- Email: [email protected]
Glutathione peroxidase and reductase, glucose-6-phosphate dehydrogenase and catalase activities in multiple sclerosis
Journal of the Neurological Sciences, 1984, 63:45 53
45
Elsevier
GLUTATHIONE PEROXIDASE AND REDUCTASE, GLUCOSE-6-PHOSPHATE DEHYDROGENASE AND CATALASE ACTIVITIES IN MULTIPLE SCLEROSIS
G.E. JENSEN and J. CLAUSEN
The Laboratory of Biochemistry and Toxicology, Institute of Biology and Chemistry, University of Roskilde, D K-4000 Roskilde and The Neurochemical Institute, 58 R~tdmandsgade, D K-2200 Copenhagen N (Denmark) (Received 9 May, 1983) (Revised, received 2 August, 1983) (Accepted 17 August, 1983)
SUMMARY
Our previous studies have demonstrated a decreased glutathione peroxidase (GSH-Px) activity of erythrocytes and leucocytes from multiple sclerosis (MS) patients. In the present communication these activities were compared with the activities of associated enzymes (glutathione reductase (GSSG-RD), glucose-6phosphate dehydrogenase (G-6-PD) and catalase). All enzymic activities were compared between MS patients, other neurologic patients (ON patients) and normal control individuals. Compared to data of ON patients and normal controls, in MS the ratio of GSHPx/GSSGRD in lympho- and granulocytes was significantly decreased (2a ~<0.05) by 35~ and 51~o, respectively. The significant correlation between GSSG-RD and the GSH-Px activity (2a ~<0.05, r = 0.501) found in control lymphocytes was not present in MS lymphocytes. However, the lymphocyte GSH-Px activities of controls as well as of MS correlated with the corresponding serum selenium levels (2~ ~<0.05, r = 0.594 and 2~ ~<0.01, r = 0.967, respectively). The G-6-PD activity was insignificantly increased by 41 ~o in MS lymphocytes compared to normal control. Catalase activity was unchanged in lymphocytes but decreased 50~o in MS granulocytes compared to normal control. No significant differences were found between MS and the ON group. The catalase activity of MS erythrocytes was increased by 63~ (2a ~<0.05) in comparison with both the normal control and ON data.
This work was financially supported by a grant from the "Kobmand Sven Hansen og Hustru lna Hansens Fond". 0022-510X/84/$03.00 © 1984 Elsevier Science Publishers B.V.
46
Key words: Catalase Glucose-6-phosphate dehydrogenase Glutathione reductase Multiple sclerosis
Glutathione peroxidase
INTRODUCTION Previous investigations comparing some enzymic activities in normal controls and multiple sclerosis (MS) patients demonstrated a significantly decreased glutathione peroxidase (GSH-Px) activity in MS erythrocyte haemolysate and in MS cytosol of lymphocytes and granulocytes (Jensen et al. 1980). Therefore, GSH-Px, which counteracts autoperoxidations (Porta et al. 1977), may play a central role in the development of pathological changes in MS patients. Although selenium is an integral component of GSH-Px (Rotruck et al. 1973) the serum selenium level in MS patients is only insignificantly decreased (Jensen et al. 1980). GSH-Px detoxicates hydrogen peroxide via the GSH-Px pathway: G-6-P ~
~
NADP ~
G-6-PD 6-PG ~
~
(~*
OSSG-RD
~ " NADPH + H + -'')
C
2 GSH
S
H,O,
GSH-Px
Gsso J
C-
where G-6-P and 6-PG are glucose 6-phosphate and 6-phosphogluconate, respectively. GSSG and GSH are oxidized and reduced forms of glutathione, and finally G-6-PD and GSSG-RD are glucose-6-phosphate dehydrogenase and glutathione reductase, respectively. However, the enzyme catalase also detoxicates H:O~. Therefore, it was tempting to relate the selenium level and the activity of GSHPx with that of GSSG-RD, G-6-PD and catalase in haematogenous cells. MATERIALSAND METHODS
Patient material Venous blood samples from MS patients were collected from the Department of Neurology, County Hospital of Roskilde, Denmark. Twelve patients were clinically stable and 2 were in acute relapse. No patient was on cortisone theraphy. The diagnosis of MS was made on clinical criteria (Schumacher et al. 1965) by local neurologists and confirmed by Dr. K. Hyllested. Control material The control material comprised of healthy volunteers and patients with neurological disease other than MS. These included patients with cerebral haemorrhage (2), Parkinson's disease (1), trauma (1), ataxia (1), lumbar and cervical disc prolapse (4) and others (6). The control individuals as well as the MS patients all originated from the Danish island Zealand.
47
Methods Isolation of serum and erythrocytes from venous blood and preparation of erythrocyte haemolysate were made as previously described (Jensen et al. 1978). Lymphocytes and granulocytes were isolated by their specific gravity by the lymphoprep method (Nyggtrd and Comp. A/S, Oslo, Norway) (B6yum 1968). Remnants of erythrocytes were eliminated by osmotic shocking in distilled water. 1.3 × 108 cells were suspended in 2 ml 175 mmol/1 KC1. Determination of haemoglobin, total protein and selenium These assays were made as previously described (Lous et al. 1959; Jensen et al. 1978). Assay of microhematocrit was made by conventional routine procedures in a "high speed centrifuge" (Bauer et al. 1974). Enzymic assay G-6-PD (E.C. 1.1.1.49), GSSG-RD (E.C. 1.6.4.2), GSH-Px (E.C. 1.11.1.9) and catalase (E.C. 1.11.1.6) were assayed under optimal kinetic conditions (Kirkman 1959; Pinto and Bartley 1969; Cohen et al. 1970; Jensen et al. 1980). The blanks used were in all cases the reagent mixtures with distilled water instead of source of enzyme. The units of the GSH-Px pathway enzymes were expressed as units ofkat (1 kat = 1 mol coenzyme transformed per second (IUPAC-IUB 1974) specific activities were expressed as kat units/g protein. The specific unit of catalase was expressed in terms of the first-order reaction rate constant k, e.g. k/g protein according to Cohen et al. (1970). Chemicals All chemicals were of highest obtainable purity from British Drug Houses, Poole, Dorset, England. Enzymes were from Sigma Chemical Company, Saint Louis, U.S.A., or from Boehringer, Mannheim, F.R.G. Statistical assays The median, 10 and 90~o deciles were computed. The level of significance was assayed by means of Wilcoxon's non-parametric method (Geigy 1965). Spearman Rank's correlation test was used for the correlation purpose. The level of significance was set at 5~o. When lines on those best fit were drawn, they were estimated by regression analysis (Croxton 1959). RESULTS
Lymphocytes Table 1A shows the specific activities of GSH-Px and associated enzymes (catalase, G-6-PD and GSSG-RD) of crude lymphocyte homogenate. In MS the G-6-PD activity was increased by 41~o compared to the median activity of normal controls [not significant (NS)]. The GSSG-RD and GSH-Px activities were decreased
48 TABLE 1 DATA FROM N O R M A L CONTROLS (NC), MS PATIENTS (MS) A N D OTHER N E U R O L O G I C A L PATIENTS (ON)
(,4) Catalase, G-6-PD, GSH-Px, GSSG-RD activities in crude lymphocyte (L Y) and granuloeyte (GR) homogenate Catalase activity (k/g protein)
G-6-PD activity (,ukat/g protein )
GSSG-RD activity (/~kat/g protein)
GSH-Px activity (#kat/g protein)
GSH-PxGSSG-RD ratio
NC LY
n Median 10 90% decile
17 0.59 0.36 0.96
13 0.74 0.38 1.58
15 0.72 0.51 0.86
18 1.91 0.63 3.23
12 2.65 1.72 4.04
GR
n Median 0 90% decile
17 1.55 0.66 3.07
12 1.01 0.20 2.25
15 0.64 0.39 0.91
20 0.63 0.21 1.01
13 0.78 0.25 1.11
LY
n Median 10 90% decile
14 0.47 0.20 1.27
15 0.92 0.52 1.69
l0 0.91 0.44 1.30
14 1.92 0.49 2.07
10 2.12 1.57 3.54
GR
n Median 10 90% decile
13 0.94 0.56 1.48
II 0.97 0.45 2.18
9 0.59 0.39 0.91
13 0.71 (I.28 1.45
9 0.51 0.25 1.56
LY
n Median 10 90%decile
l0 0.45 0.16 1.19
8 1.04 0.39 1.79
9 0.66 0.22 0.87
14 (I.72 0.51 2.31
9 1.73 0.40 3.18
GR
n Median 10 90%decile
7 0.69 0.14 1 . 2 5
7 0.90 0.14~1.76
9 0.47 0.10 1.13
9 0.14 0.06 0.42
9 0.38 0.06 0.76
ON
MS
(B) Catalase activity in ervthrocytes and selenium level in serum Catalase activity (k x 106/tool Hgb)
Selenium level (,ug/1)
NC
n Median 10 90% decile
23 13.5 9.5 29.0
21 95 84~ 140
ON
n Median 10 901'o decile
10 16.2 10.0 19.(/
10 78 65 104
MS
n Median 10 90!,, decile
8 22.0 9.0 29.5
7 81 60 99
49
~5
A
_~ z,.0 ~o [3.
'
3.0.
20.
o • . t ~ o
o /
o
--°_o/~/ ~ o
o
o
• •
o.'s 0;7
11o
1.~
2b
GSS6-RD-activitY0Jkat ~ protein) c
05
B >,
>_ 3.0
8 ~ 2.0. i
I
e
c.D
1.0. • O
•
e o
• o
o's
o.~
i.o
1.'s
2'.o "~ GSSG-RD - activity (~kat,~ protein)
F i g . 1. T h e relationship between gh,.,athione reductase activity (#kat/g protein) (X) and glutathione peroxidase activity ~kat/g protein) (Y) in crude lymphocyte homogenate. A : C o n t r o l s . • = normal controls; O = other neurological patients. Regression equation: Y = 1.07 + 0 . 9 6 X (2~ ~< 0.05, r = 0.501). B : MS patients. @ = Patients in stable p h a s e ; @ = Patients in acute phase (no significant correlation).
by 8% (NS) and 51% (2a ~< 0.01), respectively. GSH-Px: GSSG-RD-ratio was 2.65 for the normal control group but 1.73 for the MS group (2~ ~ 0.05). When MS data were compared to data from other neurological patients, similar differences as indicated above were found. In controls the individual GSH-Px activities correlated with the corresponding G S S G - R D activities (2~ ~< 0.05, r = 0.501). In contrast no significant correlation was found in MS (Fig. 1). The GSH-Px activities of controls and MS correlated with the corresponding serum selenium level (2~ ~< 0.05, r = 0.594 and 2 , ~<0.01, r = 0.967, respectively, Fig. 2). No significant difference in catalase activity was found.
50
A o
z,.o >
|
3.0,
o
~ 2.o
!
/
1.0.
/
o
•
/
o
Selenium value (lug/l)
c
B ~.o >,
>
3.o~
i
2.0_
e
o 1.0.
/
50
7.~
16o
l~s Selenium volue (lu9/1)
Fig. 2. The relation between serum selenium value (#g/l) (X) and glutathione peroxidase activity (/~kat/g) (Y) in crude lymphocyte homogenate. A: Controls. • = normal controls; O = Other neurological patients. Regression equation: Y = -0.46 + 0.025X (2~ ~<0.05, r = 0.594). B: MS patients. @ = Patients in stable phase; d~ = Patients in acute phase. Regression equation: Y = -1.38 + 0.039X (2~ ~<0.01, i"- 0.967).
Granulocytes T a b l e 1A also shows the specific activities o f G S H - P x a n d associated enzymes of crude granulocyte h o m o g e n a t e . The G S S G - R D a n d G S H - P x activities were decreased in MS by 27% (not significant) a n d by 78% (2~ ~<0.01), respectively. G S H - P x : G S S G - R D ratio was 0.78 for the n o r m a l c o n t r o l group b u t 0.38 for the MS group (2~ ~< 0.05). W h e n MS data were c o m p a r e d to data from other neurological patients, similar differences as indicated above were found. The catalase activity was decreased by 56!~,; (2~ ~< 0.05) in MS patients c o m p a r e d to n o r m a l c o n t r o l values.
51
Comparison of lymphocyte- and granulocyte data The median lymphocyte value ofcatalase was lower than that of granulocytes. The activity of G-6-PD in lymphocytes was almost identical with that of granulocytes. On the other hand the activities of GSH-Px and GSSG-RD were higher in lymphocytes than in granulocytes (Table 1A).
Erythrocyte data In contrast to the lymphocyte and granulocyte data Table IB shows that the specific activities of catalase in erythrocyte haemolysate was increased by 52~o (27 ~ 0.05) in MS compared to the activity of normal controls. Compared to other neurological patients the activity was increased by 24~o (not significantly). No significant differences in hematocrit values were found.
Serum selenium values The serum selenium level in 7 MS patients was decreased by 15~ compared to the level in normal controls (Table 1B). However, the selenium values in the two MS patients in acute phase were decreased by 51~o and 28~o. DISCUSSION GSH-Px activity is distributed on at least two separate enzyme species (Lawrence and Burk 1976; Jensen et al. 1978). The major GSH-Px activity is due an enzyme containing four molecules of selenium per mole enzyme (Lyons et al. 1978). The minor GSH-Px activity is in preparative studies associated with glutathione transferase B (Prohaska and Ganther 1977). These two enzymes exhibit different affinity towards H202 and to organic peroxides. Therefore, assay of the total GSH-Px activity should not be performed at identical substrate concentrations for H202 and organic peroxides but as used previously by us and others, the assay procedure with organic peroxide should be performed at molarities about 5 times higher than that for the saturation limit for H202 (Jensen et al. 1978; Bergad et al. 1982). The present data confirm our previous finding of decreased GSH-Px activities in MS lymphocyte- and granulocyte cytosol and erythrocyte haemolysate (Jensen et al. 1980). Since that there are experimental evidences that inactivation of GSH-Px gives rise to an increased rate of peroxidation (Noguchi et al. 1973; Omaye and Tappel 1975) the MS cells with low GSH-Px activity may show an increased peroxidation rate. The two enzymes, GSSG-RD and G-6-PD, catalyse the formation of GSH and NADPH which are cofactors for the GSH-Px pathway. In severe peroxidative stress the activities of GSH-Px, GSSG-RD and G-6-PD are linearily correlated (Chow and Tappel 1972). However, in MS lymphocytes the decreased GSH-Px activity was associated with an increased G-6-PD activity. Similar enzymic changes are found in essential fatty acid (EFA) deficiency (Jensen and Clausen 1981; Dresser et al. 1982). Linoleic and arachidonic acids have indeed been found to be decreased in hematogenous cells of MS patients (Baker et al. 1963; Gul et al.
52
1970; Thompson 1973). The low GSH-Px activity may well give rise to peroxidative decomposition of EFA. This may secondarily change prostaglandin synthesis and enhance platelet aggregation (Nugteren and Hazelhof 1973; Srivastava et al. 1975; Trapp and Bernsohn 1978; Neu et al. 1982; Seregi et al. 1982). The changes found in catalase activity are inconclusive because decreased activity was found in granulocytes but increased levels in erythrocytes. However, the decreased granulocyte activity may be related to the specific role of these cells in the phagocytosis of foreign antigens (Johnston and Lehmeyer 1977). Perhaps the hypochlorite ions formed in the active phagocytosis in MS due to myetoperoxidase (Johnston and Lehmeyer 1977; Segal and Peters 1977) may inactivate cytosolic enzymes such as catalase and GSH-Px. According to our previous studies, a high intracellular selenium content in MS erythrocytes was associated with a decreased serum selenium level (Jensen et al. 1980). Thus a factor influencing the selenium release may be changed in MS. The finding of a significant correlation between lymphocyte GSH-Px activity and serum selenium level in controls as well as in the 7 MS patients argues for a direct incorporation of serum selenium into lymphocyte GSH-Px. ACKNOWLEDGEMENTS
Gratitude is expressed to Chief Physician K. Hyllested and to Mrs. B. Miiller, Mrs. I. Nielsen and Mrs. M. Bjornholt, The Department of Neurology, County Hospital of Roskilde. Technical assistance from Miss Lise Mfi.rup is gratefully acknowledged. REFERENCES Baker, R. W. R., R. H. S. T h o m p s o n and K.J. Zilkha (1963) Fatty acids composition of brain lecithins in multiple sclerosis, Lancet, i: 26. Bauer, J.D., P.G. Ackerman and G. Toro (1974) Clinical Laboratory Methods, The Mosby Comp., St. Louis, MO. Bergad, P.L., W.B. Rathbun and W. Linder (1982) Glutathione peroxidase from bovine lens A selenoenzyme, Exp. £:ve Res., 34:131 144. B6yum, A. (1968) Isolation of mononuclear cells and granulocytes from human blood, Stand. J. Clin. Lab. Invest., 21 (Suppl.): 77. Chow, C. K. and A. L. Tappel (1972) An enzymatic protective mechanism against lipid peroxidation damage to lungs of ozone exposed rats, Lipids, 7:518 524. Cohen, G., D. Dembiec and J. Marcus (1970) Measurement of catalase activity in tissue extracts, Anal. Biochem., 34:39 45. Croxton, F.E. (1959) Elementary Statistics with Applieations in Medieille and the Biological Sciences, Dover Publications, Inc.. New York. NY. Dresser, B. L., P.T. Russell and T . M . Ludwick (1982) Effects of l;at-free diet on fetal and maternal glucose-6-phosphate dehydrogenase and the timing of parturition in the mouse, Biol. Neonate, 41 : 252-257. Geigy (1965) Documenta Ge~y, Seient(/ic Tables, Geigy, Basel. Gul, S., A. D. Smith, R. H. S. T h o m p s o n , H. Payling Wright and K.J. Zilkha (1970) Fatty acid composition ofphospholipids from platelets and erythrocytes in multiple sclerosis, J. Neurol. Neurosurg. Po~chiat., 33:506 510. I U P A C - I U B (1974) Commission on Biochemical Nomenclature (CBN), Units o~" Enzymic Activity, Europ. J. Biochem., 45:1 3.
53 Jensen, G.E. and J. Clausen (1981) Glutathione peroxidase activity in vitamin E and essential fatty acid deficient rats, Nutr. Metab., 25:27 37. Jensen, G.E., V. K. S. Shukla, G. Gissel-Nielsen and J. Clausen (1978) Biochemical abnormalities in Batten's syndrome, Scand. J. Clin. Lab. Invest., 38: 309-318. Jensen, G.E., G. Gissel-Nielsen and J. Clausen (1980) Leucocyte glutathione peroxidase activity and selenium level in multiple sclerosis, J. Neurol. Sci., 48:61 67. Johnston, Jr., R. B. and J. E. Lehmeyer (1977) The involvement of oxygen metabolites from phagocytic cells in bactericidal activity and inflammation. In : A. M. Michelson, J. M. McCord and I. Fridovich (Eds.), Superoxide and Superoxide Dismutases, Academic Press, London, pp. 291-305. Kirkman, H.N. (1959) Glucose-6-phosphate dehydrogenase and human erythrocytes, Nature (Lond.), 184: 1291-1292. Lawrence, R.A. and R.F. Burk (1976) Glutathione peroxidase activity in selenium-deficient rat liver, Biochem. Biophys. Res. Comm., 71 : 952 958. Lous, P., C.M. Plum and M. Schou (1959) In: P. Astrup, K. Brochner-Mortensen and M. Faber (Eds.), Klinisk Laboratorie Teknik, August Bangs Forlag, Copenhagen, p. 316. Lyons, D. W., C.W. Hawkes, J.W. Forstrom, J. Zakowski, C.J. Dillard, D.E. Litov and A. L. Tappel (1978) Selenium-glutathione peroxidase - - Incorporation of selenium selenocysteine as the catalytic site and effect on in vivo lipid peroxidation, Fed. Proc., 37: 133%1341. Neu, I.S., M. Prosiegel and V. Pfaffenrath (1982) Platelet aggregation and multiple sclerosis, Aeta Neurol. Scand., 66:497 504. Noguchi, T., A.H. Cantor and M.L. Scott (1973) Mode of action of selenium and vitamin E in prevention of exudative diathesis in chicks, J. Nutr., 103:1502 1511. Nugteren, D.H. and E. Hazelhof (1973) Isolation and properties of intermediates in prostaglandin biosynthesis, Bioehim. Biophys. A cta, 326: 448461. Omaye, S.T. and A. L. Tappel (1975) Effect of cadmium chloride on the rat testicular soluble selenoenzyme, glutathione peroxidase, Res. Commun. Chem. Path. Pharmacol., 12:695 711. Pinto, R. E. and W. Bartley (1969) The effect of age and sex on glutathione reductase and glutathione peroxidase activities and on aerobic glutathione oxidation in rat liver homogenates, Biochem. J., 112:109 115. Porta, E.A., B.K.F. Ching and N.S. Joun (1977) Glutathione peroxidase system and microsomal lipoperoxidation in prenecrotic stages of dietary hepatic necrosis in rats, J. Nutr., 107: 1852-1858. Prohaska, J. R. and H.E. Ganther (1977) Glutathione peroxidase activity of glutathione-S-transferase purified from rat liver, Biochem. Biophys. Res. Commun., 86: 437-445. Rotruck, J. T., A. L. Pope, H. E. Ganther, A. B. Swanson, D.G. Hagerman and W.G. Hoekstra (1973) Selenium -- Biochemical role as a component of glutathione peroxidase, Science, 179: 558-560. Schumacher, G.A., G. Beebe, R.F. Kibler, L.T. Kurland, J.F. Kurtzke, F. McDowell, B. Nagler, B. Sibley, W. W. Tourtellotte and T. L. Willman (1965) Problems of experimental trials of therapy in multiple sclerosis (Report by the Panel on the Evaluation of Experimental Trials of Therapy in Multiple Sclerosis), Ann. N.Y. Acad. Sei., 122: 552-568. Segal, A.W. and T.J. Peters (1976) Analytical subcellular fractionation of human granulocytes with special reference to the localization of enzymes involved in microbicidal mechanisms, Clin. Sei. Mol. Med., 52:429 442. Seregi, A., P. Serf6z6, Z. Margl and A. Schaefer (1982) On the mechanism of the involvement of monoamine oxidase in catecholamine-stimulated prostaglandin Biosynthesis in particulate fraction of rat brain homogenates : role of hydrogen peroxide, J. Neurochem., 38 : 2(~27. Srivastava, K. C., T. Fog and J. Clausen (1975) The synthesis of prostaglandins in platelets from patients with multiple sclerosis, Acta Neurol. Seand., 51: 193-199. Thompson, R. H. S. (1973) Fatty acid metabolism in multiple sclerosis, Biochem. Soc. Symp., 35:103 111. Trapp, B. D. and J. Bernsohn (1978) Essential fatty acid deficiency and CNS myelin Biochemical and morphological observations, J. Neurol. Sei., 37:249 266.
45
Elsevier
GLUTATHIONE PEROXIDASE AND REDUCTASE, GLUCOSE-6-PHOSPHATE DEHYDROGENASE AND CATALASE ACTIVITIES IN MULTIPLE SCLEROSIS
G.E. JENSEN and J. CLAUSEN
The Laboratory of Biochemistry and Toxicology, Institute of Biology and Chemistry, University of Roskilde, D K-4000 Roskilde and The Neurochemical Institute, 58 R~tdmandsgade, D K-2200 Copenhagen N (Denmark) (Received 9 May, 1983) (Revised, received 2 August, 1983) (Accepted 17 August, 1983)
SUMMARY
Our previous studies have demonstrated a decreased glutathione peroxidase (GSH-Px) activity of erythrocytes and leucocytes from multiple sclerosis (MS) patients. In the present communication these activities were compared with the activities of associated enzymes (glutathione reductase (GSSG-RD), glucose-6phosphate dehydrogenase (G-6-PD) and catalase). All enzymic activities were compared between MS patients, other neurologic patients (ON patients) and normal control individuals. Compared to data of ON patients and normal controls, in MS the ratio of GSHPx/GSSGRD in lympho- and granulocytes was significantly decreased (2a ~<0.05) by 35~ and 51~o, respectively. The significant correlation between GSSG-RD and the GSH-Px activity (2a ~<0.05, r = 0.501) found in control lymphocytes was not present in MS lymphocytes. However, the lymphocyte GSH-Px activities of controls as well as of MS correlated with the corresponding serum selenium levels (2~ ~<0.05, r = 0.594 and 2~ ~<0.01, r = 0.967, respectively). The G-6-PD activity was insignificantly increased by 41 ~o in MS lymphocytes compared to normal control. Catalase activity was unchanged in lymphocytes but decreased 50~o in MS granulocytes compared to normal control. No significant differences were found between MS and the ON group. The catalase activity of MS erythrocytes was increased by 63~ (2a ~<0.05) in comparison with both the normal control and ON data.
This work was financially supported by a grant from the "Kobmand Sven Hansen og Hustru lna Hansens Fond". 0022-510X/84/$03.00 © 1984 Elsevier Science Publishers B.V.
46
Key words: Catalase Glucose-6-phosphate dehydrogenase Glutathione reductase Multiple sclerosis
Glutathione peroxidase
INTRODUCTION Previous investigations comparing some enzymic activities in normal controls and multiple sclerosis (MS) patients demonstrated a significantly decreased glutathione peroxidase (GSH-Px) activity in MS erythrocyte haemolysate and in MS cytosol of lymphocytes and granulocytes (Jensen et al. 1980). Therefore, GSH-Px, which counteracts autoperoxidations (Porta et al. 1977), may play a central role in the development of pathological changes in MS patients. Although selenium is an integral component of GSH-Px (Rotruck et al. 1973) the serum selenium level in MS patients is only insignificantly decreased (Jensen et al. 1980). GSH-Px detoxicates hydrogen peroxide via the GSH-Px pathway: G-6-P ~
~
NADP ~
G-6-PD 6-PG ~
~
(~*
OSSG-RD
~ " NADPH + H + -'')
C
2 GSH
S
H,O,
GSH-Px
Gsso J
C-
where G-6-P and 6-PG are glucose 6-phosphate and 6-phosphogluconate, respectively. GSSG and GSH are oxidized and reduced forms of glutathione, and finally G-6-PD and GSSG-RD are glucose-6-phosphate dehydrogenase and glutathione reductase, respectively. However, the enzyme catalase also detoxicates H:O~. Therefore, it was tempting to relate the selenium level and the activity of GSHPx with that of GSSG-RD, G-6-PD and catalase in haematogenous cells. MATERIALSAND METHODS
Patient material Venous blood samples from MS patients were collected from the Department of Neurology, County Hospital of Roskilde, Denmark. Twelve patients were clinically stable and 2 were in acute relapse. No patient was on cortisone theraphy. The diagnosis of MS was made on clinical criteria (Schumacher et al. 1965) by local neurologists and confirmed by Dr. K. Hyllested. Control material The control material comprised of healthy volunteers and patients with neurological disease other than MS. These included patients with cerebral haemorrhage (2), Parkinson's disease (1), trauma (1), ataxia (1), lumbar and cervical disc prolapse (4) and others (6). The control individuals as well as the MS patients all originated from the Danish island Zealand.
47
Methods Isolation of serum and erythrocytes from venous blood and preparation of erythrocyte haemolysate were made as previously described (Jensen et al. 1978). Lymphocytes and granulocytes were isolated by their specific gravity by the lymphoprep method (Nyggtrd and Comp. A/S, Oslo, Norway) (B6yum 1968). Remnants of erythrocytes were eliminated by osmotic shocking in distilled water. 1.3 × 108 cells were suspended in 2 ml 175 mmol/1 KC1. Determination of haemoglobin, total protein and selenium These assays were made as previously described (Lous et al. 1959; Jensen et al. 1978). Assay of microhematocrit was made by conventional routine procedures in a "high speed centrifuge" (Bauer et al. 1974). Enzymic assay G-6-PD (E.C. 1.1.1.49), GSSG-RD (E.C. 1.6.4.2), GSH-Px (E.C. 1.11.1.9) and catalase (E.C. 1.11.1.6) were assayed under optimal kinetic conditions (Kirkman 1959; Pinto and Bartley 1969; Cohen et al. 1970; Jensen et al. 1980). The blanks used were in all cases the reagent mixtures with distilled water instead of source of enzyme. The units of the GSH-Px pathway enzymes were expressed as units ofkat (1 kat = 1 mol coenzyme transformed per second (IUPAC-IUB 1974) specific activities were expressed as kat units/g protein. The specific unit of catalase was expressed in terms of the first-order reaction rate constant k, e.g. k/g protein according to Cohen et al. (1970). Chemicals All chemicals were of highest obtainable purity from British Drug Houses, Poole, Dorset, England. Enzymes were from Sigma Chemical Company, Saint Louis, U.S.A., or from Boehringer, Mannheim, F.R.G. Statistical assays The median, 10 and 90~o deciles were computed. The level of significance was assayed by means of Wilcoxon's non-parametric method (Geigy 1965). Spearman Rank's correlation test was used for the correlation purpose. The level of significance was set at 5~o. When lines on those best fit were drawn, they were estimated by regression analysis (Croxton 1959). RESULTS
Lymphocytes Table 1A shows the specific activities of GSH-Px and associated enzymes (catalase, G-6-PD and GSSG-RD) of crude lymphocyte homogenate. In MS the G-6-PD activity was increased by 41~o compared to the median activity of normal controls [not significant (NS)]. The GSSG-RD and GSH-Px activities were decreased
48 TABLE 1 DATA FROM N O R M A L CONTROLS (NC), MS PATIENTS (MS) A N D OTHER N E U R O L O G I C A L PATIENTS (ON)
(,4) Catalase, G-6-PD, GSH-Px, GSSG-RD activities in crude lymphocyte (L Y) and granuloeyte (GR) homogenate Catalase activity (k/g protein)
G-6-PD activity (,ukat/g protein )
GSSG-RD activity (/~kat/g protein)
GSH-Px activity (#kat/g protein)
GSH-PxGSSG-RD ratio
NC LY
n Median 10 90% decile
17 0.59 0.36 0.96
13 0.74 0.38 1.58
15 0.72 0.51 0.86
18 1.91 0.63 3.23
12 2.65 1.72 4.04
GR
n Median 0 90% decile
17 1.55 0.66 3.07
12 1.01 0.20 2.25
15 0.64 0.39 0.91
20 0.63 0.21 1.01
13 0.78 0.25 1.11
LY
n Median 10 90% decile
14 0.47 0.20 1.27
15 0.92 0.52 1.69
l0 0.91 0.44 1.30
14 1.92 0.49 2.07
10 2.12 1.57 3.54
GR
n Median 10 90% decile
13 0.94 0.56 1.48
II 0.97 0.45 2.18
9 0.59 0.39 0.91
13 0.71 (I.28 1.45
9 0.51 0.25 1.56
LY
n Median 10 90%decile
l0 0.45 0.16 1.19
8 1.04 0.39 1.79
9 0.66 0.22 0.87
14 (I.72 0.51 2.31
9 1.73 0.40 3.18
GR
n Median 10 90%decile
7 0.69 0.14 1 . 2 5
7 0.90 0.14~1.76
9 0.47 0.10 1.13
9 0.14 0.06 0.42
9 0.38 0.06 0.76
ON
MS
(B) Catalase activity in ervthrocytes and selenium level in serum Catalase activity (k x 106/tool Hgb)
Selenium level (,ug/1)
NC
n Median 10 90% decile
23 13.5 9.5 29.0
21 95 84~ 140
ON
n Median 10 901'o decile
10 16.2 10.0 19.(/
10 78 65 104
MS
n Median 10 90!,, decile
8 22.0 9.0 29.5
7 81 60 99
49
~5
A
_~ z,.0 ~o [3.
'
3.0.
20.
o • . t ~ o
o /
o
--°_o/~/ ~ o
o
o
• •
o.'s 0;7
11o
1.~
2b
GSS6-RD-activitY0Jkat ~ protein) c
05
B >,
>_ 3.0
8 ~ 2.0. i
I
e
c.D
1.0. • O
•
e o
• o
o's
o.~
i.o
1.'s
2'.o "~ GSSG-RD - activity (~kat,~ protein)
F i g . 1. T h e relationship between gh,.,athione reductase activity (#kat/g protein) (X) and glutathione peroxidase activity ~kat/g protein) (Y) in crude lymphocyte homogenate. A : C o n t r o l s . • = normal controls; O = other neurological patients. Regression equation: Y = 1.07 + 0 . 9 6 X (2~ ~< 0.05, r = 0.501). B : MS patients. @ = Patients in stable p h a s e ; @ = Patients in acute phase (no significant correlation).
by 8% (NS) and 51% (2a ~< 0.01), respectively. GSH-Px: GSSG-RD-ratio was 2.65 for the normal control group but 1.73 for the MS group (2~ ~ 0.05). When MS data were compared to data from other neurological patients, similar differences as indicated above were found. In controls the individual GSH-Px activities correlated with the corresponding G S S G - R D activities (2~ ~< 0.05, r = 0.501). In contrast no significant correlation was found in MS (Fig. 1). The GSH-Px activities of controls and MS correlated with the corresponding serum selenium level (2~ ~< 0.05, r = 0.594 and 2 , ~<0.01, r = 0.967, respectively, Fig. 2). No significant difference in catalase activity was found.
50
A o
z,.o >
|
3.0,
o
~ 2.o
!
/
1.0.
/
o
•
/
o
Selenium value (lug/l)
c
B ~.o >,
>
3.o~
i
2.0_
e
o 1.0.
/
50
7.~
16o
l~s Selenium volue (lu9/1)
Fig. 2. The relation between serum selenium value (#g/l) (X) and glutathione peroxidase activity (/~kat/g) (Y) in crude lymphocyte homogenate. A: Controls. • = normal controls; O = Other neurological patients. Regression equation: Y = -0.46 + 0.025X (2~ ~<0.05, r = 0.594). B: MS patients. @ = Patients in stable phase; d~ = Patients in acute phase. Regression equation: Y = -1.38 + 0.039X (2~ ~<0.01, i"- 0.967).
Granulocytes T a b l e 1A also shows the specific activities o f G S H - P x a n d associated enzymes of crude granulocyte h o m o g e n a t e . The G S S G - R D a n d G S H - P x activities were decreased in MS by 27% (not significant) a n d by 78% (2~ ~<0.01), respectively. G S H - P x : G S S G - R D ratio was 0.78 for the n o r m a l c o n t r o l group b u t 0.38 for the MS group (2~ ~< 0.05). W h e n MS data were c o m p a r e d to data from other neurological patients, similar differences as indicated above were found. The catalase activity was decreased by 56!~,; (2~ ~< 0.05) in MS patients c o m p a r e d to n o r m a l c o n t r o l values.
51
Comparison of lymphocyte- and granulocyte data The median lymphocyte value ofcatalase was lower than that of granulocytes. The activity of G-6-PD in lymphocytes was almost identical with that of granulocytes. On the other hand the activities of GSH-Px and GSSG-RD were higher in lymphocytes than in granulocytes (Table 1A).
Erythrocyte data In contrast to the lymphocyte and granulocyte data Table IB shows that the specific activities of catalase in erythrocyte haemolysate was increased by 52~o (27 ~ 0.05) in MS compared to the activity of normal controls. Compared to other neurological patients the activity was increased by 24~o (not significantly). No significant differences in hematocrit values were found.
Serum selenium values The serum selenium level in 7 MS patients was decreased by 15~ compared to the level in normal controls (Table 1B). However, the selenium values in the two MS patients in acute phase were decreased by 51~o and 28~o. DISCUSSION GSH-Px activity is distributed on at least two separate enzyme species (Lawrence and Burk 1976; Jensen et al. 1978). The major GSH-Px activity is due an enzyme containing four molecules of selenium per mole enzyme (Lyons et al. 1978). The minor GSH-Px activity is in preparative studies associated with glutathione transferase B (Prohaska and Ganther 1977). These two enzymes exhibit different affinity towards H202 and to organic peroxides. Therefore, assay of the total GSH-Px activity should not be performed at identical substrate concentrations for H202 and organic peroxides but as used previously by us and others, the assay procedure with organic peroxide should be performed at molarities about 5 times higher than that for the saturation limit for H202 (Jensen et al. 1978; Bergad et al. 1982). The present data confirm our previous finding of decreased GSH-Px activities in MS lymphocyte- and granulocyte cytosol and erythrocyte haemolysate (Jensen et al. 1980). Since that there are experimental evidences that inactivation of GSH-Px gives rise to an increased rate of peroxidation (Noguchi et al. 1973; Omaye and Tappel 1975) the MS cells with low GSH-Px activity may show an increased peroxidation rate. The two enzymes, GSSG-RD and G-6-PD, catalyse the formation of GSH and NADPH which are cofactors for the GSH-Px pathway. In severe peroxidative stress the activities of GSH-Px, GSSG-RD and G-6-PD are linearily correlated (Chow and Tappel 1972). However, in MS lymphocytes the decreased GSH-Px activity was associated with an increased G-6-PD activity. Similar enzymic changes are found in essential fatty acid (EFA) deficiency (Jensen and Clausen 1981; Dresser et al. 1982). Linoleic and arachidonic acids have indeed been found to be decreased in hematogenous cells of MS patients (Baker et al. 1963; Gul et al.
52
1970; Thompson 1973). The low GSH-Px activity may well give rise to peroxidative decomposition of EFA. This may secondarily change prostaglandin synthesis and enhance platelet aggregation (Nugteren and Hazelhof 1973; Srivastava et al. 1975; Trapp and Bernsohn 1978; Neu et al. 1982; Seregi et al. 1982). The changes found in catalase activity are inconclusive because decreased activity was found in granulocytes but increased levels in erythrocytes. However, the decreased granulocyte activity may be related to the specific role of these cells in the phagocytosis of foreign antigens (Johnston and Lehmeyer 1977). Perhaps the hypochlorite ions formed in the active phagocytosis in MS due to myetoperoxidase (Johnston and Lehmeyer 1977; Segal and Peters 1977) may inactivate cytosolic enzymes such as catalase and GSH-Px. According to our previous studies, a high intracellular selenium content in MS erythrocytes was associated with a decreased serum selenium level (Jensen et al. 1980). Thus a factor influencing the selenium release may be changed in MS. The finding of a significant correlation between lymphocyte GSH-Px activity and serum selenium level in controls as well as in the 7 MS patients argues for a direct incorporation of serum selenium into lymphocyte GSH-Px. ACKNOWLEDGEMENTS
Gratitude is expressed to Chief Physician K. Hyllested and to Mrs. B. Miiller, Mrs. I. Nielsen and Mrs. M. Bjornholt, The Department of Neurology, County Hospital of Roskilde. Technical assistance from Miss Lise Mfi.rup is gratefully acknowledged. REFERENCES Baker, R. W. R., R. H. S. T h o m p s o n and K.J. Zilkha (1963) Fatty acids composition of brain lecithins in multiple sclerosis, Lancet, i: 26. Bauer, J.D., P.G. Ackerman and G. Toro (1974) Clinical Laboratory Methods, The Mosby Comp., St. Louis, MO. Bergad, P.L., W.B. Rathbun and W. Linder (1982) Glutathione peroxidase from bovine lens A selenoenzyme, Exp. £:ve Res., 34:131 144. B6yum, A. (1968) Isolation of mononuclear cells and granulocytes from human blood, Stand. J. Clin. Lab. Invest., 21 (Suppl.): 77. Chow, C. K. and A. L. Tappel (1972) An enzymatic protective mechanism against lipid peroxidation damage to lungs of ozone exposed rats, Lipids, 7:518 524. Cohen, G., D. Dembiec and J. Marcus (1970) Measurement of catalase activity in tissue extracts, Anal. Biochem., 34:39 45. Croxton, F.E. (1959) Elementary Statistics with Applieations in Medieille and the Biological Sciences, Dover Publications, Inc.. New York. NY. Dresser, B. L., P.T. Russell and T . M . Ludwick (1982) Effects of l;at-free diet on fetal and maternal glucose-6-phosphate dehydrogenase and the timing of parturition in the mouse, Biol. Neonate, 41 : 252-257. Geigy (1965) Documenta Ge~y, Seient(/ic Tables, Geigy, Basel. Gul, S., A. D. Smith, R. H. S. T h o m p s o n , H. Payling Wright and K.J. Zilkha (1970) Fatty acid composition ofphospholipids from platelets and erythrocytes in multiple sclerosis, J. Neurol. Neurosurg. Po~chiat., 33:506 510. I U P A C - I U B (1974) Commission on Biochemical Nomenclature (CBN), Units o~" Enzymic Activity, Europ. J. Biochem., 45:1 3.
53 Jensen, G.E. and J. Clausen (1981) Glutathione peroxidase activity in vitamin E and essential fatty acid deficient rats, Nutr. Metab., 25:27 37. Jensen, G.E., V. K. S. Shukla, G. Gissel-Nielsen and J. Clausen (1978) Biochemical abnormalities in Batten's syndrome, Scand. J. Clin. Lab. Invest., 38: 309-318. Jensen, G.E., G. Gissel-Nielsen and J. Clausen (1980) Leucocyte glutathione peroxidase activity and selenium level in multiple sclerosis, J. Neurol. Sci., 48:61 67. Johnston, Jr., R. B. and J. E. Lehmeyer (1977) The involvement of oxygen metabolites from phagocytic cells in bactericidal activity and inflammation. In : A. M. Michelson, J. M. McCord and I. Fridovich (Eds.), Superoxide and Superoxide Dismutases, Academic Press, London, pp. 291-305. Kirkman, H.N. (1959) Glucose-6-phosphate dehydrogenase and human erythrocytes, Nature (Lond.), 184: 1291-1292. Lawrence, R.A. and R.F. Burk (1976) Glutathione peroxidase activity in selenium-deficient rat liver, Biochem. Biophys. Res. Comm., 71 : 952 958. Lous, P., C.M. Plum and M. Schou (1959) In: P. Astrup, K. Brochner-Mortensen and M. Faber (Eds.), Klinisk Laboratorie Teknik, August Bangs Forlag, Copenhagen, p. 316. Lyons, D. W., C.W. Hawkes, J.W. Forstrom, J. Zakowski, C.J. Dillard, D.E. Litov and A. L. Tappel (1978) Selenium-glutathione peroxidase - - Incorporation of selenium selenocysteine as the catalytic site and effect on in vivo lipid peroxidation, Fed. Proc., 37: 133%1341. Neu, I.S., M. Prosiegel and V. Pfaffenrath (1982) Platelet aggregation and multiple sclerosis, Aeta Neurol. Scand., 66:497 504. Noguchi, T., A.H. Cantor and M.L. Scott (1973) Mode of action of selenium and vitamin E in prevention of exudative diathesis in chicks, J. Nutr., 103:1502 1511. Nugteren, D.H. and E. Hazelhof (1973) Isolation and properties of intermediates in prostaglandin biosynthesis, Bioehim. Biophys. A cta, 326: 448461. Omaye, S.T. and A. L. Tappel (1975) Effect of cadmium chloride on the rat testicular soluble selenoenzyme, glutathione peroxidase, Res. Commun. Chem. Path. Pharmacol., 12:695 711. Pinto, R. E. and W. Bartley (1969) The effect of age and sex on glutathione reductase and glutathione peroxidase activities and on aerobic glutathione oxidation in rat liver homogenates, Biochem. J., 112:109 115. Porta, E.A., B.K.F. Ching and N.S. Joun (1977) Glutathione peroxidase system and microsomal lipoperoxidation in prenecrotic stages of dietary hepatic necrosis in rats, J. Nutr., 107: 1852-1858. Prohaska, J. R. and H.E. Ganther (1977) Glutathione peroxidase activity of glutathione-S-transferase purified from rat liver, Biochem. Biophys. Res. Commun., 86: 437-445. Rotruck, J. T., A. L. Pope, H. E. Ganther, A. B. Swanson, D.G. Hagerman and W.G. Hoekstra (1973) Selenium -- Biochemical role as a component of glutathione peroxidase, Science, 179: 558-560. Schumacher, G.A., G. Beebe, R.F. Kibler, L.T. Kurland, J.F. Kurtzke, F. McDowell, B. Nagler, B. Sibley, W. W. Tourtellotte and T. L. Willman (1965) Problems of experimental trials of therapy in multiple sclerosis (Report by the Panel on the Evaluation of Experimental Trials of Therapy in Multiple Sclerosis), Ann. N.Y. Acad. Sei., 122: 552-568. Segal, A.W. and T.J. Peters (1976) Analytical subcellular fractionation of human granulocytes with special reference to the localization of enzymes involved in microbicidal mechanisms, Clin. Sei. Mol. Med., 52:429 442. Seregi, A., P. Serf6z6, Z. Margl and A. Schaefer (1982) On the mechanism of the involvement of monoamine oxidase in catecholamine-stimulated prostaglandin Biosynthesis in particulate fraction of rat brain homogenates : role of hydrogen peroxide, J. Neurochem., 38 : 2(~27. Srivastava, K. C., T. Fog and J. Clausen (1975) The synthesis of prostaglandins in platelets from patients with multiple sclerosis, Acta Neurol. Seand., 51: 193-199. Thompson, R. H. S. (1973) Fatty acid metabolism in multiple sclerosis, Biochem. Soc. Symp., 35:103 111. Trapp, B. D. and J. Bernsohn (1978) Essential fatty acid deficiency and CNS myelin Biochemical and morphological observations, J. Neurol. Sei., 37:249 266.