Metabolism of glycosaminoglycans in rats during methionine deficiency and administration of excess methionine

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1 J. Biosci., Vol. 4, Number 1, March 1982, pp Printed in India. Metabolism of glycosaminoglycans in rats during methionine deficiency and administration of excess methionine G. MURALEEDHARA KURUP and P. A. KURUP Department of Biochemistry, University of Kerala, Trivandrum MS received 7 July 1981; revised 8 August 1981 Abstract. Methionine deficiency in rats caused significant decrease in the concentration of many sulphated glycosaminoglycans in the aorta and other tissues, while administration of excess methionine caused an increase in these constituents. The activity of some important biosynthetic enzymes decreased in methionine deficiency and increased on administration of excess methionine. No uniform pattern was observed in the changes in the activity of enzymes concerned with degradation of glycosaminoglycans. The concentration of 3'-phosphoadenosine 5'-phosphosulphate and the activities of the sulphate activating system and sulphotransferase were decreased in methionine deficiency, while feeding excess methionine did not affect these parameters as compared to controls. Keywords. Methionine deficiency; glycosaminoglycans; sulphate-activating enzymes; sulphotransferases. Introduction The quality and quantity of protein in the diet have been reported to affect the metabolism of glycosaminoglycans (GAG) (Leelamma and Kurup, 1978; Menon and Kurup, 1975). This effect may in part be related to the amino acid composition of the protein. So far there have been very few studies on the relationship of methionine to the metabolism of glycosaminoglycans. Udapa et al. (1956) reported a decreased accumulation of mucopolysacharides during wound healing in rats on protein free diet and when supplemented with methionine, the accumulation and subsequent disappearance of mucoplysacharides were.normal. Srinivasan et al. (1972) showed that methionine prevented the decrease in intestinal mucopolysccharides in rats caused by ethionine feeding. Excessive amounts of sulphur containing aminoacids (methionine and cysteine) have been reported to cause accumulation of liver lipids (Aoyama et al., 1969; Aoyama et al., 1978). It is known that the lipid accumulation in the aorta is the result of interaction between serum lipoproteins (LDL and VLDL) and GAG in the aorta (Iverius, 1973) and depends on the concentration of aortic GAG as well as that of serum LDL plus VLDL. It was therefore considered necessary to study the Abbreviations used: GAG, glycosaminoglycans; VLDL or LDL, VDL, very low, or low or high density lipoproteins; PAP,3 -phosphoadenosine-5' phosphosulphate. 95

2 96 Kurup and Kurup metabolism of GAG in rats fed with methionine-deficient and methionine-excess diets. The concentration of different GAG fractions in the aorta and other tissues, and the activities of some key enzymes such as glucosamine phosphate isomerase UDPG-dehydrogenase, β-glucuronidase, β-n-acetyl glucosaminidase; hyaluro noglucosidase, as well as sulphation have been studied. The concentration of cholesterol in serum HDL and LDL plus VLDL and that of cholesterol and triglycerides in the aorta have also been studied in rats fed on excess methionine containing diet. The results are reported in this paper. Materials and methods Male albino rats (Sprague-Dawley strain, body weight g) were divided into 4 groups as in table 1, with 15 rats in each group. Table 1. Diet formulation (in g) Composition of the amino acid mixture (g) Correspond to that present in 16% casein Hawk's physiological chemistry, ed. User (1965): L-Histidine 0.5, L-valine 1.15, L-glutamic acid L-Leucine L tryplophan L-lysine 1.3, L-threonine L-asparlic acid L-tryosine L-serine 1.0. L- glycine 0.43, L-phenylalanine 0.8. L-proline L-alanine L-arginine L-cystine and L-isoleucine * Menon and Kurup (1976) The methionine-deficient group thus received only l/20th of the amount of methionine given to the control group. Since dietary consumption in the methionine-deficient group was lower, the control group (group 2) was fed the same amount of methionine-adequate diet so as to maintain a comparable caloric intake to facilitate comparison between these two groups. The methionine-excess group received 100% more methionine than the adequatediet group. The dietary consumption was more or less the same among the rats of groups 3 and 4.

3 Methionine and metabolism of glycosaminoglycans 97 The rats in both experiments were maintained on their respective diets for a period of 14 days. At the end of this period, the rats in each group were deprived of food overnight, stunned and killed by decapitation. The tissues were quickly removed to ice cold containers for various estimations. Estimation of lipids Extraction of the tissues for lipds and estimation of cholesterol and triglycerides were carried out as described before (Sandhyavathy Bai and Kurup, 1976). Serum HDL, LDL and VLDL were separated by the heparin-manganese precipitation method described by Warnick and Albers (1972). Estimation of glycosaminoglycans GAG were estimated in the tissues as described before (Sudhakaran and Kurup, 1974). Papain digest of the defatted tissue was passed through a column of cellulose and the different fractions-hyaluronic acid, heparin sulphate, chondroitin- 4-sulphate, chondroitin-6-sulphate, dermatan sulphate and heparin were eluted according to the procedure of Svejcar and Robertson (1967). The individual fractions were quantitated by the estimation of uronic acid by the method of Bitter and Muir (1962). The identity of the GAG fractions in each case was confirmed by comparison with standard GAG preparations. (Made available by the courtesy of Prof. M. B. Mathews, University of Chicago, Chicago, Illinois, USA). Estimation of enzyme activities The activity of glucosamine phosphate isomerase (glutamine forming) (EC ) was estimated by the method of Pogell and Gryder (1957) and that of UDPG dehydrogenase (EC ) by the method of Strominger et al. (1957). β- Glucuronidase (EC ), β-n-acetyl glucosaminidase (EC ) and hyaluronoglucosidase (EC ) were estimated according to the procedure of Kawai and Anno (1971) and Cathepsin-D (EC ) by using 4% haemoglobin in 0.1 M acetate buffer PH 4.5 as the substrate and determining the amount of tyrosine liberated by the method of Folin and Ciocalteu (1927). Sulphate metapolsim The estimation of the concentration of 3'-phosphoadenosine-5'-phosphosulphate (PAPS) and the activity of sulphate activating system [which includes sulphate adenyl transferase (EC ) and adenyl sulphate kinase (EC )] and aryl sulphotransferase (EC ) of the liver were carried out as described before (Sudhakaran and Kurup; 1974) according to the procedure of Vankempen and Jansen (1972, 1973) using methyl umbelliferone. Protein was estimated in the enzyme extract after trichloroacetic acid precipitation by the method of Lowry et al. (1951). Statistical analysis The data given in the tables are the mean for the number of animals used in each case ±SEM. Statistical significance was calculated using Students t-test (Bennet and Franklin, 1967).

4 98 Kurup and Kurup Results and discussion Methionine-deficient rats showed lower gain in body wt. (8.2 ± 1.9 g) as compared to the corresponding pair-fed control (21.6 ±2.4 g). The gain in body wt. in the methionine-excess group (16.8 ±2.9 g) was also lower than that in the corresponding control group (23.6 ±2.2 g). Concentration of cholesterol and triglycerides in the serum and aorta in rats fed with excess methionine There was significant increase in the concentration of cholesterol and triglycerides in rats fed with excess methionine diet as compared to those fed with just an adequate dose. HDL cholesterol showed significant decrease in these rats while LDL plus VLDL cholesterol increased (table 2). Table 2. Concentration of cholesterol and triglycerides in serum and aorta in rats fed with excess methionine diet. Average of the values from 6 rats in each group ±SEM, values expressed as mg/100g wet tissue except for serum where it is mg/100 ml ±SEM a p less than 0.01 b p between 0.01 and 0.05 HDL high density lipoprotein LDL low density lipoprotein VLDL very low density lipoprotein. Concentration of GAG and activity of some enzymes of GAG metabolism in the aorta In the methionine deficient group, there was a significant decrease in chondroitin-4 and -6 sulphate in the aorta as compared to the pair-fed control group, while there was no significant alteration in heparin, heparin sulphate and dermatan sulphate. However, an increase in hyalauronic acid content was observed. On the other hand, rats fed with excess methionine diet showed significant increase in all the GAG fractions in the aorta except heparin, as compared to the corresponding control group. Heparin was not significantly altered (table 3). Glucosamine-6-phosphate isomerase activity decreased in methionine-deficient rats as compared to the pair-fed control but in rats fed with excess methionine diet, the enzyme activity increased significantly.

5 Methionine and metabolism of glycosaminoglycans 99

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7 Methionine and metabolism of glycosaminoglycans 101 Hyaluronoglucosidase and β-glucuronidase activities were not significantly altered in the methionine-deficient rats while β-n acetyl glucosaminidase and Cathepsin D activity decreased. In rats fed with excess methionine diet the activities of Cathepsin-D and β-glucuronidase increased in the aorta while those of β-n-acetyl glucosaminidase and hyaluronoglucosidase were not significantly altered. The aorta is particularly important both from the point of view of lipid accumulation in atherosclerosis and the high concentration of GAG present. Excess methionine caused increase in LDL plus VLDL cholesterol in the serum and in aortic cholesterol and triglycerides. It is known that serum lipoproteins (LDL plus VLDL) interact with aortic GAG forming insoluble complexes and this interaction has been considered to be responsible for lipid accumulation in the arterial wall (Iverius 1973). This interaction involves molecular seiving effect (Laurent et αl., 1963; Iverius, 1970), stearic exclusion effect (Laurent, 1968; Iverius, 1968) and ionic interaction (Srinivasan et al., 1972) and has been well studied. The decreased concentration of many GAG fractions in the aorta in methionine deficiency may result in decreased interaction with the serum lipoproteins with consequent decrease in lipid accumulation in the aorta. Decreased lipid accumulation has been reported in tissues in methionine deficiency (Aoyama and Ashida, 1972; Aoyama et al., 1969). In rats fed with excess methionine diet, the increased concentration of GAG in the aorta, along with the increase in serum LDL plus VLDL may result in their increased interaction resulting in increased accumulation of lipids in this tissue. The increased concentration of cholesterol and triglycerides in the aorta obtained in rats fed with excess methionine is in support of this view. The synthesis of GAG is regulated by glucosamine phosphate isomerase as well as UDPG dehydrogenase. The isomerase provides glucosamine-6-phosphate which is the precursor of hexosamines in GAG synthesis, while UDPG dehydrogenase makes available UDP glucuronic acid, the precursor of uronic acid moieties. The activity of the former enzyme is regulated by feed back inhibition by UDP-N-acetylglucosamine and that of the latter by UDP-xylose. UDPG dehydrogenase activity could not be studied in the aorta but the decreased activity of the isomerase in methionine deficient rats would result in decreased availability of hexosarnine precursors for GAG synthesis. The decreased concentration of GAG in the aorta inmethionine deficiency may therefore be due to their decreased synthesis. In rats fed with excess methionine, the isomerase activity is increased making more glucosamine-6-phosphate available for GAG synthesis but in the absence of information on the activity of UDPG dehydrogenase, it is not possible to speculate whether the synthesis of GAG is increased in the aorta in this case. The pattern of change in the GAG degrading enzymes is not uniform in the aorta. In methionine deficiency, the activities of some enzymes decreased while those of others were not significantly altered. In methionine-excess group, some enzyme activities increased while the activities of others were not significantly altered. Since the concerted action of all these enzymes would be required for the degradation of GAG, it is not possible to say how the overall degradation of GAG is affected by methionine deficiency or administration of excess methionine.

8 102 Kurup and Kurup Concentration of GAG and activity of some enzymes of GAG metabolism in the liver The concentration of hyaluronic acid, heparin sulphate, chondroitin-6-sulphate and heparin was decreased in the liver in methionine deficiency while chondroitin- 4-sulphate and dermatan sulphate were not altered. In rats fed excess methionine, heparin sulphate, chondroitin-4 and -6 sulphate and dermatan sulphate increased while heparin and hyaluronic acid were not significantly altered. (Table 4). The activity of glucosamine phosphate isomerase decreased in the liver in methionine deficiency while UDPG dehydrogenase was not affected. Both these enzyme activities increased in the liver in rats fed excess methionine. The decreased activity of isomerase in the liver in methionine deficiency even though UDPG dehydrogenase was not affected, may result in decreased synthesis of GAG in these tissues and the decreased concentration of many GAG observed in methionine deficiency may be due to this. The increased activity of both these enzymes in the liver in rats fed excess methionine may result in increased GAG synthesis in agreement with the increased concentration of GAG observed. Activity of hyaluronoglucosidase, cathepsin D and β-glucuronidase was not affected in methionine deficiency while that of β-n-acetyl glucosaminidase decreased. But in rats fed excess methionine the activities of cathepsin D, β-nacetyl glucosaminidase and hyaluronoglucosidase increased while that of β- glucuronidase was not affected. Thus, the pattern of change in these enzyme activities in the liver, as in the case of aorta, is not uniform and therefore it is not possible to say how the degradation of GAG is affected in the liver by methionine. Sulphate metabolism Methionine deficiency resulted in significant decrease in the concentration of PAPS in the liver as also in the activity of sulphate activating system and sulphotransferase. But feeding excess methionine did not alter these parameters from their normal values. The decrease in the concentration of PAPS in methionine deficiency is due to the decrease in the activity of sulphate activating system (including sulphate adenyl transferase and adenyl sulphate kinase) which generate PAPS. Decreased sulphate metabolism can result in decreased sulphation of glycosaminoglycans. The decrease in some of the sulphated GAG observed in methionine deficiency in the liver, may also be due to the decreased sulphate metabolism. Even, though the sulphotransferase now studied is aryl-sulphotransferase, PAPS is also the sulphate donor for sulphation of GAG. Thus the decreased concentration of PAPS would affect sulphation of GAG also. It is possible that methionine also provides sulphate for the formation of PAPS and in its deficiency, sulphate metabolism is decreased (table 5). Changes in the concentration of GAG in the heart and kidney In methionine deficient rats, hyaluronic acid, heparin sulphate and chondroitin-6- sulphate decreased in the heart while chondroitin-4 sulphate, dermatan sulphate and heparin were not significantly altered. In the kidney, only hyaluronic acid showed decrease While other GAG fractions were not affected.

9 Methionine and metabolism of glycosaminoglycans 103 Table 5. Concentration of PAPS, activity of sulphate activating system and Sulphotransferase in the liver of rats fed with methionine deficient and methionine excess diet. * µmol of methylumbellifuone sulphate formed/h/g protein. Foot note same as for table 3. On the other hand, rats fed excess methionine showed increase in chondroitin-4 sulphate, chondroitin-6 sulphate and dermatan sulphate in the heart and kidney, Heparin sulphate increased in the heart but was not affected in the kidney. Hyaluronic acid increased in the kidney but was not altered in the heart. Heparin decreased in the heart, but was not altered in the kidney. Table 6. Concentration of glycosaniinoglycans in the heart and kidney. Average values from 6 rats in each group ±SEM. Comparison of groups same as in table 3. a P<0.01 b P between 0.01 and (Values expressed as mg of uronic acid/g dry defatted tissue ±SEM) Foot note same as for table 3.

10 104 Kurup and Kurup Thus metabolism of GAG is significantly affected by the level of methionine in the diet. This effect is seen at all levels viz., biosynthesis of precursors, degradation of GAG and sulphation. References Aoyama, Y. Yoshida, A. and Ashida, K. (1969) J. Nutr., 97, 348. Aoyama, Y. and Ashida, K. (1972) J. Nutr., 102, Aoyama, Y. and Ashida, K. (1978) J. Nutr. Rep. Inst., 17, 463. Bennet, C. A. and Franklin, N. L. (1967) Statistical Analysis in Chemistry and Chemical Industry (New York: John Wiley and Sons, Inc.) Bitter, J. and Muir, H. M. (1962) Anal. Biochim., 4,330. Folin, O. and Ciocalteu, V. (1927) J. Biol. Chem., 73, 627. Iverius, P. H. (1968)J. Biol. Chem., 73, 627. Iverius, P. H. (1968) Clin. Chem. Aota., 20, 261. Iverius, P. H. (1970) in Chemistry and Molecular Biology of Intercellular Matrix, ed. E. A. Balate (London: Acadamic Press), 3, Iverius, P. H. (1973) Possible role of glycosaminoglycans in the genesis of atherosclerosis, in Atherosclerosis: Initiation factors, Ciba foundation symposium 12, Elsevier Amsterdam 185. Kawai, Y. and Anno, K. (1971) Biochem. Biophys. Acta, 242, 428. Laurent, T. C, Bjork, I. Pietruszkiewiez, A. and Person, H. (1963) Biochem. Biophys. Acta, 78, 351. Laurent, T. C. (1968) in The Chemical Physiology of the Mucopolysaccharides, ed. G. Quintarelit (Boston: Little, Brown), 153. Leelamma, S. and Kurup, P. A. (1978) Indian J. Exp. Biol, 16, 36. Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem., 193, 265. Menon, P. V. G. and Kurup, P. A. (1975) Indian J. Biochem. Biophys., 12, 389. Menon, P. V. G. and Kurup, P. A. (1976) Artery, 21,219. Menon, P. V. G. and Kurup, P. A. (1976) Indian J. Biochem. Biophys., 13, 146. Oser, B. L. (1965) in Hawks Physiological Chemistry, (New York: McGraw Hill, 132. Pogell, B. M. and Gryder, R. M. (1957)J. Biol. Chem., 228, 701. Sandhyavathy Bai, and Kurup, P. A. (1976) Indian J. Exp. Biol., 14, Srinivasan, H., Srinivaas, L. and Rao, P. B. R. (1972) Nutr. Rep. Int., 6, 145. Srinivasan, S. R., Dolan, P., Radhakrishna Murthy, B. and Berenson, G. S. (1972) Atherosclerosis, Strominger, J. L., Maxwell, E. S., Axelford, J. and Kalckar, H. M. (1957) J. Biol. Chem., 224, 79. Sudhakaran, P. R. and Kurup, P. A. (1974) J. Nutr., 104, 871. Svejear, J. and Robertson, W. E. B. (1967) Anal. Biochem., 18, 333. Udupa, K. N., Woessner, J. F. and Dunphy, J. E. (1956) Surg. Gynecol. Obset., 102, 639. Vankempen, G. M. J. and Jansen, G. C. I. M. (1972) Anal. Biochem., 46, 438. Vankempen, G. M. J. and Jansen, G. S. I. M. (1973) Anal. Biochem., 51, 324. Warnick, R. G. and Albers, J. T. (1978) J. Lipid. Res., 19, 65.