoxide dismutase. Increased catalase activity in the erythrocytes and decreased glutathione peroxidase activity (GSHpx) has been reported in smokers. 2,3 However, it is difficult to obtain groups of smokers and non-smokers who have comparable genetic expression of these enzymes. Selenium is an essential trace element and a component of glutathione peroxidase in blood.’ In rat serum the major pool of selenium has been shown to be bound to selenoprotein P.5 Decreased levels of plasma selenium have been reported in smokers6,7 and have also been associated with pulmonary disease.’ Further, a significant inverse relationship between plasma selenium levels and the degree of formation of atherosclerosis has been reported in a study of hospitalized patients in Finland.’
The aims of the present study were to investigate the variability in activity of the antioxidative enzymes y-glutathione peroxidase and catalase in erythrocytes and to investigate the effect of smoking on them. Plasma selenium levels were measured in order to investigate a possible influence on GSH-px activity in erythrocytes. The study of the twin group was accepted by the local ethical committee.
Methods
’
Material Ten monozygotic twin pairs, seven female and three male, age 36-64 (mean 49.5) years, discordant for smoking in 1973 and 1990 were selected from the Swedish twin registry. 10 Their smoking habits and intake of selenium are shown in Table 1. Monozygozity was confirmed by a DNA ’fingerprinting’ analysis using the minisatellite DNA probe MZ 1.3; 11,12 the chance of false monozygosity with this test is less than 10 - 4.
Correspondence: Lars Björkman.
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342
L.
Table 1 Sex, age, smoking habits of the and intake of selenium supplement.
smoking twin
UV-160A). The NADPH consumption
was
also
determined when substituting deionized water for haemolysate and subtracted from the NADPH consumption in the haemolysate assay. One unit was classified as the activity that brings about the oxidation of 1 gmol NADPH min - I. All samples were assayed twice and the mean value
was
calculated.
Catalase assay:
Catalase
activity in erythro-
cytes was assayed by a method described by Aebi. &dquo; Of the haemolysate, 10 pl was diluted with phosphate buffer (50 mmol 1-’, pH 7.0) to final volume of 5.0 ml. Catalase activity was measured by adding 1.0 ml hydrogen peroxide (30 mmol 1-’, Merck, Germany) to 2.0 ml diluted haemolysate and reading the decrease in absorbance at 240 nm between 20 and 30s with a
S
=
smoker, NS non-smoker =
~
’
Methods Venous blood was collected using Vacutainer tubes (Becton Dickinson) with heparin (143 USP units) as anticoagulant and stored at 4°C until the next day when plasma and erythrocytes were separated by centrifugation at 3000 rpm for 5 min. Plasma samples were stored in polyethylene tubes at - 70°C until analyses were performed. The erythrocytes were washed three times in cold isotonic NaCI and haemolysates were prepared by mixing 0.5 ml of the erythrocyte suspension and 2.0 ml deionized water with a Whirlmixer (Fisons Scientific Apparatus, Loughbourough, Leicestershire, UK). The haemoglobin concentration of the haemolysates was determined by a standard method (Sigma Kit no 525, Sigma Chemical Co, St Louis, MO, USA). Catalase was assayed the day after sample collection ; GSH-px was analysed after storing of the haemolysates in polyethylene tubes at - 70°C.
Glutathione peroxidase assay: The activity of
GSH-px in erythrocytes was analysed by a coupled method using tert. butyl hydroperoxide as a substrate. 13 The haemolysates were diluted with deionized water to a haemoglobin content of 3 g Hb 1- and treated with excess of cyanide and hexacyanoferrate. Of this transformed haemolysate, 0.5 ml was mixed with 0.2 ml glutathione reductase (Sigma Chemical Co, St
Louis, MO, USA) (5 U ml -’ potassium phosphate buffer pH 7.0), 0.1 ml reduced glutathione (10 mmol 1-’ , Merck, Germany) and incubated for 12 min at 37 ° C. After incubation, 0. I ml NADPH (2.5 mmol 1 ~ ’ , Sigma Chemical Co, St Louis, MO, USA) and 0.1 ml tert. butyl hydroperoxide (12 mmol 1-’, Merck, Germany) were added. To the blank, 0.1 ml distilled water was added in place of the substrate. The consumption of NADPH at 25°C was registered at 366 nm&dquo; using a spectrophotometer (Shimadzu
spectrophotometer (Shimadzu UV-160A) against a blank consisting of 2.0 ml diluted haemolysate and 1.0 ml deionized water at 25°C. Each sample was assayed three times and the mean of the first order rate constants, k, was a
calculated. Final results, corrected for the dilution and related to the calculated haemoglobin concentration in the reaction mixture (U g-’1 Hb), were expressed as follows:
g - Hb =kx (CHb)-1 x 7.5 x 105
Units catalase
Clb is concentration of haemoglobin (g Hb 1-’ ) in the undiluted haemolysate. The constant 7.5 x 10s is calculated from equations 4 5, 6, and 7 in the used reference.&dquo; where
.
Analysis of selenium in plasma: Selenium was determined by graphite furnace atomic absorption spectrophotometry with Zeeman background correction using a Perkin Elmer 5000 equipped with an electrothermal atomization unit (Perkin Elmer HGA-500 programmer), automatic sample injector (Perkin Elmer AS 40), a laboratory computer (Perkin Elmer 7500 Laboratory Computer), and a printer (PR-210). An electrodeless discharge lamp (EDL) and standard graphite tube with L’Vov platform were used. The sample treatment was slightly modified after a method described by Alfthan and Kumpulainen. 15 Plasma (50 ~1) was mixed with 450 u1 matrix modifier containing nickel nitrate (42.6 mmol 1-’, Merck, Germany), Triton-X (1.852 mg ml-’, Roth, Germany) in 0.072 mol 1-’ nitric acid (suprapur, Merck, Germany) and stored overnight at 4 ° C. The following day the treated samples were mixed again and analysed. All samples were assayed twice in duplicate. Standard curves were determined using a standard addition method. For quality control
Downloaded from het.sagepub.com at Purdue University on June 26, 2015
343
Seronorm 105 (Nycomed Co, analysed as a reference.
Norway)
was
As a test of the significance between means the Student’s t-test was used. To test the differences between smokers and nonsmokers Wilcoxon’s signed rank sum test was used. To determine the variation between smokers and non-smokers, and the variation between pairs two-way ANOVA was performed by using the mean results from the assays of each sample. To determine the variation between samples one-way ANOVA was performed, using the results from the separate assays.
Statistical methods: _
’
Activity of glutathione peroxidase in erythas U g-’ Hb for the twin pairs (n=10). Enzyme activity of the smoking twin is plotted against the enzyme activity of the non-smoking twin. Calculated regression y= - 0.82 + 1.26x (r=0.74; P < 0.02) A=Pair with no selenium supplement, ·=intake of selenium supplement by nonsmoking twin, ·=Intake of selenium supplement by smoking twin. Figure
Results
1
rocytes, expressed
The results from the analyses are listed in Table 2. The activity of GSH-px in erythrocytes (mean and s.d.) for smokers was 4.1 ± l.l U g-’ Hb (range 2.3-6.4) and for non-smokers 3.9 ± 0.6 U g - Hub (range 2.9-4.9). In Figure 1 the activities of GSH-px in erythrocytes for smokers are plotted against those for non-smokers. To determine the variation between smokers and nonsmokers, and between pairs, variance analysis (two-way ANOVA) was performed. It showed that 2% of the total variation was explained by variation within the pairs, and 81 % was explained by variation between the pairs (Table 3). Variation between samples was determined by one-way ANOVA as seen in Table 4. Of the total variation 95% was explained by variation between the samples. Mean activity of catalase in erythrocytes for smokers was 270 ± 30 U g - Hb (range 230300) and for non-smokers 260 ± 30 U g ~ ’ Hb
(range 220-320). The correlation of catalase activity in erythrocytes between smokers and non-smokers is shown in Figure 2. Of the total variation 2% was explained by variation within the pairs, and 89% by variation between the pairs (two-way ANOVA). Of the total variation of the first-order rate constants, 96% was explained as variation between the samples (oneway ANOVA). The mean ± s.d. selenium concentration in plasma for smokers was 98 ± 16 Jig 1-’ plasma (range 66-123) and for non-smokers 111 ± 16 Jig 1’’I (range 93-143). The difference between
Table 2 Activity of glutathione peroxidase (GSH-px) and catalase in in plasma, expressed as U g-’Hb and pg Se 1 -’, respectively.
erythrocytes,
and selenium concentration
S = smoker, NS = non-smoker.
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344
Analysis of variation in activity of glutathione peroxidase (GSH-px) in erythrocytes between smokers and non-smokers and between twin pairs (two-way Table 3
ANOVA). .o
T/nrinfj/)JI1
T/nyjnYJ/&dquo;o
Figure 2 Activity of catalase in erythrocytes, expressed U catalase g-’ Hb for the twin pairs (n=10). Enzyme activity of the smoking twin is plotted against
as
Analysis of variation in activity of glutathione peroxidase (GSH-px) in erythrocytes between samples
Table 4
(one-way ANOVA).
smokers and non-smokers is
the enzyme activity of the non-smoking twin. Calculated regression y=89 + 0.68x (r=0.84; P < 0.01 ) A=Pair with no selenium supplement, . = Intake of selenium supplement by non-smoking twin, 8 = Intake of selenium supplement by smoking twin.
non-significant
0.05, Wilcoxon’s signed rank sum test). When pairs with an intake of supplementary selenium are excluded (pairs number 5, 6, 7, and 9) the difference is still non-significant. Mean plasma selenium for the remaining pairs (n=6) (P >
was
98:!: 19 Jig I -
for smokers and
109:!: 13 Jig
1- for non-smokers. The correlation of plasma selenium concentration between smokers and non-smokers is shown in Figure 3. Analysis of variance was performed in the same way as for the GSH-px determinations and showed that 14% of the total variation could be explained by variation within the pairs, and 50% by variation between the pairs (two-way ANOVA). One-way ANOVA showed that 96% of the total variation was explained as variation between samples. If twin pairs with intake of selenium supplement were excluded 11 % of the total variation was explained by variation within the pairs, and 73% was explained by variation between the pairs
(two-way ANOVA).
Quality control 105) Analysis of the reference sample (Seronorm revealed a figure of 99.6:!: 9.2 Jig 1-I plasma
Figure 3 Concentration of selenium in plasma expressed as pg 1-’ for the twin pairs (ii = 10). Plasma selenium concentration for the smoking twins plotted against the non-smoking twins. A = Pair with no selenium supplement, .= Intake of selenium supplement by non-smoking twin, 8 = Intake of selenium supplement by smoking twin. Calculated regression for pairs with no selenium supplement y= - 17.73 + 1.06x (r=0.7; n=6; p > 0.1)
(mean ± s. d., n = 4). The certified mean ± s. d. is 90.6±6ugl&dquo;’ serum.’ The blood collection tubes were tested for selenium content by filling with 10 ml nitric acid (0.03 mol 1-’ ) (suprapur, Merck, Germany). Selenium concentration was subsequently analysed and found to be below the detection limit (~ 21 ng Se g -’ nitric acid). Of the total variation between plasma samples
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345
only 4% could not be explained by variation between samples. This indicates a low variation due to analysis. The influence of the type of anticoagulant used in the blood collection tubes on GSH-px activity has been discussed. 13 A test was thus made by collecting three tubes of blood with 143 USP units heparin as anticoagulant (Vacutainer, Becton Dickinson) and three tubes of blood with K,EDTA as anticoagulant (Vacutainer, Becton Dickinson) from a voluntary blood donor. Haemolysates were prepared as described above. No difference between the heparin- and K,
~
’
EDTA-treated samples was found. The mean±s.d. (for n=3) of the heparin-treated sample was 5.05 ± 0.06 U g -’ Hb and for the K,EDTA-treated sample 5.29 ± 0.11 U g - He.
Discussion
’
Studies of exposure discordant monozygotic twins provide an unique opportunity to distinguish the influence of exposure factors on, e.g. enzyme systems from genetic factors. In this study it was possible to investigate effects of smoking on the activity of glutathione peroxidase (GSH-px) and catalase in 10 pairs of monozygotic twins discordant for smoking. There were no effects of smoking on erythrocyte catalase or GSH-px activity in the present study. Conflicting results have been reported concerning the effects of smoking on these enzymes. Decreased3,7 levels of GSH-px activity and increased’ levels of catalase, as well as no effects of GSH-pX2 or catalase’ activity, in erythrocytes from smokers have been reported. Lloyd et al.6 found lower GSH-px levels in smoking men over the age of 30. On the other hand no differences in GSH-px levels between smokers and non-smokers were found for women. Increased levels of catalase and decreased GSH-px levels have been reported in human alveolar macrophages from smokers.&dquo; However, anti-oxidative enzyme levels in airway secretions may be altered but the change not reflected in the circulating blood cells. A change in local enzyme activity could be important for the protection of cell membranes in the airways. GSH-px in erythrocytes is selenium dependent and maximal activity has been shown to be achieved at selenium concentrations exceeding 140 gg 1-’ erythrocytes. 18 A strong association has been found between the whole blood selenium concentration and GSH-px activity when whole blood selenium concentrations were below 1.26 jmol 1 ~ ’ (99.5 Jig 1- 1).19 Using data from the study by Rea et al.’ 99.5 gig Se I - ’I whole blood would correspond to a selenium concentration in plasma of approximately 60-
80 Jig 1-’ plasma. Consequently, the plasma selenium levels (66-143 Jig 1-’ ) of the twins in the present study reflect a sufficiently high intake of selenium to maintain maximal activity of GSHpx. The variation in GSH-px and catalase activity in erythrocytes was only 2% within pairs and a significant correlation within pairs was found for the activity of the enzymes. Between pairs the variation was 81 % and 89%, respectively. This
indicates a large individual variation which can be explained by either genetic influence or by the influence of a shared environment. Consequently smoking habits are not likely to be a major determinant of the activity of these enzymes. The unexplained variation (variation due to other factors than differences between samples, e.g. random and variation in the analytical methods) for the analyses was low (4-5%). Generally, selenium levels in the blood or serum are considered to reflect the dietary intake of selenium.’8°2o The selenium levels of smokers seemed to be lower than those of the nonsmokers as has been found in previous studies. 6, When the four twin pairs taking selenium supplements were excluded, the difference between the remaining six pairs was in the same range. An interesting
finding is that when twin pairs taking selenium supplements are excluded from the study, analysis of variance shows that the variation of plasma selenium concentrations within the pairs is less than between the pairs. This could be result of similar dietary habits. Unfortunately, there is no detailed dietary information. To conclude, oxidative stress in the form of cigarette smoking does not significantly influence the activity of the antioxidant enzymes GSH-px or catalase and the bulk of individual variation is due to genetic expression or shared environment. Smoking habits are not a major determinant for the activity of these enzymes in erythrocytes. Plasma selenium seems to be lower in smokers than non-smokers and the dietary intake of selenium in both groups was high enough to achieve maximal GSH-px activity in erythrocytes. However, a smoke-associated decrease in selenium can possibly be important for GSH-px enzyme activity during relative or manifest selenium deficiency. If these enzymes are important large individual variation in susceptibility to disease due to genetic determinants could be expected.
Acknowledgement Brita Palm and Bo Nilsson are gratefully acknowledged for their skilful laboratory assistance. Grants
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346
from the Karolinska Institutet and National Asthma and Allergy Association are acknowledged. The Swed-
ish Twin Registry is supported by funds from the John D. and Catherine T. MacArthur Foundation.
References 1 Church DF &
Pryor WA. Free radical chemistry of smoke and its toxicological implications. Environmental Health Perspectives 1985; 64: 111-26. 2 Toth KM, Berger EM, Beehler CJ & Repine JE. Erythrocytes from cigarette smokers contain more glutathione and catalase and protect endothelial cells from hydrogen peroxide better than do erythrocytes from nonsmokers. American Review of Respiratory Disease 1986; 134: 281-4. 3 Duthie GG, Arthur JR & James WPT. Effects of smoking and vitamin E on blood antioxidant status. American Journal of Clinical Nutrition 1991; 53: 1061S-3S. 4 Thomson CD. Selenium-dependent and non-seleniumdependent glutathione peroxidase in human tissue of New Zealand residents. Biochemistry International 1985; 10: 673-9. 5 Read R, Bellew T, Yang J-G et al Selenium and amino acid composition of selenoprotein P, the major selenoprotein in rat serum. Journal of Biological Chemis17899-905. try 1990; 29: 6 Lloyd B, Lloyd RS & Clayton BE. Effect of smoking, alcohol, and other factors on the selenium status of a healthy population. Journal of Epidemiology and Community Health 1983; 37: 213-7. 7 Ellis N, Lloyd B, Lloyd RS & Clayton BE. Selenium and vitamin E in relation to risk factors for coronary heart disease. Journal of Clinical Pathology 1984; 37: 200-6. 8 Stone J, Hinks LJ, Beasley R, Holgate ST & Clayton BA. Reduced selenium status of patients with asthma. Clinical Science 1989; 77: 495-500. 9 Moore JA, Noiva R & Wells IC. Selenium concentration in plasma of patients with arteriographically defined coronary atherosclerosis. Clinical Chemistry 1984; 30: 1171-3. 10 Medlund P, Cederlof R, Floderus-Myrhed B et al. A new Swedish twin registry containing environmental and medical base line data from about 14,000 same-sexed pairs born 1926-58. Acta Medica Scandinavia 1977; (suppl), 600. 11 Hill AVS & Jeffreys AJ. Use of minisatellite DNA probes
cigarette
for determination of twin zygosity at birth. Lancet 1985; 1394-5. 21: Schacker U, Schneider PM, Holtkamp B et al. Isolation of the DNA minisatellite probe MZ 1.3 and its application to DNA ’fingerprinting’ analysis. Forensic Science International 1990; 44: 209-24. ’ 3 Günzler WA, Kremers H & Flohe L. An improved coupled test procedure for glutathione peroxidase (EC 1.11.1.9) in blood. Zeitschrirt für Klinische Chemie und Klinische Biochemie 1974: 12: 444-8. 14 Aebi H. Catalase. In: Methods of Enzymatic Analyses, ed Bergmeyer HU, Vol II pp. 673-84. Weinheim: VerlagChemie, 1974. 15 Alfthan G & Kumpulainen J. Determination of selenium in small volumes of blood plasma and serum by electrothermal atomic absorption spectrometry. Analytica Chimica Acta 1982; 165: 221-7. 16 Christensen JM, Ihnat M, Stoeppler M et al. Human body fluids - IUPAC proposed reference materials for trace elements analysis. Fresenius Zeitschrift für Analytische Chemie 1987; 326: 639-42. 17 Hoidal J. Selective increase of McCusker KT & antioxidant enzyme activity in the alveolar macrophages from cigarette smokers and smoke-exposed hamsters. American Review of Respiratory Disease 1990; 141: 67882. 18 Rea HM, Thomson CD, Campbell DR & Robinson MF. Relation between erythrocyte selenium concentrations and glutathione peroxidase (EC 1.11.1.9) activities of New Zealand residents and visitors to New Zealand. British Journal of Nutrition 1979; 42: 201-8. 19 Lloyd B, Robson E, SmithI & Clayton BE. Blood selenium concentrations and glutathione peroxidase activity. Archives of Disease in Childhood 1989; 64: 352-6. 20 Alfthan G. Longitudinal study on selenium status of healthy adults in Finland during 1975-84. Nutrition Research 1988; 8: 467-76. 12
(Receined 22 November 1991; accepted
Downloaded from het.sagepub.com at Purdue University on June 26, 2015
29
January 1992)
Smoking Lars Björkman, 1,2 1 Magnus Svartengren Department 1
of Environmental
&
Monica
1 Nordberg
Department of Occupational Hygiene, Karolinska Institutet & 2
Health, Karolinska Hospital, S-104 01 Stockholm, Sweden 1 Cigarette smoke contains free radicals. The enzymes glutathione peroxidase (GSH-px) and catalase are important parts of the anti-oxidative protecting system. 2 Ten pairs of monozygotic twins, who were discordant for smoking, were analysed in order to determine their erythrocyte glutathione peroxidase and catalase activities and their plasma concentrations of selenium. 3 Analysis of variance (ANOVA) revealed that the difference in activities of catalase and glutathione peroxidase was much less within pairs than between pairs, indicating a large individual variation due to genetic expression or shared environment and no major effect from smoking. 4 The plasma selenium levels of the investigated twins revealed sufficient intake of selenium to maintain maximal activity of GSH-px in erythrocytes. The mean±s.d. -1 and for non-smokers selenium concentration in plasma for smokers was 98 ± 16 μg 1 111 ± 16 μg . -1 There was no correlation between plasma selenium and glutathione 1 peroxidase in erythrocytes.
Introduction
Cigarette smoke contains free radicals’ which may initiate lipid peroxidation and biological damage. The enzymes glutathione peroxidase (GSH-px) and catalase are important parts of the antioxidative protecting system, in addition to vitamin E and superoxide dismutase. Increased catalase activity in the erythrocytes and decreased glutathione peroxidase activity (GSHpx) has been reported in smokers. 2,3 However, it is difficult to obtain groups of smokers and non-smokers who have comparable genetic expression of these enzymes. Selenium is an essential trace element and a component of glutathione peroxidase in blood.’ In rat serum the major pool of selenium has been shown to be bound to selenoprotein P.5 Decreased levels of plasma selenium have been reported in smokers6,7 and have also been associated with pulmonary disease.’ Further, a significant inverse relationship between plasma selenium levels and the degree of formation of atherosclerosis has been reported in a study of hospitalized patients in Finland.’
The aims of the present study were to investigate the variability in activity of the antioxidative enzymes y-glutathione peroxidase and catalase in erythrocytes and to investigate the effect of smoking on them. Plasma selenium levels were measured in order to investigate a possible influence on GSH-px activity in erythrocytes. The study of the twin group was accepted by the local ethical committee.
Methods
’
Material Ten monozygotic twin pairs, seven female and three male, age 36-64 (mean 49.5) years, discordant for smoking in 1973 and 1990 were selected from the Swedish twin registry. 10 Their smoking habits and intake of selenium are shown in Table 1. Monozygozity was confirmed by a DNA ’fingerprinting’ analysis using the minisatellite DNA probe MZ 1.3; 11,12 the chance of false monozygosity with this test is less than 10 - 4.
Correspondence: Lars Björkman.
Downloaded from het.sagepub.com at Purdue University on June 26, 2015
342
L.
Table 1 Sex, age, smoking habits of the and intake of selenium supplement.
smoking twin
UV-160A). The NADPH consumption
was
also
determined when substituting deionized water for haemolysate and subtracted from the NADPH consumption in the haemolysate assay. One unit was classified as the activity that brings about the oxidation of 1 gmol NADPH min - I. All samples were assayed twice and the mean value
was
calculated.
Catalase assay:
Catalase
activity in erythro-
cytes was assayed by a method described by Aebi. &dquo; Of the haemolysate, 10 pl was diluted with phosphate buffer (50 mmol 1-’, pH 7.0) to final volume of 5.0 ml. Catalase activity was measured by adding 1.0 ml hydrogen peroxide (30 mmol 1-’, Merck, Germany) to 2.0 ml diluted haemolysate and reading the decrease in absorbance at 240 nm between 20 and 30s with a
S
=
smoker, NS non-smoker =
~
’
Methods Venous blood was collected using Vacutainer tubes (Becton Dickinson) with heparin (143 USP units) as anticoagulant and stored at 4°C until the next day when plasma and erythrocytes were separated by centrifugation at 3000 rpm for 5 min. Plasma samples were stored in polyethylene tubes at - 70°C until analyses were performed. The erythrocytes were washed three times in cold isotonic NaCI and haemolysates were prepared by mixing 0.5 ml of the erythrocyte suspension and 2.0 ml deionized water with a Whirlmixer (Fisons Scientific Apparatus, Loughbourough, Leicestershire, UK). The haemoglobin concentration of the haemolysates was determined by a standard method (Sigma Kit no 525, Sigma Chemical Co, St Louis, MO, USA). Catalase was assayed the day after sample collection ; GSH-px was analysed after storing of the haemolysates in polyethylene tubes at - 70°C.
Glutathione peroxidase assay: The activity of
GSH-px in erythrocytes was analysed by a coupled method using tert. butyl hydroperoxide as a substrate. 13 The haemolysates were diluted with deionized water to a haemoglobin content of 3 g Hb 1- and treated with excess of cyanide and hexacyanoferrate. Of this transformed haemolysate, 0.5 ml was mixed with 0.2 ml glutathione reductase (Sigma Chemical Co, St
Louis, MO, USA) (5 U ml -’ potassium phosphate buffer pH 7.0), 0.1 ml reduced glutathione (10 mmol 1-’ , Merck, Germany) and incubated for 12 min at 37 ° C. After incubation, 0. I ml NADPH (2.5 mmol 1 ~ ’ , Sigma Chemical Co, St Louis, MO, USA) and 0.1 ml tert. butyl hydroperoxide (12 mmol 1-’, Merck, Germany) were added. To the blank, 0.1 ml distilled water was added in place of the substrate. The consumption of NADPH at 25°C was registered at 366 nm&dquo; using a spectrophotometer (Shimadzu
spectrophotometer (Shimadzu UV-160A) against a blank consisting of 2.0 ml diluted haemolysate and 1.0 ml deionized water at 25°C. Each sample was assayed three times and the mean of the first order rate constants, k, was a
calculated. Final results, corrected for the dilution and related to the calculated haemoglobin concentration in the reaction mixture (U g-’1 Hb), were expressed as follows:
g - Hb =kx (CHb)-1 x 7.5 x 105
Units catalase
Clb is concentration of haemoglobin (g Hb 1-’ ) in the undiluted haemolysate. The constant 7.5 x 10s is calculated from equations 4 5, 6, and 7 in the used reference.&dquo; where
.
Analysis of selenium in plasma: Selenium was determined by graphite furnace atomic absorption spectrophotometry with Zeeman background correction using a Perkin Elmer 5000 equipped with an electrothermal atomization unit (Perkin Elmer HGA-500 programmer), automatic sample injector (Perkin Elmer AS 40), a laboratory computer (Perkin Elmer 7500 Laboratory Computer), and a printer (PR-210). An electrodeless discharge lamp (EDL) and standard graphite tube with L’Vov platform were used. The sample treatment was slightly modified after a method described by Alfthan and Kumpulainen. 15 Plasma (50 ~1) was mixed with 450 u1 matrix modifier containing nickel nitrate (42.6 mmol 1-’, Merck, Germany), Triton-X (1.852 mg ml-’, Roth, Germany) in 0.072 mol 1-’ nitric acid (suprapur, Merck, Germany) and stored overnight at 4 ° C. The following day the treated samples were mixed again and analysed. All samples were assayed twice in duplicate. Standard curves were determined using a standard addition method. For quality control
Downloaded from het.sagepub.com at Purdue University on June 26, 2015
343
Seronorm 105 (Nycomed Co, analysed as a reference.
Norway)
was
As a test of the significance between means the Student’s t-test was used. To test the differences between smokers and nonsmokers Wilcoxon’s signed rank sum test was used. To determine the variation between smokers and non-smokers, and the variation between pairs two-way ANOVA was performed by using the mean results from the assays of each sample. To determine the variation between samples one-way ANOVA was performed, using the results from the separate assays.
Statistical methods: _
’
Activity of glutathione peroxidase in erythas U g-’ Hb for the twin pairs (n=10). Enzyme activity of the smoking twin is plotted against the enzyme activity of the non-smoking twin. Calculated regression y= - 0.82 + 1.26x (r=0.74; P < 0.02) A=Pair with no selenium supplement, ·=intake of selenium supplement by nonsmoking twin, ·=Intake of selenium supplement by smoking twin. Figure
Results
1
rocytes, expressed
The results from the analyses are listed in Table 2. The activity of GSH-px in erythrocytes (mean and s.d.) for smokers was 4.1 ± l.l U g-’ Hb (range 2.3-6.4) and for non-smokers 3.9 ± 0.6 U g - Hub (range 2.9-4.9). In Figure 1 the activities of GSH-px in erythrocytes for smokers are plotted against those for non-smokers. To determine the variation between smokers and nonsmokers, and between pairs, variance analysis (two-way ANOVA) was performed. It showed that 2% of the total variation was explained by variation within the pairs, and 81 % was explained by variation between the pairs (Table 3). Variation between samples was determined by one-way ANOVA as seen in Table 4. Of the total variation 95% was explained by variation between the samples. Mean activity of catalase in erythrocytes for smokers was 270 ± 30 U g - Hb (range 230300) and for non-smokers 260 ± 30 U g ~ ’ Hb
(range 220-320). The correlation of catalase activity in erythrocytes between smokers and non-smokers is shown in Figure 2. Of the total variation 2% was explained by variation within the pairs, and 89% by variation between the pairs (two-way ANOVA). Of the total variation of the first-order rate constants, 96% was explained as variation between the samples (oneway ANOVA). The mean ± s.d. selenium concentration in plasma for smokers was 98 ± 16 Jig 1-’ plasma (range 66-123) and for non-smokers 111 ± 16 Jig 1’’I (range 93-143). The difference between
Table 2 Activity of glutathione peroxidase (GSH-px) and catalase in in plasma, expressed as U g-’Hb and pg Se 1 -’, respectively.
erythrocytes,
and selenium concentration
S = smoker, NS = non-smoker.
Downloaded from het.sagepub.com at Purdue University on June 26, 2015
344
Analysis of variation in activity of glutathione peroxidase (GSH-px) in erythrocytes between smokers and non-smokers and between twin pairs (two-way Table 3
ANOVA). .o
T/nrinfj/)JI1
T/nyjnYJ/&dquo;o
Figure 2 Activity of catalase in erythrocytes, expressed U catalase g-’ Hb for the twin pairs (n=10). Enzyme activity of the smoking twin is plotted against
as
Analysis of variation in activity of glutathione peroxidase (GSH-px) in erythrocytes between samples
Table 4
(one-way ANOVA).
smokers and non-smokers is
the enzyme activity of the non-smoking twin. Calculated regression y=89 + 0.68x (r=0.84; P < 0.01 ) A=Pair with no selenium supplement, . = Intake of selenium supplement by non-smoking twin, 8 = Intake of selenium supplement by smoking twin.
non-significant
0.05, Wilcoxon’s signed rank sum test). When pairs with an intake of supplementary selenium are excluded (pairs number 5, 6, 7, and 9) the difference is still non-significant. Mean plasma selenium for the remaining pairs (n=6) (P >
was
98:!: 19 Jig I -
for smokers and
109:!: 13 Jig
1- for non-smokers. The correlation of plasma selenium concentration between smokers and non-smokers is shown in Figure 3. Analysis of variance was performed in the same way as for the GSH-px determinations and showed that 14% of the total variation could be explained by variation within the pairs, and 50% by variation between the pairs (two-way ANOVA). One-way ANOVA showed that 96% of the total variation was explained as variation between samples. If twin pairs with intake of selenium supplement were excluded 11 % of the total variation was explained by variation within the pairs, and 73% was explained by variation between the pairs
(two-way ANOVA).
Quality control 105) Analysis of the reference sample (Seronorm revealed a figure of 99.6:!: 9.2 Jig 1-I plasma
Figure 3 Concentration of selenium in plasma expressed as pg 1-’ for the twin pairs (ii = 10). Plasma selenium concentration for the smoking twins plotted against the non-smoking twins. A = Pair with no selenium supplement, .= Intake of selenium supplement by non-smoking twin, 8 = Intake of selenium supplement by smoking twin. Calculated regression for pairs with no selenium supplement y= - 17.73 + 1.06x (r=0.7; n=6; p > 0.1)
(mean ± s. d., n = 4). The certified mean ± s. d. is 90.6±6ugl&dquo;’ serum.’ The blood collection tubes were tested for selenium content by filling with 10 ml nitric acid (0.03 mol 1-’ ) (suprapur, Merck, Germany). Selenium concentration was subsequently analysed and found to be below the detection limit (~ 21 ng Se g -’ nitric acid). Of the total variation between plasma samples
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only 4% could not be explained by variation between samples. This indicates a low variation due to analysis. The influence of the type of anticoagulant used in the blood collection tubes on GSH-px activity has been discussed. 13 A test was thus made by collecting three tubes of blood with 143 USP units heparin as anticoagulant (Vacutainer, Becton Dickinson) and three tubes of blood with K,EDTA as anticoagulant (Vacutainer, Becton Dickinson) from a voluntary blood donor. Haemolysates were prepared as described above. No difference between the heparin- and K,
~
’
EDTA-treated samples was found. The mean±s.d. (for n=3) of the heparin-treated sample was 5.05 ± 0.06 U g -’ Hb and for the K,EDTA-treated sample 5.29 ± 0.11 U g - He.
Discussion
’
Studies of exposure discordant monozygotic twins provide an unique opportunity to distinguish the influence of exposure factors on, e.g. enzyme systems from genetic factors. In this study it was possible to investigate effects of smoking on the activity of glutathione peroxidase (GSH-px) and catalase in 10 pairs of monozygotic twins discordant for smoking. There were no effects of smoking on erythrocyte catalase or GSH-px activity in the present study. Conflicting results have been reported concerning the effects of smoking on these enzymes. Decreased3,7 levels of GSH-px activity and increased’ levels of catalase, as well as no effects of GSH-pX2 or catalase’ activity, in erythrocytes from smokers have been reported. Lloyd et al.6 found lower GSH-px levels in smoking men over the age of 30. On the other hand no differences in GSH-px levels between smokers and non-smokers were found for women. Increased levels of catalase and decreased GSH-px levels have been reported in human alveolar macrophages from smokers.&dquo; However, anti-oxidative enzyme levels in airway secretions may be altered but the change not reflected in the circulating blood cells. A change in local enzyme activity could be important for the protection of cell membranes in the airways. GSH-px in erythrocytes is selenium dependent and maximal activity has been shown to be achieved at selenium concentrations exceeding 140 gg 1-’ erythrocytes. 18 A strong association has been found between the whole blood selenium concentration and GSH-px activity when whole blood selenium concentrations were below 1.26 jmol 1 ~ ’ (99.5 Jig 1- 1).19 Using data from the study by Rea et al.’ 99.5 gig Se I - ’I whole blood would correspond to a selenium concentration in plasma of approximately 60-
80 Jig 1-’ plasma. Consequently, the plasma selenium levels (66-143 Jig 1-’ ) of the twins in the present study reflect a sufficiently high intake of selenium to maintain maximal activity of GSHpx. The variation in GSH-px and catalase activity in erythrocytes was only 2% within pairs and a significant correlation within pairs was found for the activity of the enzymes. Between pairs the variation was 81 % and 89%, respectively. This
indicates a large individual variation which can be explained by either genetic influence or by the influence of a shared environment. Consequently smoking habits are not likely to be a major determinant of the activity of these enzymes. The unexplained variation (variation due to other factors than differences between samples, e.g. random and variation in the analytical methods) for the analyses was low (4-5%). Generally, selenium levels in the blood or serum are considered to reflect the dietary intake of selenium.’8°2o The selenium levels of smokers seemed to be lower than those of the nonsmokers as has been found in previous studies. 6, When the four twin pairs taking selenium supplements were excluded, the difference between the remaining six pairs was in the same range. An interesting
finding is that when twin pairs taking selenium supplements are excluded from the study, analysis of variance shows that the variation of plasma selenium concentrations within the pairs is less than between the pairs. This could be result of similar dietary habits. Unfortunately, there is no detailed dietary information. To conclude, oxidative stress in the form of cigarette smoking does not significantly influence the activity of the antioxidant enzymes GSH-px or catalase and the bulk of individual variation is due to genetic expression or shared environment. Smoking habits are not a major determinant for the activity of these enzymes in erythrocytes. Plasma selenium seems to be lower in smokers than non-smokers and the dietary intake of selenium in both groups was high enough to achieve maximal GSH-px activity in erythrocytes. However, a smoke-associated decrease in selenium can possibly be important for GSH-px enzyme activity during relative or manifest selenium deficiency. If these enzymes are important large individual variation in susceptibility to disease due to genetic determinants could be expected.
Acknowledgement Brita Palm and Bo Nilsson are gratefully acknowledged for their skilful laboratory assistance. Grants
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from the Karolinska Institutet and National Asthma and Allergy Association are acknowledged. The Swed-
ish Twin Registry is supported by funds from the John D. and Catherine T. MacArthur Foundation.
References 1 Church DF &
Pryor WA. Free radical chemistry of smoke and its toxicological implications. Environmental Health Perspectives 1985; 64: 111-26. 2 Toth KM, Berger EM, Beehler CJ & Repine JE. Erythrocytes from cigarette smokers contain more glutathione and catalase and protect endothelial cells from hydrogen peroxide better than do erythrocytes from nonsmokers. American Review of Respiratory Disease 1986; 134: 281-4. 3 Duthie GG, Arthur JR & James WPT. Effects of smoking and vitamin E on blood antioxidant status. American Journal of Clinical Nutrition 1991; 53: 1061S-3S. 4 Thomson CD. Selenium-dependent and non-seleniumdependent glutathione peroxidase in human tissue of New Zealand residents. Biochemistry International 1985; 10: 673-9. 5 Read R, Bellew T, Yang J-G et al Selenium and amino acid composition of selenoprotein P, the major selenoprotein in rat serum. Journal of Biological Chemis17899-905. try 1990; 29: 6 Lloyd B, Lloyd RS & Clayton BE. Effect of smoking, alcohol, and other factors on the selenium status of a healthy population. Journal of Epidemiology and Community Health 1983; 37: 213-7. 7 Ellis N, Lloyd B, Lloyd RS & Clayton BE. Selenium and vitamin E in relation to risk factors for coronary heart disease. Journal of Clinical Pathology 1984; 37: 200-6. 8 Stone J, Hinks LJ, Beasley R, Holgate ST & Clayton BA. Reduced selenium status of patients with asthma. Clinical Science 1989; 77: 495-500. 9 Moore JA, Noiva R & Wells IC. Selenium concentration in plasma of patients with arteriographically defined coronary atherosclerosis. Clinical Chemistry 1984; 30: 1171-3. 10 Medlund P, Cederlof R, Floderus-Myrhed B et al. A new Swedish twin registry containing environmental and medical base line data from about 14,000 same-sexed pairs born 1926-58. Acta Medica Scandinavia 1977; (suppl), 600. 11 Hill AVS & Jeffreys AJ. Use of minisatellite DNA probes
cigarette
for determination of twin zygosity at birth. Lancet 1985; 1394-5. 21: Schacker U, Schneider PM, Holtkamp B et al. Isolation of the DNA minisatellite probe MZ 1.3 and its application to DNA ’fingerprinting’ analysis. Forensic Science International 1990; 44: 209-24. ’ 3 Günzler WA, Kremers H & Flohe L. An improved coupled test procedure for glutathione peroxidase (EC 1.11.1.9) in blood. Zeitschrirt für Klinische Chemie und Klinische Biochemie 1974: 12: 444-8. 14 Aebi H. Catalase. In: Methods of Enzymatic Analyses, ed Bergmeyer HU, Vol II pp. 673-84. Weinheim: VerlagChemie, 1974. 15 Alfthan G & Kumpulainen J. Determination of selenium in small volumes of blood plasma and serum by electrothermal atomic absorption spectrometry. Analytica Chimica Acta 1982; 165: 221-7. 16 Christensen JM, Ihnat M, Stoeppler M et al. Human body fluids - IUPAC proposed reference materials for trace elements analysis. Fresenius Zeitschrift für Analytische Chemie 1987; 326: 639-42. 17 Hoidal J. Selective increase of McCusker KT & antioxidant enzyme activity in the alveolar macrophages from cigarette smokers and smoke-exposed hamsters. American Review of Respiratory Disease 1990; 141: 67882. 18 Rea HM, Thomson CD, Campbell DR & Robinson MF. Relation between erythrocyte selenium concentrations and glutathione peroxidase (EC 1.11.1.9) activities of New Zealand residents and visitors to New Zealand. British Journal of Nutrition 1979; 42: 201-8. 19 Lloyd B, Robson E, SmithI & Clayton BE. Blood selenium concentrations and glutathione peroxidase activity. Archives of Disease in Childhood 1989; 64: 352-6. 20 Alfthan G. Longitudinal study on selenium status of healthy adults in Finland during 1975-84. Nutrition Research 1988; 8: 467-76. 12
(Receined 22 November 1991; accepted
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January 1992)
