Acta Neiirol. Scnndinav. 56, 542-550, 1977

* The Laboratory of Biochcmistry and Toxicology, The Institute of Life Sciences and Chemistry, Roskilde University, Roskilde and * The Neurochemical Institute, Copenhagen, Denmark.

EKYTHKOCYTE GLUTATHIONE PEROXIDASE DEFICIENCY I N MULTIPLE SCLEROSIS

v. K. s. SHUKLA *, GUKDEEGESKOV JENSEN*

and

JBRGEN CLAUSEN

ABSTRACT The present study demonstrates a significant decrease i n glutathione pcroxidase activity in erythrocytes of 24 patients with multiple sclerosis (MS) when t h e data are expressed as enzymic units per mg hemoglobin and compared to data from normal controls without known family history of demyelinating diseases, Since selenium is an essential part of glutathione peroxidase, this study also compares the topographic diffcrenccs in selenium availability (expressed a s selenium content of forage) with the prevalence and death rates of MS in the USA. The comparison cannot exclude the possibility of a relationship betwecn low selenium content and high prevalence of MS. The data are discussed in relationship to current theories on the pathogenesis of MS.

Pathochemical studies during recent years have shown that multiple sclerosis (11s) is associated with decreased serum linoleate level (Baker et al. 1961, Tichy e f al. 1969) and changes in the fatty acid pattern of the brain lipids (Clausen & Hansen 1970, Gerstl et al. 1970, Alling et al. 1971). Recently, Thoiripson (1973) has elaborated these biochemical abnormalities in relation to the pathogenesis of the disease. It has been shown that the erythrocytes are also altered in this disease. Thus Lasslo (1961) reported a highly significant increase in osmotic fragility of red blood cells, a finding confirmed by Caspary et al. (1967). PZum & Fog (1959) showed increase in mean erythrocyte diameter in niultiple sclerosis. This was confirmed by Prineas (1968) who correlated the increase with the activity of the disease. This phenomenon may be explained by membrane changes and may well be due to abnormal peroxidalion processes in the erythrocytes. Glutathione peroxidase ( GSH-Px) protects biological membranes

543 against oxidative challenge (Flohe & Zimmermann 1970). Awashty et al. (1975) have shown selenium to be a n essential nutrient for man and they have demonstrated that selenium functions as an integral part OP glutathione peroxidase (GSH-Px) . Recently Westermarck & Sandholm ( 1977) showed decreased erythrocyte glutathione peroxidase ( GSH-Px) activity in Finnish type of iieuronal ceroid lipofuscinosis (NCL) which was shown to be corrected by selenium supplementation. Wikstriim et al. (1976) reported low selenium content of whole blood in MS patients from the high risk areas of Finland. These above-mentioned facts promptrd us to study the glutathione peroxidase (GSH-Px) activity in M S erythrocytes u s h g three different peroxides.

MATERIALS AND METHODS Patient material: Blood samples were collected from 24 MS patients (from the MS rehabilitation Centre, Haslev, Denmark) comprising 1 2 men and 12 women aged 31-61 gears. The diagnosis of M S was made on the basis of the clinical criterias previously used (Offner et al. 1974). The diagnoses were made by local neurologists and confirmed by the consultant doctors a t the Haslev centre: Chief Physicians T. Fog (M.D.) and Vivc Larsen. Confrol material: The control material comprised healthy volunteers totalling 18 pcrsons, 10 mcn and 8 women aged 15-76 years, with no family background of demyelination diseases. The control individuals as well as the MS patients originated from the Danish Island of Zealand. Enz1ime sourre anti assag o f hemoglobin : Preparation of erythrocyte hemolysate i n Drahkins reagent as well a s assay of hemoglobin was followed as described earlier ( E g e s k o o Jensen rt al. 1978). Assag of glutathione peroxidase (GSH-Px) (E.C.l.li.1.9). Enzyme activity was measured under optimal conditions by a modification of t h e procedure of Paglia h Valentine (1967): Enzyme sourcc (0.1 ml) was added t o 2.80 m l cold reaction mixture (0") which consisted of 2.58 m l 0.05 hI sodium phosphate buffer (pH 7.4, containing 0.005 M EDTA), 0.010 m l 1.125 hi Nah',, 0.10 m l 0.15 M GSH (rcduccd glutathione), 0.01 m l glutathione reductase (Hoecht, Germany, 1 E.U. per mg p r o t e i d p e r 1 ml) and 0.100 ml 0.0084 M NADPH. The reaction mixture was allowed to incubate 5 min a t 22" C in the quartz cuvette (1 cm path length) of a Bcckman DU spectrophotometcr, Acta C I11 equipped with an automatic recorder. The initiation of the reaction was followed by the addition of 0.10 ml peroxide solution (50 mM cumcme h>dropcroxide, 90 mM t-hutyl hydropcroxide or 2.2 mM H 2 0 2 (Lawrence & Burk 1976, Egcd;oo Jensen et al. 1978). Absorbance a t 340 nm was recorded for 10 min. The enzgmic activity was calculated from the change in extinction per min. The extinction of t h e blank consisted of t h e mixture where t h e enzyme source was replaced by distilled water was substracted. The enzyme activity was calculated by means of the molar extinction coefficient E = 6.22 cm%/,,molc and was expressed a s n k a t / m g hemoglobin where 1 kat = 1 mole/sec of KADPH oxidized. A l l assayes were done i n triplicate. Chemicals. All chemicals were of highest obtainable purity from British Drug Houses, Poole, Dorset, England and enzymes and coenzymes were from Sigma

544 Chemical Company, USA and Boehringer, Manheim, West Germany. Organic peroxides were kindly provided by Peroxid-Chemie GmbH D-8023 HBllriegelskreuth bei Miinchen, West Germany. Statistical assay: The mean, standard deviation, median and 10 and 90 per cent deciles were computed. The level of significance was assayed by means of Wilcoxon’s nan-parametric method (Geigy 1965). The level of significance was set at 5 per cent level. RESULTS

The results of the assay of glutathione peroxidase (GSH-Px) activity using three different perixides in patients and in controls are presented in Tables 1-3. The glutathione peroxidase ( GSH-PX) activities were significantly decreased in erythrocytes of MS patients as compared to controls with all three peroxides (for H,O,: 2 a 5 0.01, cumenehydroperoxide: 2 (Y 5 0.05 and t-butylhydroperoxide: 2 (Y 5 0.01). Related to median values the glutathione peroxidase ( GSH-Px) activities were decreased about 40 per cent with hydrogen peroxide, 16 per cent with cumene hydroperoxide and 24 per cent with t-butylhydroperoxide. Furthermore the patients showed a lower range than controls in all the three cases. DISCUSSION

It has been hypothesized that selenide (Se--) is an active part of nonheme iron containing proteins in cellular membranes (Frost 1973). Glutathione peroxidase seems to be the enzyme which catalyzes the elimination of organic hydroperoxides such as those formed by oxidative damage (Necheles 1974, Flohe et al. 1976). A low peroxidase activity in MS may give rise to decreased stability of membrane structure and may thus contribute to the pathological changes found in MS paients. The following alternative explanations for the low erythrocyte peroxidase in MS patients may be given: 1. MS patients may possess a low uptake of selenium, an essential part of the glutathione peroxidase molecule, similar to the low peroxidase activity in selenium deficient mammals ( S f o p f o r d e f al. 1976, Rotruck et al. 1972, 1973), where it gives rise to hepato- and myelopathies (Gissel-Nielsen 1973, Floht et al. 19761, but testes and pancreas may also be affected. 2. An increased urinary excretion of selenium of unknown reasons, 3. The low peroxidase level may be related to a persistent (vinis) infection associated with MS. In relationship to this last point epidemiological studies (Adams & Zmagawa 1962, Panilius 1969, A m m i t z b d l &

545 Table I . llssay o f glutathione peroxide (GSH-Px) activity in erythrocytes. Substrate: Hydrogen peroxide. Activity expressed as n kat/mg hemoglobin x 48.1.

Normal values Age Sex'

Code

N-1 N-2 N-3 N-4 N-5 N-6 N-7 N-8 N-9

N-1 0 N-11 N-12 N-13 N-14 N-15 N-16 N-17 N-18

45 28 26 21 20 19 21 22 22 20 19 15 32 76 71 42 37 46

GSH-Px

M M

16.0 13.3 9.1 7.7 8.5 9.5 8.3 7.6 7.9 8.6 8.4 1.3 8.0 6.8

M M F F M F

M M F

F F M F F M M

Mean + SD Range Median Decile 10 70 90 7 0

6.0

8.3 11.2 7.4

8.9 f 2.4 6.0 16.0 8.3 6.8 13.3

-

Code P-1 P-2 P-3 P-4 P-5 P-6 P-7

P-8 P-9 P-10 P-1 1 P-12 P-13 P-14 P-15 P-16 P-17 P-18 P-19 P-20 P-21 P-22 P-23 P-24

Patient values Age Sex' 39 61

61 57

M M M

M

61

F

46 48 45 47 58 54 52 45 41 31 45 57 60 32 56 56 54 52 48

F F

M M F M F

F F M F F F

M F M F M M

GSH-Px 3.9 4.5 6.0 5.5 6.4 4.3 4.5 4.7 5.5 6.8 3.1 6.3 6.7 5.7 5.1 4.8 5.0 4.8 4.9 4.5 6.5 7.3 4.5 5.6 5.3 -c 1.0 1.3 3.7 5.0 4.3 6.7

-

M : male, F :female.

Clausen 19'12, Beck h Clausen 1977) have revealed increased measles virus antibody liters in serum of MS patients, associated with oligoclonal immune reactions of the spinal fluid (Link 1967, Clausen, Fog & Garde 1973). These data, taken together with the demonstrated abnormalities in the lymphocyte response against measles virus ( O f f n e r , Konat & Clausen 1974, Levy, Auerbach & Hayes 1976) may indicate MS to be associated with abnormalities in the immune system especially in 36

ACTA NEUROL. SCAND.

56, 6

546 Table 2. A s s a ~of glutathione peroride (GSH-PI) a c t i v i t y i n erythrocytes. Substrate : Cumene hydroperoxide. Aotivity expressed as n kat/mg hemoglobin X 48.1.

Code

Normal values Age Sex*

N-1 N-2 N-3 N-4 N-5 N-6 N-7 N -8 N-9 N-1 0 N-1 1 N-12 N-13 N-14 N-15 N-16 N-17

N-18

45 28 26 21 20 19 21 22 22 20 19 15 32 76 71 42 37 46

Mean -C SD Range Median Decile 10 % 90 7 0

M ni

M M

F F M F M M

F

F F M F

F M

M

GSH-Px

Code

14.8 12.4 8.2 7.1 9.4 7.7 9.0 7.7 6.7 9.1 8.5 6.9 9.1 9.2 8.2 6.7 8.1 6.6

P-1 P-2 P-3 P-4 P-5 P-6 P-7 P-8 P-9 P-1 0 P-1 1 P-12 P-13 P-14 P-15 P-16 P-17 P-18 P-19 P-20 P-21 P-22 P-23 P-24

8.6 -+ 2.1 6.6 14.8 8.2

-

6.7

12.4

Patient values Age Sex'

39 61 61 57 61 46 48 45 47 58 54 52 45 41 31 45 57 60 32 56 56 54 52 48

M M M M F F F M

M F M F F F M F F

F M

F M F M

M

GSH-Px 3.1 4.3 8.3 5.1 4.7 6.0 4.0 5.0 9.7 10.7 2.2 8.1 9.9 9.4 6.2

6.7 8.6 5.9 7.0 6.2 12.3 9.4 6.9 7.1 7.0 +. 2.5 2.2 -12.3 6.9 4.0 9.9

M: male, F: female.

the delayed hypersensitivity reactions. Since infectious diseases associated with delayed hypersensitivity, i.e. leprosy and tuberculosis are characterized by low peroxidase activities (Hokamo et al. 1974), the decreased peroxidase in M S may be secondary to the immunological abnormalities. Comparison of epidemiological studies on the occurrence of M S in USA registered either as prevalence rate or death rate (Kurtzke 1973)

547 Table 3. A s s a ~of glutathione peroxidase (GSH-Px) activity in eruthrocutes. Subs’trate: t - l u t y l hydroperoxide. Activity expressed as n kat/mg hemoglobin x 48.1.

Code

Normal values Age Sex‘

N-l N-2 N-3 N-4 N-5 N-6 N-7 N-8 N-9 N-l 0 N-l 1

N-12 N-13 N-14 N-15 N-16 N-17 N-18

45 28 26 21 20 19 21 22 22 20 19 15 32 76 71 42 37 46

M M M M F

F M F M M

F F F M

F F M

M

GSH-Px 14.5 13.0 9.6 8.3 7.0 8.8 9.5 7.8 6.3 9.2 8.5 6.7 10.4 10.3 8.4 6.8 9.3 8.4

Code

P-1 P-2 P-3 P-4 P-5 P-6 P-7

P-8 P-9

P-1 0 P-11 P-12 P-13 P-14 P-15 P-16 P-17 P-18 P-19 P-20

P-21 P-22 P-23 P-24 Mean -C SD Range hfedian Decile 10 qo 90 %

9.0 f 2.1 6.3 - 14.5 8.8 6.7 13.0

Patient values Age Sex’

39 61 61 57 61 46 48 45 47 58 54 52 15 41 31 45 57 60 32 56 56 54 52 48

M M M

M F F F

M

M F M

F

F F M

F F F M F M F M M

GSH-Px 4.9 6.2 9.9 5.6 4.3 3.8 5.4 6.7 8.4 10.0 3.5 8.9 7.8 9.5 7.1 8.0 6.7 5.8 7.7 5.1

12.4 8.1 6.5 6.1 7.0 f 2.2 3.5 - 12.4 6.7 4.3 9.9

M: male, F: female.

with data on the “native” selenium content of crops in USA (Kubota et al. 1967) (cf. Table 4 ) , revealed that the selenium deficient areas of the USA do not correspond to areas with high death rates of MS. Thus northwest and southwest areas of the USA are deficient in selenium, but although Kurtzke indicated the northwest part of the USA to be a zone with annual death rate of MS on 1.1-1.3 MS cases/100,000 individuals, this is indeed not the case with the southeastern areas, 36’

548 Table 4 . Coniparison of selenium content of forage with death rates and preuatence of MS in different areas o f the USA. Median eonc. (mg/kg forage) (Kubota et al.

Arizona Florida Idaho Georgia Maine New Hampshire New England (Boston’) New York Tennessee North Dakota South Dakota Nebraska Oklahoma Texas (Houston’) Philadelphia Louisiana (New Orleans’) Colorado (Denver*) Michigan Kansas

1967)

MS age adjusted death rates by state of residence by death

0.09 0.02 0.09

0.7 0.6 1.2

0,06

0.9

0.05 0.05 0.05

1.2

0.05 0.05 0.26 0.26 0.26 0.26 0.26 0.05 0.26 0.26

0.8

0.10

0.26

rate pr. 100,000 p

~

(Kurtzke 1973)

0.7 1.1

0.5 1.3 1.1 1.1 0.6 0.4 0.8 0.2 1.1 0.9 0.8

43

(1944)

7 50 11 37 90

(1959) (1960) (1949) (1949) (1951)

indicates prevalence rates of capital i n the corresponding state.

where the prevalence rates are around 0.4 to 0.6/100,000 individuals. On the other hand, the few data available on M S prevalence rates per 100,000 individuals with probable multiple sclerosis in the USA (Kurtzlce 1973) seem in states with a homogenous selenium distribution (Kubota e t QZ. 1967) to reveal a trend such that high prevalence rate is associated with low selenium level in forage (Table 4 ) . These data thus seem not to exclude selenium deficiency as a pathogenic factor in MS. ACKNOWLEDGEMENTS These studies have been supported by a grant from “Kebmand Sven Hansen og Hustru Ina Hansens foundation”. Gratitude for the support available i s hereby expressed. V. K. S. Shulrla thanks the Danish International Development Agency (DANIDA) for the award of a post-doctoral fellowship.

~

54.9 REFERENCES Adams, J. & D. Imagawa (1962): Measles antibodies in multiple sclerosis. Proc. Soc. exp. Biol. Med. 11 1, 562-566. Alling, C., M. T. Vanier & I,. Svennerholm (1971): Lipid alterations i n apparently normal white matter i n multiple sclerosis. Brain Res. 35, 325-336. Ammitzhsll, T. & J. Clausen (1972): Measles antibody in serum of multiple sclerosir patients, their children, siblings and parents. $eta neurol. scand. 48, 47-56. Awasthy, Y. C., E. Beutlcr 8: S. K. Srivastava (1975) : Purification and properties of human erythrocyte glutathione peroxidase. J. Biol. Chem. 250, 5144-5149. Baker, It. W. R., H. H. S. Thompson & K. Zilkha (1964): Serum fatty acid i n multiple sclerosis. I. Neurol. Neurosurg. Psychiat. 27, 408-414. Batten, F. E. (1903): Cerebral dcgcncration with symmetrical changes in the maculae in two members of a family. Trans. ophthalmol. Soe. U.K. 23, 386. Beck, H. W.8i. J. Clausen (1977) : A comparative epidemiologieal study on measles and para-influenza type 1 antibody titers i n multiple sclerosis, systemic lupus and rheumatoid arthritis. Z. Bakt. Parasitenk. Infektionsk. IIyg. A 238: 431-443. Caspars, E. A., F. Sewcll & E. J. Field (1967): Red blood cell fragility in multiple sclerosis. Brit. mcd. J. 2, 610-611. Clausen, J. 8: I. B. Hansen (1970): Myelin constituents of human central nemous system. Acta neurol. scand. 46, 1-17. Clausen, J., T. Fog & K. Garde (1972): Spinalvacskeundersegelsers praktiskc vaerdi ved diagnosen dissemineret sclerose, med szrligt henblik pB agargel-clektroforctisk fraktionering af globulinerne. Ugcsltr. Lzg. 134, 872-877. Flohe, L. B It. Zimmermann (1970): The role of glutathione i n protecting the membrane of r a t liver mitochondria. Biochim. Biophys. Acta 223, 210-213. Flohe, L., W. A. Gunzler & R. Ladenstein (1976): Glutathione peroxidasc. I n : Glutathione metabolism and function. (Arias, I. M. 8: W. B. Jacoby ed.s). Pp. 115-135. Ravcn Press, New York. Frost, D. T’. (1973): Thc selenium CJXAC and health. Ann. Meeting Air Pollution Control Ass. Chicago. June 2-28. Geigy (1965): Documcnta Geigy, Scientific tables, Gcigy. Basel, Switzerland. Gerstl, B., L. F. Eng, BI. Tavaststjerna, K. J. Smith & S. L. Kruse (1970): Lipids and proteins i n multiple sclerosis white matter. J. Neurochem. 17, 677-689. Gissel-Nielsen, G. (1973): Ecological effects of selenium application to field crops. Ambio 2, 114-118. Hokama, Y., L. €1. Kimura, T. Y. Kobara, S. Perreira, D. V. P. Su, C. Torikawa R: N. Oishi (1974): Variation i n peroxisomal enzyme levels i n peripheral leucocytes of rancer, leprosy and tuberculosis patients. Cancer Res. 34, 2784-2789. Jensen, Gunde Egeskov, V. K. S. Shukla, G. Gissel-NieIsen & J. CIausen (1977): Biochemical abnormalities in Batten’s syndrome. Scand. J. Clin. 1,ab. Invest. (in press). Kubota, J., W. H. Allaway, D. L. Carter, E. E. Cary 8: V. A. Lazar (1967): Selenium i n crops i n the United States i n relation to selenium responsive diseases of animals. J. Agr. Food Chcm. 15, 448-453. Kurtzke, J. F. (1973): An overview of the epidcmiology of multiple sclerosis. Proc. Internal. Symp. Aetiolog. Pathog. Demyelinating Dis. Kyoto 1973. Laszlo, S. (1964): Fragilite osmotique des globules rouges dans la sclerose en plaques. Acta Neurol. Belg. 64, 529-533.

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550 Lawrence, H. A. & R. F. Burk (1976) : Glutathione peroxidase activity i n selenium deficient r a t liver. Biochem. Biophys. Res. Comm. 71 ( 4 ) , 952-958. Levy, N. Id., P. S. Auerbach & E. C. Hayes (1976) : A blood test for multiple sclerosis based on the adhcrence of lymphocytes t o mcaslcs-infected cells. New Engl. J. Med. 294, 1423-1427. Link, 11. (1967) : Immunoglobulin G and low molecular weight proteins i n human cerebrospinal fluid. Acta neurol. scand. 43 (Suppl. 28), 1-135. Nechelcs, T. F. (1974) : The clinical spectrum of glutathione peroxidase deficiency: Glutathione. (Flohe, L., H. Ch. Benohr, H. Sies, H. D. Waller & A. G. Wendel). Pp. 173-180. Thieme Verlag, Stuttgart. Offner, H., G . Konal & J. Clausen (1974): Effect of phytohemagglutinin, basic protein and measles antigen on myo-(23H)-inositol incorporation into phosphatidylinositol of lymphocytes from patient% with multiple sclerosis. Acta neurol. scand. 50, 791. Paglia, D. E. & W. N. Valentine (1970) : Studies on the qualitative characterization of erythrocyte glutathione peroxidase. J. Lab. Clin. Med. 70: 158-169. Panelius, M. (1969) : Studies on epidemiological, clinical and etiological aspects of multiple sclerosis. Acta neurol. scand. 45 (Suppl. 39), 1-82. Plum, C. A[. & T. Fog (1959) : Studies i n multiple sclerosis. Acta psychiat. scand 34, Suppl. 128. Prineas, J. (1968) : Red blood cell size i n multiple sclerosis. Acta neurol. scand. 44, 81-90. Rotruck, J. T., W. G. Hoekstra, A. L. Pope, H. Ganther, A. Swanson & D. Hafeman (1972) : Relationship of selenium to GSH peroxidase. Fed. Proc. 31, 691. Rotruck, J. T., A. L. Pope, H. E. Ganther, A. B. Swanson, D. Hafeman & W. G. Hoekstra (1973) : Selenium: Biochemical role as a component of glutathione peroxidase. Science 179, 588-590. Stopford, W., D. H. Donovan, M. B. Abou Donia & D. B. Menzel (1976) : Glutathione peroxidase deficiency and mercury allergy : Amelioration with selenium supplementation. Symposium abstracts on selenium-tellurium i n t h e environment. Centre for continuing education, University of Notre Dame, Notre Dame, Indiana. Thompson, R. H. S. (1973) : Fatty acid metabolism i n multiple sclerosis. Biochemical Society Symposium No. 35. Tichy, J., J. Vymazal & C. Michalec (1969): Serum lipoproteins, cholesterol esters and phospholipids i n multiple sclerosis. Acta neurol. scand. 45, 32-40. Westermark, T. & M. Sandholm (1977) : Decreased erythrocyte glutathione perodixase activity i n neuronal Lipofuscinosis (NCL)-corrected with selenium supplementation. Acta pharmacol. e t toxicol. 40, 70-74. WikstrBm, J., T. Westermarck & J. Palo (1976) : Selenium, vitamin E and copper in multiple sclerosis. Acta neurol. scand. 54, 287-290. Received June 21, accepted July 20, 1977

Prof. J. Clausen Institute of Life Sciences and Chemistry Roskilde University P.O. Box 260 DK-4000 Roskilde Denmark

Erythrocyte glutathione perioxidase deficiency in multiple sclerosis.

Acta Neiirol. Scnndinav. 56, 542-550, 1977 * The Laboratory of Biochcmistry and Toxicology, The Institute of Life Sciences and Chemistry, Roskilde Un...
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