EFFECT OF VITAMINS C AND E ON THE LEVELS OF CARBOHYDRATE COMPONENTS OF GLYCOPROTEINS IN COLLAGEN INDUCED ARTHRITIS

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1 Chapter 4 EFFECT OF VITAMINS C AND E ON THE LEVELS OF CARBOHYDRATE COMPONENTS OF GLYCOPROTEINS IN COLLAGEN INDUCED ARTHRITIS 4.1. INTRODUCTION Rheumatoid Arthritis is a chronic inflammatory disease categorized under autoimmune disorders. Initially it affects the joints, typically in feet, wrists, ankles and knees. But soon it spreads to the entire body and even affects organs like heart, liver, kidney, eyes and brain. Rheumatoid Arthritis (RA) is a classical example of multifactorial disease with diverse reasons like abnormal life styles and nutritional factors, food allergies, microorganisms, oxidative stress and genetic factors. Several kinds of biochemical studies also have been done to explore the disease mechanism as well as the line of therapy. Recent analyses have revealed that the glycoprotein metabolism is altered in RA. The study of carbohydrate components of glycoproteins is relevant once their function in the proteoglycan is identified. The biological role of glycoprotein is also assessed on the basis of concentration of individual carbohydrate components in it. The autoantigenic property of glycoproteins in RA has been localized to the constant region (C 2 domains) of IgG. The IgG isolated from normal individuals and from RA patients differed in the distribution of asparagine-linked, bi-antennary complex type glycans. The IgG of RA patients was found to be agalactosylated (IgG-GO) [71, 11]. Gillard and

2 122 Chapter-4 Lowther [117] reported loss of proteoglycans from the articular cartilage in carrageenin induced arthritis. An increase in the metabolic turn over of glycoproteins was reported by Exer et al. [118]. Lombart et al. [166] explained an increased incorporation of N-acetylglucosamine, galactose and sialic acid together with an enhanced activity of glycosyltransferases in rat liver inflammation. Significant increase in the contents of total hexoses, fucoses, hexosamines and sialic acids was observed by Reddy and Dhar [133] in adjuvant induced arthritis (AIA) in rats. Glycosidases are lysosomal enzymes involved in the trimming of oligosaccharide chains at the time of glycoprotein synthesis. They also play roles in degrading the glycoprotein. These enzymes include α-mannosidase, β D glucosidase, β D galactosidase, α L-fucosidase, N-Acetyl β-d-hexosaminidase etc. Variations in the levels of glycosidase activity have been reported in many diseases including RA [14, 35, 193, 198, 21]. These variations in activity are accepted as workers in the diagnosis of the disease. The destruction of joint cartilage in RA is related to the degradation of proteoglycans by glycosidases. At the same time, accumulation of glycoprotein (Advanced Glycosylated Proteins-AGPs) in tissues is explained as a defense mechanism against excessive loss of proteoglycans. There exists a paradoxical relationship between the amount of glycoprotein and the level of activity of glycosidases in inflammatory conditions. The effect of drugs for reducing the symptoms may be attributed to their capacity to stabilize lysosomal enzymes. Drug design and delivery are targeting glycohydrolases with the view that the altered proteoglycan metabolism could be rectified. Allopathic and ayurvedic treatments for

3 Effect of Vitamins C and E on the levels of Carbohydrate components of Glycoproteins in Collagen Induced Arthritis 123 RA are available. Physicians prescribe vitamin E formulations for RA therapy. At the same time, no work has been carried out with viamin C for this treatment. So in this section, an attempt has been made to describe the role of vitamin C and vitamin E since these vitamins come under the class of neutraceuticals which have antioxidant potential too. This part of the work was aimed at estimating the variations in the carbohydrate components of the glycoprotein in CIA rats and correlating this with simultaneous variations in selected glycosidases. The sugars selected were hexoses, fucoses, hexosamines and sialic acid whereas the enzymes included α-mannosidase, β-d-glucosidase, β-d-galactosidase, α- L-fucosidase, and N-Acetyl- β-d-hexosaminidase. The results were compared between normal and CIA rats. Also, the effects of supplementing vitamin C and vitamin E on these parameters were studied as detailed in the materials and methods and discussed MATERIALS AND METHODS As described elsewhere in Chapter RESULTS Effects on protein bound Hexose levels and related Glycosidase activity Protein bound hexoses The levels of Protein bound hexoses in various tissues of normal, arthritic and treated rats are given in Table 4.1. The percentage variation in their concentration from the normal is given in parenthesis.

4 124 Chapter-4 Table 4.1. Effects of Vit. C and Vit. E on the level of Hexoses in the glycoproteins in tissues of CIA rats GROUP Control RA Vit. C Vit. E Concentration of Hexoses in tissues ( mg per g weight of defatted tissue ) SERUM LIVER HEART KIDNEY TESTIS BRAIN a b 4.7 (135.97) # d (172.73) # c (153.44) p s (166.41) r (143.89) q (134.41) b a (9.4) b (96.51) b (95.53) p r (159.8) q (114.51) r (156.8) b a 2.47 (67.59) # b (1.17) b (92.32) p r 4.64 (129.59) q (12.17) p (13.69) Levels of Significance Paired t $ (2-tailed) --- P <.1 P <.1 P <.1 # Values above the limit. Values are mean SEM of 5 animals. variation of RA and treated groups from Control is shown in parenthesis. Statistical analysis by ANOVA followed by DMRT at 99% level. Column figures with the same superscript do not differ significantly from each other. Others differ at P <.1. $ Significant difference between Control and RA as well as RA and each Treatment. The levels of protein bound hexoses in different tissues except heart and testis were found to be increased significantly in CIA rats. There was significant decrease in protein bound hexoses in the heart and testis of arthritic rats. Both the vitamins C and E treatments further enhanced the hexose levels of serum significantly beyond those of arthritic condition. Liver of treated rats showed significant reduction in the protein bound hexose content. Kidney of CIA rats experienced a significant decrease in protein bound hexose content

5 Effect of Vitamins C and E on the levels of Carbohydrate components of Glycoproteins in Collagen Induced Arthritis only under vitamin C treatment. There was no significant variation in vitamin E treatment. The hexose fraction of glycoproteins in the brain of vitamin C treated CIA rats was regulated towards normal by vitamin C and almost normalized by vitamin E. The levels of protein bound hexoses in vitamins C and E treated heart and testis increased significantly and attained normalcy. The concentration of protein bound hexoses increased significantly in arthritic group (P<.1). Both vitamin C and vitamin E generally produced significant decrease in hexose level in CIA rats (P <.1) except for similar increase in heart and testis Glycosidase activity The specific activities of mannosidase, glucosidase and galactosidase in normal, arthritic and vitamin treated rats are tabulated respectively in Tables 4.2, 4.3 and 4.4. The percentage variation in activity from the normal is given in parenthesis in each case α-d Mannosidase activity There was significant increase in the activity of mannosidase in all the tissues of CIA rats other than heart. with both vitamins C and E could not lower the activity of mannosidase in serum significantly. Mannosidase activity of arthritic liver was normalized by vitamin C whereas vitamin E reduced it further down. Levels of activity in kidney, testis and brain were significantly decreased towards normal by both these vitamins. Vitamin C brought the mannosidase activity to normalcy in the brain. However, heart of arthritic rats recorded a reduced level of mannosidase activity which was regulated slightly by vitamin C. But vitamin E further deteriorated the situation with a lesser activity of this enzyme. 125

6 126 Chapter-4 The arthritic group showed significant increase from the control (P <.1). Both the treatments showed significant decrease from arthritic group (P <.1). GROUP Control RA Vit. C Vit. E Table 4.2. Effects of Vit. C and Vit. E on α-mannosidase activity in tissues of CIA rats Specific activity of α-mannosidase in tissues (µmoles / min / mg protein ) x 1 SERUM LIVER HEART KIDNEY TESTIS BRAIN 1.39 a b.16 (237.59) b.17 (195.42) 3.55 b.62 (233.38) 5.27 q r.192 (218.38) q.342 (132.1) * p.49 (69.92) 4.15 c a b.12 (36.91) 2.74 b.18 (5.52) * a.199 (32.59) p s (921.51) r.239 (561.94) q.225 (3.33) a d (42.5) b.518 (253.12) c.628 (367.92) p r.467 (423.9) p.163 (125.99) q.242 (246.73) Levels of Significance Paired t $ (2-tailed) --- P <.1 P <.1 P <.1 * Values below the limit. Values are mean SEM of 5 animals. variation of RA and treated groups from Control is shown in parenthesis. Statistical analysis by ANOVA followed by DMRT at 99% level. Column figures with the same superscript do not differ significantly from each other. Others differ at P <.1. $ Significant difference between Control and RA as well as RA and each Treatment β-d- Glucosidase activity The activity of glucosidase was lowered significantly in serum, heart and testis of CIA rats and raised in the liver, kidney and brain. Both the vitamins C and E normalized the serum glucosidase activity. The enzyme activity was

7 Effect of Vitamins C and E on the levels of Carbohydrate components of Glycoproteins in Collagen Induced Arthritis decreased significantly in liver treated with vitamin C and reached normalcy. Vitamin E did not produce any marked change. Arthritic kidney also experienced a reduction in glucosidase activity attaining normalcy under these two vitamins. Brain of CIA rats behaved differently towards these vitamins: glucosidase activity was regulated by vitamin E while it was not at all affected by vitamin C. In the heart, both the vitamins raised the glucosidase activity to normal level. The reduced levels of activity in testis of CIA rats was elevated beyond the normal limit by both the vitamins. These changes were highly significant. GROUP Control RA Vit. C Vit. E Table 4.3. Effects of Vit. C and Vit. E on β-d-glucosidase activity in tissues of CIA rats Specific activity of β-d-glucosidase in tissues (µmoles / min / mg protein ) x 1 SERUM LIVER HEART KIDNEY TESTIS BRAIN.15 b.4.84 a.7 (56.).135 b.2 (9.).137 b.2 (91.33).827 p q.173 (28.1) p.45 (142.9) 1.74 q.126 (26.) 5.22 b a.285 (33.64) b.114 (82.1) b.26 (94.41) p q (32.29) p.628 (11.87) p ( b a.165 (68.68) # c.84 (124.93) # c.237 (12.56) 3.52 p r.41 (22.36) r.95 (198.63) 5.24 q.5 (143.46) 127 Levels of Significance Paired t $ (2-tailed) --- P <.5 P <.5 P <.5 # Values above the limit. Values are mean SEM of 5 animals. variation of RA and treated groups from Control is shown in parenthesis. Statistical analysis by ANOVA followed by DMRT at 99% level. Column figures with the same superscript do not differ significantly from each other. Others differ at P <.1. $ Significant difference between Control and RA as well as RA and each Treatment.

8 128 Chapter-4 β-d glucosidase activity in arthritic group showed significant increase (P <.5) in liver, kidney and brain and significant decrease in the other tissues compared to the control. Both vitamin C and vitamin E produced significant regulatory effect at (P <.5) in enzyme activity in CIA rats β-d Galactosidase activity Significant increase was noticed in the activity β-galactosidase in serum, testis and brain of CIA rats. Level of β- galactosidase was reduced significantly in liver, heart and kidney of arthritic rats. with both vitamins C and E normalized the activity of β- galactosidase in serum, heart and kidney of arthritic rats. Enzyme activity in treated liver was reduced significantly towards normal by both these vitamins. Vitamin C regulated significantly the enzyme level of arthritic testis while vitamin E almost normalized the activity. There was no significant effect for either of these vitamins on the brain of CIA rats. CIA group showed significant increase in galactosidase activity in serum, testis and brain and decrease in liver, heart and kidney (P<.5). Vitamins C and E regulated these alterations significantly (P<.5).

9 Effect of Vitamins C and E on the levels of Carbohydrate components of Glycoproteins in Collagen Induced Arthritis 129 Table 4.4. Effects of Vit. C and Vit. E on β-d-galactosidase activity in tissues of CIA rats GROUP Control RA Vit. C Vit. E Specific activity of β-d-galactosidase in tissues (µmoles / min / mg protein ) x 1 SERUM LIVER HEART KIDNEY TESTIS BRAIN.785 a b.478 (165.22).93 a.36 (115.3).888 a.31 (113.12) r p.716 (44.11) q.275 (63.97) q.255 (61.38) b a.232 (42.93) 4.89 b.91 (92.14) b.7 (89.18) q p (52.35) q (91.94) q 8.16 (98.15) a c (154.74) b (127.9) a b.812 (111.4) p q (197.65) q.849 (186.21) q.777 (179.36) Levels of Significance Paired t $ (2-tailed) --- P <.5 P <.5 P <.5 Values are mean SEM of 5 animals. variation of RA and treated groups from Control is shown in parenthesis. Statistical analysis by ANOVA followed by DMRT at 99% level. Column figures with the same superscript do not differ significantly from each other. Others differ at P <.1. $ Significant difference between Control and RA as well as RA and each Treatment. Tissue specific variations in protein bound hexoses and related glycosidases of normal, arthritic and treated rats are illustrated graphically in Figure 4.1.

10 13 Chapter-4 Fig. 4.1.Variations in protein bound Hexose level and activity of related Glycosidases in CIA rats treated with vitamins C and E 25 SERUM 25 LIVER Con RA Vit.C Vit.E Con RA Vit.C Vit.E 12 HEART 1 KIDNEY Con RA Vit.C Vit.E Con RA Vit.C Vit.E TESTIS Con RA Vit.C Vit.E BRAIN Con RA Vit.C Vit.E

11 Effect of Vitamins C and E on the levels of Carbohydrate components of Glycoproteins in Collagen Induced Arthritis Effects on Fucose levels and Fucosidase activity Fucose levels The amounts of fucose in glycoproteins of various tissues of normal, arthritic and treated rats are given in Table 4.5. The percentage variation in Fucose content from the normal is presented in parenthesis. Table 4.5. Effects of Vit. C and Vit. E on the level of Fucose in the glycoproteins in tissues of CIA rats GROUP Control RA Vit. C Vit. E Concentration of Fucose in tissues ( mg per g weight of defatted tissue ) SERUM LIVER HEART KIDNEY TESTIS BRAIN a b.38 (186.45) # c.569 (298.17) # d.572 (369.8) p s.394 (16.99) r.511 (134.68) q.386 (123.93) a b.384 (177.17) a.394 (113.99) b.825 (144.91) p q.411 (151.15) p.362 (11.43) q.479 (144.35) d a.648 (67.44) b.63 (75.38) c.516 (88.16) p r.525 (152.7) q.736 (127.29) q.62 (125.31) Levels of Significance Paired t $ (2-tailed) --- P <.1 NS NS # Values above the limit. Values are mean SEM of 5 animals. NS: Not Significant variation of RA and treated groups from Control is shown in parenthesis. Statistical analysis by ANOVA followed by DMRT at 99% level. Column figures with the same superscript do not differ significantly from each other. Others differ at P <.1. $ Significant difference between Control and RA as well as RA and each Treatment.

12 132 Chapter-4 Fucose content of different tissue proteins was increased significantly in arthritic rats except in testis where it was reduced significantly. The serum of CIA rats treated with vitamin C and vitamin E showed a further increase in fucose content. These changes were highly significant. Fucose level was regulated towards normal in liver treated with both the vitamins. In heart, the level of fucose was normalized by vitamin C. Fucose level in vitamin C treated arthritic kidney was reduced significantly to normal whereas vitamin E had no significant effect. Both the vitamins up-regulated the fucose levels significantly in testis. In brain it was down-regulated significantly towards normal. Fucose levels in arthritic rats was significantly increased (P <.1). At the same time, treatments with vitamins C and E did not produce any significant effect on arthritic rats Fucosidase activity Fucosidase activity in various tissues of normal, arthritic and treated rats is presented in Table 4.6 with the percentage variation in activity compared to normal in parenthesis. Significant elevation in the activity of fucosidase was noticed in all the tissues of CIA rats except brain where it declined remarkably. Vitamin C and E treatments lowered the enzyme level significantly in serum and liver of CIA rats. In the heart, vitamin C lowered the activity of fucosidase significantly towards normal. But vitamin E had no effect. Fucosidase levels in kidney and testis of CIA rats were regulated significantly towards normal by both the vitamins. Brain of vitamin C treated rats recorded significant elevation in fucosidase activity while that under vitamin E treatment approached normalcy. It could be seen that Vitamin E has highly significant role in maintaining the level of fucose by reducing the activity of fucosidase.

13 Effect of Vitamins C and E on the levels of Carbohydrate components of Glycoproteins in Collagen Induced Arthritis 133 GROUP Control RA Vit. C Vit. E Table 4.6. Effects of Vit. C and Vit. E on α-l Fucosidase activity in tissues of CIA rats Specific activity of α-l Fucosidase in tissues (µmoles / min / mg protein ) x 1 SERUM LIVER HEART KIDNEY TESTIS BRAIN 1.56 a c.42 (45.45) 4.42 b.195 (283.33) 3.86 b.286 (247.44) p r (322.26) q.536 (237.45) p q.645 (173.59).514 a c.27 (732.88) b.26 (413.81) c.157 (65.) p r (169.6) q 5.3 (134.92) q (121.8) a c (117.63) b (18.77) b 8.14 (11.29) r p.41 (6.7) 6.26 q.221 (75.93) q r.315 (83.49) Levels of Significance Paired t $ (2-tailed) --- P <.1 P <.5 P <.1 Values are mean SEM of 5 animals. variation of RA and treated groups from Control is shown in parenthesis. Statistical analysis by ANOVA followed by DMRT at 99% level. Column figures with the same superscript do not differ significantly from each other. Others differ at P <.1. $ Significant difference between Control and RA as well as RA and each Treatment. The arthritic group showed significant increase in fucosidase activity over the control (P <.1). Activity was found to be decreased significantly (P <.5) in vitamin C group and more significantly (P <.1) in vitamin E group. Tissue specific variations in fucose level and fucosidase activity of normal, arthritic and treated rats are represented graphically in Figure 4.2.

14 134 Chapter-4 Fig Variations in Fucose level and activity of Fucosidase in CIA rats treated with vitamins C and E SERUM CON RA GlcNA c LIVER CON RA Glc NAc HEART CON RA GlcNA c KIDNEY CON RA GlcNAc TESTIS CON RA GlcNAc BRAIN CON RA GlcNA c

15 Effect of Vitamins C and E on the levels of Carbohydrate components of Glycoproteins in Collagen Induced Arthritis Effects on Hexosamine levels and Hexosaminidase activity Hexosamines Hexosamine content of glycoproteins in various tissues of normal, arthritic and treated rats are presented in Table 4.7.The percentage variation of values from the normal is given in parenthesis. Table 4.7. Effects of Vit. C and Vit. E on the level of Hexosamines in the glycoproteins in tissues of CIA rats GROUP Control RA Vit. C Vit. E Concentration of Hexosamines in tissues ( mg per g weight of defatted tissue ) SERUM LIVER HEART KIDNEY TESTIS BRAIN a c.664 (123.66) c.711 (122.27) b.667 (11.3) p q r.619 (142.54) # r.958 (151.92) q.761 (134.87) a c.716 (166.9) 55.4 # d.62 (228.4) b.682 (119.3) p s 1.23 (224.2) q.956 (145.2) r.936 (161.8) c a (65.82) b (84.42) c.673 (91.) p q.576 (156.49) # r (24.84) q 1.83 (158.52) Levels of Significance Paired t $ (2-tailed) --- P <.1 NS P <.1 # Values above the limit. Values are mean SEM of 5 animals. NS: Not Significant variation of RA and treated groups from Control is shown in parenthesis. Statistical analysis by ANOVA followed by DMRT at 99% level. Column figures with the same superscript do not differ significantly from each other. Others differ at P <.1. $ Significant difference between Control and RA as well as RA and each Treatment.

16 136 Chapter-4 All the tissues except testis exhibited significant increase in hexosamine content in arthritic state compared to normal. Vitamin C could not reduce serum hexosamine level of CIA rats whereas vitamin E reduced hexosamines towards normal. In liver as well as in heart, vitamin C produced a further significant enhancement in hexosamine level. But vitamin E reduced the levels in both of these tissues significantly towards normal. Both the vitamins expressed a regulatory effect on kidney hexosamine content. The hexosamine levels in testis treated with vitamin C was regulated significantly. Vitamin E reduced the value to almost normal. In brain, vitamin C further elevated the hexosamine level significantly. Vitamin E treatment could not make any effect on brain. Hexosamine level was increased in CIA group significantly (P<.1). Vitamin E supplementation reduced hexosamine levels significantly (P<.1) whereas vitamin C treatment did not lead to any significant effect N-Acetyl-β-D- Hexosaminidase activity The activity of hexosaminidase in different tissues of normal, arthritic and treated rats is tabulated in Table 4.8 with the percentage variation in activity in parenthesis. Hexosaminidase activity was elevated in all the tissues of CIA rats. with vitamins C and E reduced the activity of hexosaminidase towards normal in the serum of CIA rats. Activity was brought below normal in liver under the influence of vitamin C while it was regulated by vitamin E. In heart, it was normalized by vitamin C with a further fall in values and was reduced significantly towards normal by vitamin E. Activity of hexosaminidase was brought below the normal by vitamin C and regulated by vitamin E in kidney of arthritic rats. Testicular hexosaminidase activity was reduced

17 Effect of Vitamins C and E on the levels of Carbohydrate components of Glycoproteins in Collagen Induced Arthritis significantly towards normal by both these vitamins. In brain, the activity was reduced beyond normal by vitamin C and not at all affected by vitamin E. GROUP Control RA Vit. C Vit. E Table 4.8. Effects of Vit. C and Vit. E on N-Acetyl β-d- Hexosaminidase activity in tissues of CIA rats Specific activity of N-Acetyl β-d-hexosaminidase in tissues (µmoles / min / mg protein ) x 1 SERUM LIVER HEART KIDNEY TESTIS BRAIN a c.582 (214.14) a b.17 (137.15) b.86 (166.57) p q s (138.73) * p (83.53) q r (114.49) b c d 1.71 (125.64) * a (58.51) c d.395 (114.75) p r 8.46 (14.39) p (94.68) q (121.99) a c (165.26) a b (126.15) a b (123.9) q r (126.53) * p (76.62) r (125.5) 137 Levels of Significance Paired t $ (2-tailed) --- P <.1 P <.1 P <.1 * Values below the limit. Values are mean SEM of 5 animals. variation of RA and treated groups from Control is shown in parenthesis. Statistical analysis by ANOVA followed by DMRT at 99% level. Column figures with the same superscript do not differ significantly from each other. Others differ at P <.1. $ Significant difference between Control and RA as well as RA and each Treatment. The increase in hexosaminidase activity in arthritic group was highly significant (P<.1). Vitamin C supplementation caused highly significant decrease (P <.1). Decrease in vitamin E group was significant (P <.1). The relationship between hexosamine content and N-Acetyl-β-Dhexosaminidase activity in these tissues in arthritic state and under treatment is depicted graphically in Figure 4.3.

18 138 Chapter-4 Fig Variations in Hexosamine level and activity of Hexosaminidase in tissues of CIA rats treated with vitamins C and E 25 2 SERUM LIVER CON RA Vit.C Vit.E CON RA Vit.C Vit.E KIDNEY HEART CON RA Vit.C Vit.E CON RA Vit.C Vit.E TESTIS BRAIN CON RA Vit.C Vit.E CON RA Vit.C Vit.E

19 Effect of Vitamins C and E on the levels of Carbohydrate components of Glycoproteins in Collagen Induced Arthritis Effects on Sialic acid levels 139 The levels of sialic acids in various tissues of normal, arthritic and vitamin treated rats are presented in Table 4.9. The percentage variation in their concentration from normal is furnished in parenthesis and represented in Figure 4.4. Table 4.9. Effects of Vit. C and Vit. E on the level of Sialic acids in the glycoprotein in tissues of CIA rats GROUP Control RA Vit. C Vit. E Concentration of Sialic acid in tissues ( mg per g weight of defatted tissue ) SERUM LIVER HEART KIDNEY TESTIS BRAIN a b (123.21) # c (141.38) # c 7.3 (145.43) p r (128.2) p (11.8) q (124.29) b a 6.11 (79.44) a (8.93) * a (77.88) p r (118.75) q r (114.6) q (111.17) c a (62.71) b (88.3) b (87.76) p q (127.18) # q (129.62) # q 6.74 (128.88) Levels of Significance Paired t $ (2-tailed) --- P <.5 NS P <.5 # Values above the limit. * Values below the limit. Values are mean SEM of 5 animals variation of RA and treated groups from Control is shown in parenthesis. Statistical analysis by ANOVA followed by DMRT at 99% level. Column figures with the same superscript do not differ significantly from each other. Others differ at P <.1. $ Significant difference between Control and RA as well as RA and each Treatment. NS: Not Significant

20 14 Chapter-4 The amount of sialic acids is significantly increased in serum, liver, kidney and brain and decreased in heart and testis of the arthritic rats when compared to control. These levels were further increased significantly in serum under treatment with both the vitamins. Vitamin C normalized the sialic acid content of liver in arthritic rats while vitamin E could only regulate it towards normal. No significant effects were observed for these two vitamins on sialic acid concentration in arthritic heart. Significant reduction in sialic acid content was experienced by kidney treated with both the vitamins. Testis showed an upregulation in sialic acid content under both the treatments. These changes were significantly different from arthritic as well as normal states. Neither of the vitamins could produce any significant change in sialic acid content of brain of CIA rats. At the same time a further increment in the level was noticed in these cases which was not significant. The CIA group expressed significant increase (P<.5) in sialic acid content. Among the treatments, vitamin E showed significant reduction (P <.5) in sialic acid level while Vitamin C did not produce any significant variation. Fig. 4.4 Variations in Sialic acid level in tissues of CIA rats treated with Vit. C and Vit. E

21 Effect of Vitamins C and E on the levels of Carbohydrate components of Glycoproteins in Collagen Induced Arthritis DISCUSSION The parameters selected for studying the biochemical alterations during arthritis were the protein bound carbohydrates like hexoses, fucose, hexosamines and sialic acids. These sugars act as determinants for specific recognition and clearance of glycoproteins with terminal galactose [313]. Though the physiological role of glycoproteins in the connective tissue has not been completely revealed, it has been suggested that they have some role in stabilizing the tissue by maintaining the structure of collagen fibrils [314]. Also, the carbohydrates in glycoproteins are primarily responsible for the antigenic property in pathological situations. Significant increase was noticed in the levels of protein bound hexoses, fucose, hexosamines and sialic acids in the serum, liver, kidney and brain of CIA rats compared to normal. But the testis in CIA rats recorded a decline in the levels of these four carbohydrate components. Interestingly, heart of infected rats showed an increase in the concentration of fucose and hexosamines whereas the levels of protein bound hexoses and sialic acid were found to be decreased in this tissue. This designated a tissue specific alteration in protein glycosylation during arthritis as has been reported by earlier workers [71, 12, 112, 166]. Organ specificity in N-glycosylation has been reported by Kobata [91]. Bisecting GlcNAc was detected in the sugar chain of kidney enzymes in man, cattle, mouse and rat, while they were absent in the liver enzymes of these animals. This indicated a suppressed expression of GnT III in liver. This might lead to the higher level of hexosamines in kidney than in liver as noticed in the present study too.

22 142 Chapter-4 Alteration of GnT III activity in RA has been explained as one reason for the increase in oligosaccharides containing bisecting GlcNAc [96]. In this study, all the tissues except testis showed increased concentration of hexosamines in arthritic state. Gillard and Lowther [117] had reported that there is significant loss of proteoglycan from articular cartilage in the initial period of carrageenin induced arthritis which was overcome later by an increased rate of synthesis above those of control. An increase in the metabolic turnover of proteoglycans in the cartilage and ligaments in adjuvant induced arthritis was reported by Exer and coworkers [118]. Increased incorporation of N-Acetyl glucosamine, galactose and sialic acids into glycopeptides consequent to enhanced activity of glycosyltransferase in turpentine induced inflammation of rat liver was suggested by Lombart et al. [166]. A similar increase in the level of carbohydrate moieties observed in this study agrees with these earlier findings. Reddy and Dhar [133] observed a significant increase in the contents of total hexoses, hexosamines, fucose and sialic acids in different tissues including skin, liver, kidney and spleen. The contents of neutral sugars like glucose, galactose, mannose and xylose were also found to be elevated in acute as well as chronic inflammation. In a rather advanced investigation, Raghav et al. [134] noticed that most of the monosaccharides including glucosamine, galactose and other unidentified ones were found to be proportionately similar in both control and RA samples. A high mannose/glucose ratio in RA patients was explained due to the presence of unprocessed oligomannose glycan structures. Elevated levels of monosaccharides in body fluids have been extensively documented in the diseases of cartilage by many authors [66-68].

23 Effect of Vitamins C and E on the levels of Carbohydrate components of Glycoproteins in Collagen Induced Arthritis Several studies have identified changes in serum glycoproteins in patients with Hepatocarcinoma [152] and other liver diseases [315]. Liver is the organ where major part of body metabolism including that of glycoproteins is taking place. Secretion of glycoproteins by the liver occurs after the assembly of carbohydrates which terminate the macromolecules. A rise in all carbohydrate moieties of glycoproteins has been reported in liver during RA [134]. Thus the increased levels of protein bound hexoses, fucose, hexosamines and sialic acids could be explained due to an increased glycoprotein metabolism in the different tissues studied, except testis. The reversal of this effect in testis needs to be investigated in detail. Three factors influence the protein glycosylation. They are: a) the overall protein confirmation, b) the effect of local confirmation and c) the available repertoire of glycosylation-processing enzymes in the particular cell type. The pattern of glycoforms is protein-specific, site-specific and tissue/cellspecific. The oligosaccharides incorporated in the glycoprotein function as recognition markers. However, they can alter the intrinsic properties of proteins to which they are attached by altering the stability, protease resistance, or quaternary structure. Abnormal glycosylation is characteristic of a number of disease states including Rheumatoid Arthritis and cancer which may result in quite different functions of oligosaccharides. It was noticed that the glycosidases behaved differently in various tissues of arthritic rats. Mannosidase activity was increased in all the tissues studied except in heart, where it was decreased significantly. β-glucosidase activity was increased in the liver, kidney and brain and decreased in serum, heart and testis of CIA rats. β-galactosidase expressed enhanced levels of activity in serum, testis and brain, but decreased activity in liver, heart and kidney. Fucosidase, on 143

24 144 Chapter-4 the other hand, showed elevated level in all the tissues except brain where the activity was reduced significantly. Activity of hexosaminidase was increased in all the tissues of CIA rats compared to normal. Results showed that the activity of glycosidases depend on the tissues involved and the metabolism of glycoprotein therein. However, an overall assessment of the activities of these enzymes in tissues of CIA rats revealed that there is significant increase in activities of all the five enzymes in CIA rats compared to normal. The activity of glycosidases is reported to be altered in various pathological conditions. Activities of β-glucuronidase, β-galactosidase, α-mannosidase and α-fucosidase were reported to be increased in senile and diabetic cataracts on man whereas β-galactosaminidase and β-glucosidase activities were not affected [22]. Similar study on glycosidase activities of kidney of alloxan induced diabetic rat revealed a decrease in β-galactosidase and α-fucosidase activities. There existed tissue specific variations in the activities of these enzymes in normal as well as disease states. Also, the responses of these enzymes of the same tissue to various diseases are not uniform. Studies on glycosidases of various tissues in Rheumatoid arthritis have shown an increase in their activities in the blood/serum, liver, kidney and heart [32,198, 316]. Vijayalakshmi et al. reported that the total and free activity of the lysosomal enzymes (β-galactosidase, β-glucuronidase and N acetyl β- glucosaminidase) were increased in plasma, liver, heart and kidney of arthritic rats with concomitant increase in plasma level of protein bound carbohydrates [32]. They attributed this to increased lysosomal membrane fragility. It was also reported that the anti-arthritic activity of the extract of Semecarpus anacardium nuts is involved in stabilizing the lysosomal

25 Effect of Vitamins C and E on the levels of Carbohydrate components of Glycoproteins in Collagen Induced Arthritis membranes. This extract reduced the glycoprotein levels significantly with subsequent reduction in glycosidase activity too. Activity of N-Acetyl β-d Hexosaminidase (NAHase) was reported to be elevated in sera and synovial fluid of RA patients with high serum NAHase activity had more corrosive cartilage in their inflamed joints. The possibility of NAHase to be a marker of erosion was inquired by these workers. Polynuclear leukocytes contained NAHase in their lysosomes. Presence of large number of polynuclear leukocytes in the synovial fluid of RA patients was suggested to be the reason for the increased NAHase activity [317]. Shikhman et al. reported that the hexosaminidase activity was increased in chondrocytes stimulated with interleukin 1β while other glycosidases did not show significant change [22]. Lin et al. reported increased activity of hexosaminidase in osteoarthritis and demonstrated that inhibition of this enzyme activity could prevent cartilage degradation [2]. The dominant activity of hexosaminidase as compared to the rest of the glycosidases in the synovial fluid was also demonstrated by them. Increased levels of hexosaminidase activity was reported by some other workers too [318, 319]. Though the anti-inflammatory and antioxidant properties of vitamin C and vitamin E have extensively been explained by various workers [264, 295, 32] no work has been done to investigate their roles in glycoprotein metabolism in arthritis. The dietary importance of vitamin C and vitamin E and their role in alleviating RA symptoms were studied by Bae et al. [258] and was related to oxidative stress. They established that short term vitamin E supplementation (3 days) did not reduce the levels of these biomarkers to normal. However, the chemical composition of glycoproteins was not analyzed under vitamin E supplementation and perhaps this might be the first report on this aspect. 145

26 146 Chapter-4 This part of the study showed that vitamin C has no significant effect in the overall metabolism of glycoproteins with regard to their carbohydrate components. Individual tissue had shown moderate but significant variation in the levels of protein bound hexoses when treated with vitamin C. But no significant changes were noticed in the levels of fucoses, hexosamines and sialic acids in most of the vitamin C treated tissues. The enhanced levels of almost all the five glycosidases in CIA rats were brought down or regulated towards normal by the treatment with vitamin C. But serum mannosidase and brain glucosidase and galactosidase activities were not influenced by vitamin C treatment. In the case of hexosaminidase, vitamin C was found to be hyperactive in reducing the elevated levels beyond the normal limits in liver, heart and brain. A similar hyperactive suppression was noticed for vitamin C on β-glucosidase acivity of testis. All other enzyme activities were either normalized or regulated by vitamin C. Vitamin E behaved differently from vitamin C on CIA rats by regulating the levels of protein bound hexoses, hexosamines and sialic acids significantly towards normal. However, the concentrations of fucoses were not affected by vitamin E. Individual tissues responded differently with regard to the levels of these sugars. Vitamin E treatment could produce significant reduction in the activity of all the glycosidases in CIA rats. When the individual tissues of treated rats are considered, activities of mannosidase in serum and heart; β-glucosidase in liver; β-galactosidase in brain; fucosidase in heart and hexosaminidase in brain were not influenced by vitamin E. Hyperactive effect of this vitamin was noticed for liver mannosidase and testis β-glucosidase. However, the overall effect of vitamin E on CIA rats is similar to that of vitamin C.

27 Effect of Vitamins C and E on the levels of Carbohydrate components of Glycoproteins in Collagen Induced Arthritis Elevation in the levels of glycosidase activity in different tissues of CIA rats may be the reflection of the deranged glycoprotein metabolism in inflamed conditions. The concentration of monosaccharides in various tissue proteins of CIA rats were also found to be increased even in spite of enhanced glycosidase activity. with vitamin C and vitamin E could reduce the degradative enzyme activity, but the carbohydrate levels remained unchanged. The reduced activities of glycosidases result in a decrease in the rate of breakdown of proteoglycans in treated animals compared to the untreated. This may lead to the accumulation of proteoglycans in various tissues. Vitamins C and E play roles in regulating the glycoprotein degradation. Vitamin E was found to be more effective in regulating the content of carbohydrate components by decreasing the degradation when compared to vitamin C CONCLUSION There occurs significant increase in the levels of protein bound hexoses, fucose, hexosamines and sialic acids in serum, liver, kidney and brain of CIA rats. The alteration in protein glycosylation during arthritis is tissue specific. The increased levels of these carbohydrates could be explained due to either an increase in synthesis or a deranged degradation. The oligosaccharides incorporated in the glycoprotein function as recognition markers, alter the intrinsic properties of proteins to which they are attached by changing their stability, protease resistance or even their quaternary structure. Abnormal glycosylation might be due to the alterations in the expression of specific glycosyltransferases. As the newly synthesized protein pass from the endoplasmic reticulum to trans-golgi, oligosaccharide chains are assembled. Specific set of enzymes called glycosidases carry out the trimming reactions 147

28 148 Chapter-4 and the glycoprotein structure in completed. Any disturbance to this membrane flow may generate alterations in protein glycosylation. Supplementation with vitamin C and vitamin E decreased degradative enzyme activities in regulating the levels of carbohydrates in the glycoproteins of the tissues thereby protecting the tissue from RA. The elevated level of activities of all the five glycosidases in CIA rats were either normalized or regulated towards normal by the treatments with Vitamin C and E. This might have resulted in the accumulation of glycoproteins in various tissues of CIA rats under treatments. Vitamin C supplementations regulated the level of protein bound hexoses only. Other components were not affected. But vitamin E regulated the levels of protein bound hexoses, hexosamines and sialic acids and did not influence fucose content of CIA rat tissues. The over all level of carbohydrates in glycoproteins in CIA rats remained unaffected even after supplementation with vitamins C and E. This reduction in the activity of glycosidases in treated rats by vitamins might have slowed down the degradation of glycoproteins. The possibility of enhancement of glycosyltransferase activity (synthetic) cannot be ruled out. Since the biosynthetic enzymes were not studied, a definite conclusion could not be arrived at. But the supplementation of vitamin C and vitamin E to some extent could protect the tissues. The effect of vitamin E is more pronounced when compared to that of vitamin C.