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Changes in aortic glycosaminoglycans and lipoprotein lipase activity in rats with age and atheroma
Atherosclerosis, 21 (1975) 1-14 0 Elsevier Scientific Publishing
Company, Amsterdam
- Printed in The Netherlands
CHANGES IN AORTIC GLYCOSAMINOGLYCANS AND LIPOPROTEIN LIPASE ACTIVITY IN RATS WITH AGE AND ATHEROMA
S. T. VIJAYAKUMAR, Department
S. LEELAMMA
of Biochemistry,
AND
P. A. KURUP
University of Kerala, Trivandrum-695001
(India)
(Received August 17th, 1973) (Revised received April 18th and August 27th, 1974) (Accepted August 28th, 1974)
SUMMARY
The changes in the individual glycosaminoglycans of the aorta and in lipoprotein lipase activity of the aorta, liver and heart have been studied at various stages in the development of mild atheroma in the rat. Three responses were seen: (a) Hyaluronic acid initially decreased, then increased; (b) Heparan sulphate and chondroitin sulphates A and C initially increased, then decreased. (c) Chondroitin sulphate-B and heparin increased with progressing lipid infiltration and decreased markedly only in the later stages. Ageing changes were also investigated in the rat aorta: total cholesterol, phospholipids and triglycerides increased progressively from weaning to 9 months of age. Hyaluronic acid decreased after weaning, reached a minimum at 6 months and then increased thereafter. Heparan sulphate and chondroitin sulphate-C reached a maximum at 6 months and then decreased thereafter. Chondroitin sulphates A and B showed a similar but less marked pattern of change with age. Heparin progressively increased with age. Aortic lipoprotein lipase activity increased in the early stages of atheroma and then decreased as the lipid infiltration became more severe. The ageing study showed that enzyme activity was quite high at weaning, decreased considerably at 3 months, but thereafter fell only slightly.
Key words : Aorta - Atherogenic diet - Cholesterol - Chondroitin sulphates - Heparan sulphate - Heparin - Hyaluronic acid - Lipoprotein lipase - Phospholipid Rat
2
S. T. VIJAYAKUMAR,
S. LEELAMMA,
P. A. KURUP
INTRODUCTION
The changes in the concentration of different glycosaminoglycans (gg) in the aorta with atherosclerosis have been the subject of many investigations. The early observations based on histochemical methods, generally suggested an increase in the ggi?s, but these methods were mainly used qualitatively. Conflicting results have been obtained in more recent studies based on biochemical separation and estimation of different ggs-10. The spectrum of reported results comprises decrease/no change in hyaluronic acid (HA) and decrease/increase in the chondroitin sulphates, dermatan sulphate and heparan sulphate. Discrepancies between various reports probably arise from the different treatments that the tissues were subjected to, the different ways in which the results were expressed, the species of animals used and also because of the difficulties inherent in the complete extraction and separation of gg fractions. These differences may also be related to variations in the severity of the atherosclerosis produced in the experimental animals used, depending on the nature of the atherogenic diet and the period during which the animals were maintained on the diet. Kumar et al.11, as a result of their studies in the aorta of Negro females, reported that an increase of acid mucopolysaccharides was characteristic of fatty streaks and a decrease characteristic of fibrous plaques, and of complicated and calcified lesions. Other reports on the changes in the gg with development of atheroscletosis include the observation of Baev et aZ.12on decreased HA and increased chondroitin sulphates in the human aorta with the development of atherosclerosis; that of Kasatikina and Pozdyuninals on the early increase of chondroitin sulphate-B (Ch S-B) and later increase of chondroitin sulphate-C (Ch S-C) in atherosclerotic rabbit aorta; that of Schmidt and Dmochowskii4 on the increase of dermatan sulphate and heparan sulphate and the decrease in HA and chondroitin sulphates in the human aorta in advanced atherosclerosis; and the observation of Seethanathan and Kurupis on the increase in HA and decrease in the sulphated gg in the aorta of rats fed a high-fat-cholesterol diet for 6 months. Because these conflicting reports may be due to variations in the severity of atherosclerosis in the experimental animals or subjects studied, the aortic gg levels have here been studied in rats maintained for different periods on an atherogenic diet. The role of lipoprotein lipase in atherosclerosis has also received considerable attention, but there are conflicting reports on the level of this enzyme. Increaseis-is, decreaselp-21 and no change 22 have all been reported. Here again the discrepancies between the various reports may be related to variations in the severity of atherosclerosis. Although there has been considerable interest in the changes of the gg in atherosclerosis, the changes in the gg with respect to age have been little studied. Meyers3 observed increase in the concentration of gg in rapidly dividing and growing tissues and decrease in older tissues, while others 24-2s failed to find any significant change in the gg with age. Bertelsen27 reviewing the results obtained by histologists, observed increased gg in both intima and media in the earlier decades, but the trend in later life was not clear. Velican and Velicanss reported an increase in the sulphated gg with age.
GLYCOSAMINOGLYCANS
AND
LIPOPROTEIN
LIPASE
3
In view of the apparent contradiction in these reports, it seemed appropriate to study in our animals the changes in gg with age. As it was difficult to get human material for controlled studies, this work has been confined to rats. MATERIALS
AND
METHODS
(a) Changes in gg and lipoprotein lipase in rats fed the atherogenic diet for diferent periods
Young male albino rats (average weight 80 g) were divided into 3 groups of 30 rats each. They were fed as follows: Group I: Normal laboratory diet (Hind Lever rat feed) supplied by M/s. Hindustan Lever Limited. Its ingredients include cereals, oilcakes, bran and other milk solids and contain 24 % protein and 4 % ether extractables. Group II: High-fat-cholesterol diet of the following composition: sucrose 57.5%; casein - 16%; hydrogenated groundnut oil - 15%; cholesterol - 5%; sodium cholate - 0.5 %; salt mixture (Hubbel, Mendel and Wakeman) - 2 %; yeast tablets - 2 %; and sharkliver oil - 2 %. Group III: High-fat-cholesterol diet (same as above) + 25 mg of potassium perchlorate/lOO g of body weight/day. Ten rats in each group were killed at the end of the lst, 3rd and 6th month of feeding. Serum, liver, aorta and heart were collected for various estimations. (b)
Changes in gg and lipoprotein lipase with age
Male albino rats used for the experiments were maintained on Hind Lever rat feed, described above. Weanling, 3, 6 and 9 month-old rats were used. The animals were fasted overnight before sacrifice and the aortas were removed for the various estimations. Only those aortas that fulfilled the criteria of having no grossly observable atherosclerosis were used for the estimations. Estimation of lipids. Total cholesterol, phospholipids and triglycerides were estimated in the serum, liver and aorra. Total cholesterol was estimated by the method of Carr and Drekteraa, phospholipids by the method of Zilversmit and Davis30 and triglycerides by the method of Van Handel and Zilversmitsi, with the modification that a Florisil column was used to remove the phospholipids. Estimation of difirent gg in the aorta. The aorta (pooled sample) was defatted by successive extractions at 60 “C with ethanol-ether (3 : 1, v/v) followed by chloroform-methanol (1 :I, v/v), each for 2 h. The dry defatted tissue was subjected to papain digestion at 65-70°C in 0.1 M phosphate buffer (pH 6.5), containing 0.005 M EDTA and 0.005 M cysteine hydrochloride according to the procedure of Laurent32. The digestion was carried out for 48 h with addition of fresh papain at the end of every 16 h. The digest was centrifuged and the clear supernatant was passed through a column of cellulose. The cellulose column (10 cm x 1 cm; microcrystalline chromatography grade, E. Merck, Germany) was washed first with distilled water and then with
4
S. T. VIJAYAKUMAR, S. LEELAMMA, P. A. KURUP
15 ml of 1% cetylpyridinium chloride (CPC). The digest was then passed through the column and washed again with 15 ml of 1% CPC. Elution was carried out successively with 15 ml of each of the following solvents in the order given below, as described by Svejcar and Robertson3s. Each eluate of 15 ml was collected separately. 1. 0.3 M NaCl in 0.05 % CPC 2. 0.28 M MgCls in 0.05 % CPC 3. Wash with 15 ml of 0.05 % CPC 4. n-Propanol-methanol-glacial acetic acid-water (40 :20: 15 :38.5, by vol.) containing 0.4 % CPC 5. Wash with 15 ml of 0.05 % CPC 6. 0.75 M MgCla in 0.1 A4 acetic acid containing 0.05 % CPC 7. 0.75 M MgCle in 0.05 % CPC 8. Wash with 15 ml of 0.05 % CPC 9. 1.25 M MgCle in 0.05 % CPC These fractions have been shown to contain the following as the major gg component@: (1) hyaluronic acid (HA); (2) heparan sulphate (HS); (4) chondroitin sulphate-A (Ch S-A); (6) chondroitin sulphate-C (Ch S-C); (7) chondroitin sulphate-B (Ch S-B); (9) heparin (H). The identity of the major component in each fraction was confirmed by comparison with standard gg*. To each fraction (15 ml), was added 10 ml of 1% CPC, diluted with 45 ml water; the mixture was allowed to stand at room temperature for 24 h and then centrifuged for 1 h at 3000 x g. The precipitate was washed by centrifugation three times for 1 h with 95 % ethanol saturated with sodium chloride. The residue was dissolved in 1.5 ml of 0.01 M NaOH and used to determine uranic acid by the modified carbazole reaction of Bitter and Muirss. Estimation of lipoprotein lipase. Lipoprotein lipase activity was estimated in the aorta, liver and heart. Chilled tissue was homogenized with 3 vol. of cold Sorenson’s phosphate buffer (pH 7.38,6.6 x 10-2M). The homogenate was centrifuged at 0°C at 2000 x g for 10 min; the supernatant was used as the enzyme source. Enzyme assay was carried out by the method of Korn, as described beforeas. Enzyme activity was expressed as micromoles of glycerol liberated/h/g of wet tissue and also, in the case of aorta, per 10 mg of protein. RESULTS I. Lipid levels of the serum, liver and aorta in rats fed the atherogenic
diet for difSerent
periods
The results are given in Tables 1 and 1A. As can be seen, the total cholesterol and triglycerides in the serum, liver and aorta, expressed on a wet weight basis, increase progressively with the duration of the atherogenic diet. In the case of aorta, a similar increase in these lipids is also observed when the results are referred to tissue * Standards of hyaluronic acid, heparan sulphate, chondroitin sulphate-A, chondroitin sulphateB, chondroitin sulphate-C and heparin were obtained from Sigma Chemicals, U.S.A.
AND LIVER
64.9 228.8 268.4
After 6 months Group I Group II Group III
+ 2.4 f 3.6 & 3.8
88.3 -+ 2.15 168.5 * 3.1 190.4 i 4.2
81.1 120.6 137.5
85.29 * 2.6 74.8 I_t 3.1 71.22 f 1.9
(mg/lOO ml i
S.E.)
FED ATHEROGENIC
phospholipids
IN RATS
7.11 i 0.89 15.83 & 1.12 18.36 & 1.56
6.9 i 0.65 12.50 & 0.85 14.60 & 1.06
6.42 & 0.64 8.49 & 1.01 9.1 & 0.93
as
FOR DIFFERENT
triglycerides glycerol
DIETS
& 4.8 & 6.4 zt 7.1
* 3.2 * 4.1 + 3.6
537.4 f 4.6 1286 I_t 7.4 1860 + 6.9
442 865 996
435 544 703 i 6.8 i 7.2 + 5.9
2841 3719 4280
i 6.2 * 7.01 & 7.6
2807.2 i 6.9 3206 i 7.2 3562 & 7.5
2715 2012 2003
phospholipids
559 i 4.8 1240 * 6.9 1460 & 7.8
523 & 5.2 868 16.4 1096 & 6.6
508 i 3.8 611 i 4.1 833 + 5.6
triglycerides glycerol (tng/lOO g of wet tissue * S.E.)
total cholesterol
Liver
PERIODS
Average of the values from 6 rats in each group. Comparison between Groups I and II, and I and III in each case. In all cases P < 0.01.
f 2.3 + 4.3 i- 3.9
60.5 * 1.98 158.28 + 2.64 179.6 xt 3.52
After 3 months Group I Group II Group III
total cholesterol
Serum
OF THE SERUM
61.25 * 2.50 93.52 + 2.25 104.5 & 3.30
LEVELS
1
After 1 month Group I Group II Group III
Period
LIPID
TABLE
as
171 * 2.05 296 & 3.1 334 * 4.3
After 6 months Group I Group II Group III
22.19 & 0.24 41.46 & 1.64 50.32 & 1.82
22.26 & 0.21 29.1 * 0.18 36.2 _c 0.24
1018 * 4.5 1640 + 6.4 1926 + 7.2
992 i 3.8 1420 * 6.8 1680 j, 7.1
983 i 4.3 989 * 5.1 b 991 + 5.4&
136 232 298
136 195 231 i 3.2 + 4.1 + 5.2
+ 2.5 & 3.2 & 3.4
139.4 It 1.4 139.6 & 1.6b 138 & 2b
mgJg of tissue protein h S.E.
2529 i 6.6 4116 YF 7.1 5210 + 8.2
2503 & 5.5 3329 zk 7.4 3706 zt 6.7
2412 + 7.6 2703 + 6.4 2967 I!Z6.8
3.6 4.2 4.6
339 f 3.8 585 + 4.6 809 zk 4.2
345 & 4.2 459 * 5.1 510 + 6.4
342 i 381 i 414 i
mgjg of tissue protein i S.E.
as glycerol
mgJlO0 g of wet tissue X!ZS.E.
Triglycerides
The aorta from 2 rats have been pooled and the value given is the average of 5 experiments from 10 rats. Comaprison between Groups I and II and I and III in each case. No symbol: P < 0.01 ; a P between 0.01 and 0.05; b P bteween 0.05 and 0.1.
166 * 1.4 212 i_ 2.2 264 * 2.6
22.41 i 0.26 22.72 * 0.31% 22.85 i 0.32
mgj100 g of wet tissue + S.E.
mg/g of tissue protein & S.E.
PERIODS
mgj100 g of wet tissue * S.E.
FOR DIFFERENT
Phospholipids
DIETS
Total cholesterol
FED ATHEROGENIC
After 3 months Group I Group II Group III
IN RATS
158 & 1.3 161 & 2.01” 162 * 1.9
LIPIDS
After 1 month Group I Group II Group III
Period
AORTIC
TABLE 1A
GLYCOSAMINOGLYCANS
AND LIPOPROTEIN
LIPASE
Fig. 1. Aorta from rats fed for 6 months on (left) atherogenic diet plus potassium perchlorate, (right) high-fat-cholesterol diet plus cholate. Frozen, Sudan black, x 40.
and
protein. Phospholipids of the serum and liver, on the other hand, decrease at the end of the 1st month as compared with the rats fed the normal diet, but progressively increase after the 3rd month. Either on a wet weight or tissue protein basis, the aortic phospholipid level is not appreciably affected at the end of the 1st month except in the case of rats of Group III (wet weight), but subsequently it increases progressively. In the rats fed the high-fat-cholesterol diet + potassium perchlorate, the increase in the lipids is much greater. The aorta in these rats shows more atheroma than in those fed the high-fat-cholesterol diet alone (Fig. 1). 2. Changes in the lipid levels of the aorta with age The results are given in Table 2. The total cholesterol, phospholipids and triglycerides show a progressive increase from weaning to 9 months of age. 3. Changes in the gg levels of the aorta in rats fed the atherogenic
diet for diferent
periods
The results are given in Table 3. HA shows a decrease in the aorta at the end of TABLE 2 CHANGES
IN AORTIC
LIPIDS
WITH
Weight
AGE IN THE RAT
AORTA
Total cholesterol
(g 3.z S.E.)
(1) (2) (3) (4)
Weanling 3 months 6 months 9 months
30 f 110 + 155 f 205 i
2.5 5.2 4.5 5.8
Phospholipids
Triglyceride
glycerol
(mg/lOO g wet tissue 3= S.E.)
142.8 + 1.58 i 166 f 171 *
1.86 2.01 2.24 2.60
852 + 983 jz 992 i1018 %
3.01 3.60 3.45 4.15
2054 2412 2503 2529
f i & +
6.05 6.46 5.80 6.71
The aorta from 2 rats have been pooled and the values given are the average of 5 experiments from 10 rats. Comparison between groups 1 and 2, 1 and 3 and 1 and 4. In all cases P < 0.01.
GLYCOSAMINOGLYCANS
IN RATS
FED ATHEROGENIC
DIETS
349 + 3.6 341 k 3.2 335 zt 2.9
325 * 1.8 337 zt 2.6 352 If 2.8
361 & 3.01 411 + 4.2 463 i 2.8
After 1 month Group I Group 11 Group III
After 3 months Group I Group II Group III
After 6 months Group I Group II Group III
1618 + 4.3 1544 i_ 4.1 1501 & 3.6
1663 + 4.8 1641 & 3.2 1611 + 3.8
1522 + 5.6 1556 % 6.1 1513 i4.9a
HS
405 + 3.9 381 * 4.1 360 i 4.0
412 k 3.8 437 & 4.2 461 i 4.6
395 * 4.5 408 i 6.1 417 * 4.9
Ch S-A
S.E.)
FOR DIFFERENT
PERIODS
1487 * 5.01 1403 i 4.0 1372 & 5.6
1509 & 5.2 1483 * 4.1 1427 ??4.6
1413 ??3.6 1425 i 4.6 1403 * 5.1
Ch S-C
346 i 2.9 295 & 3.1 237 i_ 2.6
352 * 3.6 387 zt 4.8 419 ??5.2
333 * 2.8 346 * 3.6 354 * 4.1
Ch S-B
415 * 3.0 410 f 4.6b 385 i 4.2
391 ??4.0 430 & 2.9 467 & 3.2
388 i 2.9 401 * 3.1 422 f 3.6
H
IN AORTIC
398 349 323 411
HA
& i_ + +
GG WITH
1.25 1.65 2.02 2.44
AGE IN RATS
1017 1522 1663 1618
HS
i_ & A *
2.10 2.38 3.62 3.15
Ch S-C
383 395 412 405
i_ i i i
1.05 1.02 2.11 1.87
795 & 1413 + 1509 & 1487 i
from 10 rats.
309 333 352 346
& * C +
Ch S-B tissue 31 S.E.
2.00 2.64 3.05 3.44
pg of uranic acid/g of dry defatted
Ch S-A
The aorta from 2 rats have been pooled and the values given are the average of 5 experiments Comparison between Groups 1 and 2, 1 and 3 and 1 and 4. In all cases P < 0.01.
(1) Weanling (2) 3 months (3) 6 months (4) 9 months
CHANGES
TABLE 4
2.31 2.10 1.86 1.34
319 388 391 415
H
i + & &
1.89 1.6 1.90 2.06
The aorta from 2 rats have been pooled and the values given are the average of 5 experiments from 10 rats. Comparison between Groups I and II and I and III in each case. No symbol: P < 0.01; aP between 0.01 and 0.05; b P between 0.05 and 0.1
HA
Period
(Expressed as pg of uranic acid per g of dry defatted tissue i
AORTIC
TABLE 3
GLYCOSAMINOGLYCANS
AND LIPOPROTEIN LIPASE
9
the 1st month, which is greater in the animals of Group III receiving potassium perchlorate. It increases at the end of the 3rd and 6th months, the animals of the perchlorate group showing higher values. Thus after the initial decrease, HA shows a progressive increase. In the case of HS, there is an initial increase at the end of the 1st month, followed by a decrease after the 3rd month in the rats fed high-fat-cholesterol diet. In the animals of group III receiving potassium perchlorate, however, HS decreases throughout. Ch S-A increases at the end of the 1st month; this becomes more marked after the 3rd month, but thereafter decreases at the end of the 6th month in the case of rats fed the high-fat-cholesterol diet. These changes in Ch S-A are more marked in the case of the rats receiving potassium perchlorate. In the case of Ch S-C, there is an initial increase after the 1st month, but a decrease after the 3rd and 6th months in the rats fed the high-fat-cholesterol diet. Animals of Group III receiving potassium perchlorate show a more marked decrease. Ch S-B shows a progressive increase after the 1st and 3rd months, but decreases considerably after the 6th month in the case of animals of both Groups I and III, the changes being more marked in the animals of Group III. Heparin shows a similar increase after the 1st and 3rd months in the animals of Group II. H increases more in the rats of Group III. After the 6th month there is no significant difference in the level of H in the animals of Group II compared with the controls, while there is a decrease in the animals of Group III. 4. Changes in the glycosanzinoglycans of the aorta with age The results are given in Table 4. HA concentration is high in the weanling rat aorta and decreases progressively, reaching a minimum in the 6 month-old rats and then increases afterwards. The concentration in the 9 month-old rats is higher than that in the weanling rats. HS is lowest in the weanling rat aorta and increases progressively; it reaches a maximum in the 6 month-old rats and then decreases in the 9 month-old rats. Ch S-A shows a similar pattern of change, but its extent is considerably less. Ch S-C is lowest in the weanling rat aorta, but shows a considerable increase in the 3 month-old rat. It again increases in the 6 month-old rats, but decreases in the 9 month-old rats. The change in the Ch S-B is similar to that in the case of Ch S-A. Heparin (H) on the other hand shows a progressive increase throughout. 5. Lipoprotein lipase activity of the aorta, liver and heart in rats fed the atherogenic diet for different periods
The results are given in Table 5. As can be seen, the lipoprotein lipase activity of the aorta on a wet weight basis is not appreciably affected in the animals of Groups II and III at the end of the 1st month, but increases at the end of the 3rd month. It decreases at the end of the 6th month, the changes being more marked in the animals of Group III. On a tissue protein basis, enzyme activity increases at the end of the 1st and 3rd months in animals of both groups and decreases at the end of the 6th month. In the liver, enzyme activity increases after the 1st month. At 3 months it remains more or less unaffected in Group II, but decreases in Group III. After the 6th month,
S. T. VIJAYAKUMAR, S. LEELAMMA, P. A. KURUP
10 TABLE 5 LIPOPROTEIN DIFFERENT
LIPASE
ACTIVITY
OF THE
AORTA,
LIVER
AND
HEART
IN RATS
FED
ATHEROGENIC
DIETS
FOR
PERIODS
Period
Aorta _umoIeof glycerol liberated per h per
Liver
Heart
pmole of glycerol liberated per h per g of wet tissue & S.E.
10 mg of protein f S.E.
g of wet tissue * S.E.
After 1 month Group I Group II Group III
2.06 f 0.02 2.21 i. 0.04& 2.22 f 0.02
14.54 & 1.02 15.64 i 1.60c 15.87 i 0.98b
13.53 f 1.06 16.16 xt 1.24 16.82 & 1.08
11.34 f 0.87 11.40 f l.OOC 11.41 f 1.2oc
After 3 months Group I Group II Group III
1.99 * 0.06 2.18 k 0.04 2.20 * 0.06
14.40 zt 0.96 15.81 f 1.01 15.91 * 0.99
13.52 * 0.86 13.23 h l.Olc 12.10 i 0.91a
11.26 * 0.86 11.25 5 0.92c 11.15 i 0.91c
After 6 months Group I Group II Group III
1.95 * 0.02 1.73 i 0.03 1.69 i 0.06
14.34 xt 0.92 12.16 zt 0.84 10.92 & 1.01
13.43 i 0.88 10.47 + 1.01 8.64 zt 0.96
11.16 & 0.87 8.95 + 1.01 7.68 + 0.90
Average of the value from 6 rats in each group. Comparison between Groups 1 and II and I and III in each case. No symbol: P < 0.01; aP between 0.01 and 0.05; b P between 0.05 and 0.1; c P > 0.2.
it decreases in both groups, the decrease being more marked in Group III. In the heart, there is no appreciable change in the enzyme level after the 1st and the 3rd months, but a perceptible both groups.
decrease after the 6th month
is observed
in the animals
of
6. Changes in lipoprotein lipase activity of the aorta with age The results are given in Table 6. As can be seen, the enzyme activity is quite high in the weanling rat aorta. It decreases considerably in the aorta of the 3 month-old rat, but afterwards
shows only a small tendency
to decrease.
DISCUSSION
Rats are relatively resistant to induced atherosclerosis and feeding a high-fatcholesterol diet containing sodium cholate has been observed to result only in mild lipid infiltration in the aorta 37, the extent of which increases with the duration of feeding. Administration of an antithyroid substance, such as potassium perchlorate, together with a high-fat-cholesterol-cholic acid diet for long periods has been found to result in more severe lipid infiltration in the aorta in agreement with previous reports37.
GLYCOSAMINOGLYCANS AND LIPOPROTEIN LIPASE
11
TABLE 6 CHANGES
IN AORTIC
LIPOPROTEIN
LIPASE
WITH
AGE IN RATS
Enzyme activity (pmoles of glycerol liberated/g of wet tissue per h * S. E.) (1) (2) (3) (4)
Weanling 3 months 6 months 9 months
25.84 14.54 14.40 14.34
+ f f +
2.25 0.46 0.38 0.26
Average of the values from 6 rats in each group. Comparison between Groups 1 and 2, 1 and 3 and 1 and 4. In all cases P < 0.01.
The pattern of change in the different gg fractions of the aorta varies with increased lipid infiltration. The changes in the gg fraction are similar in the animals of Group II, receiving only high fat-cholesterol diet and Group III, receiving in addition potassium perchlorate, the extent of the change being greater in the animals of the latter group. HA decreased in the early stage, but increased as the lipid infiltration became greater. HS increasedin the early stages and then decreased. Ch S-A and ChS-C increased in the early stage and then decreased. Ch S-B and heparin (H) continued to rise with increased lipid infiltration and decreased markedly only in the later stage. The nature of the changes in the individual gg now obtained is in general similar to that reported by Kumar et al.11 in the human aorta with increasing severity of atherosclerosis, with the exception of the increase in HA that we found. Kumar et al. found an initial increase for sulphated gg, but a decline in fibrous and calcified lesions. One reason for the many divergent previous results (see INTRODUCTION) might be that animals may have been used in different stages in the development of atherosclerosis. Closer examination of these results reveals that increased sulphated gg has been mostly reported in animals maintained for a short period on the atherogenic diet and also in the fatty streak of the human aorta. This increase in the early stages might represent a reaction of the arterial tissue to injury. As the lesions develop and become advanced, the capacity of the arterial tissue to synthesise gg may be affected or the activity of enzymes degrading gg may increase or both these factors may operate. The initial slight decrease and subsequent increase in HA that we observed is, however, difficult to explain. The pattern of ageing changes now observed in the rat aorta varies among the different gg. HA decreases with age, reaches a minimum in the 6 month-old rat and then increases in the 9 month-old animals. Almost all reports so far published indicate that HA slowly decreases with age in manssJs. Kumar et al.39 observed that HA tended to reach peak levels at an early age and then only gradually decreased in the female human aorta. The pattern of change - initial decrease and then increase - now reported in the rat differs from the previous reports in the human aorta.
12
S. T. VIJAYAKUMAR,
S. LEELAMMA, P. A. KURUP
HS has now been found to increase reaching a peak value in the 6 month-old rat and subsequently shows a decrease in the 9 month-old rat; this agrees with other reports on ageing changes for HS 25,38,3g.HS is widely distributed in tissues but its actual physiological function is not clear. Ch S-A and Ch S-B increased with age, reaching a peak in the 6 month-old rat, and subsequently decreased in the 9 month-old rat. Kaplan and Meyer25 reported a relative increase in Ch S-B with age. Kumar et al. 39 found that Ch S-B increased in the intima and then decreased, while Ch S-A progressively increased in the human aorta. Aortic Ch S-C in the current studies considerably increased in the 3 month-old rat when compared with the weanling, reached a peak value in the 6-month-old rat and then decreased. Kaplan and Meyer25 reported a decrease in Ch S-C with age, while the results reported by Kumar et ~1.~9 indicate an initial increase followed by a decrease, similar to our results. Heparin in our experiments continues to show an increase with age; but no other reports are available on this gg during ageing. Lipoprotein lipase activity increased in the early stages and then decreased as lipid infiltration became more severe. Increased lipolytic activity in the early stages has been reported by several workersts-1s. Seethanathan et al.36 observed considerably decreased myocardial lipoprotein lipase activity in rats fed a high-fat-cholesterol diet for 6 months. It is possible that the physiological role of this enzyme may be to prevent fat accumulation in tissues. Results obtained in several laboratories are consistent with this view. The increased lipolytic activity now reported in the early stages of lipid infiltration may represent an adaptation (enzyme induction) to increased penetration of lipids into the tissues. However the continuous influx of lipids into the tissues may promote degenerative changes; degenerating cells can no longer produce sufficient lipolytic enzyme. The pattern of change in lipoprotein lipase with progressive lipid infiltration almost parallels that in the sulphated gg. There is some evidence that lipoprotein lipase is a mucoprotein, containing a heparin-like mucopolysaccharide as an integral part of the enzyme. Heparin is known to stimulate in vivo release of this enzyme and the role of an acid mucopolysaccharide in the production of lipoprotein lipase has been clarified40. Change in this enzyme activity, parallel with that in gg concentration, is therefore understandable.
REFERENCES
1 FABER,M., The human aorta. Sulphate-containing polyuronides and the deposition of cholesterol, Arch. Pathol., 48 (1949) 342. 2 MOON, H. D. AND RINEHART, J. F., Histogenesis of coronary arteriosclerosis, Circulation, 6 (1952) 481. 3 KAPLAN, D. AND MEYER, K., Mucopolysaccharides of aorta at various ages, Proc. Sot. Exp. Biol. Med., 105 (1960) 78. 4 BERNESON,G. S., Studies of “ground substance” of the vessel wall and alterations in atherosclerosis and related diseases, J. Arheroscler. Res., 1 (1961) 386. 5 BUDDECKE,E., Chemical changes in the ground substances of the vessel wall in atherosclerosis, J. Atheroscler. Res., 2 (1962) 32.
GLWOSAMINOGLWANS
.4NfDLIPOPROTEIN LIPAS
6 BGTTCHER,C. J. F. AND KLYNSTRA, F. B., Acid mucopolysaccharides 7 8 9 10
11 12 13 14 15 16 17 18 19 20 21 22
23 24 25 26 27 28 29 30 31 32
13
in aortic tissue at different stages of atherosclerosis, Lam-et, i (1962) 1304. B~TITTCHER, C. J. F. AND KLYNSTRA, F. B., Content of acid mucopolysaccharides in human aorta, J. Atheroscler. Res., 2 (1962) 263. B~~TTCHER, C. J. F. AND KLYNSTRA, F. B., Acid mucopolysaccharides in human aortic tissues (The distribution at different stages of atherosclerosis), Lancet, ii (1963) 439. in atheroma and alloxan diabetes, ICHIDA, T. AND KALANT, N., Aortic glycosaminoglycans Can. /. Biochem., 46 (1968) 249. NAKAMURA, T., TOKITA, K., TATENO, S., KOTOKU, T. AND OHBA, T., Human aortic acid mucopolysaccharides and glycoproteins. Changes during ageing and in atherosclerosis, J. Atheroscler. Rex, 8 (1968) 891. KUMAR, V., BERENSON,G. S., RUIZ, M., DALFERES,JR., E. R. AND STRONG, J. P., Acid mucopolysaccharides of human aorta, J. Atheroscler. Res., 7 (1967) 583. BAEV, A. A., NIKIFOROVSKAYA, L. F. AND VINOGRADOVA,M. S., Acid mucopolysaccharides of the human aorta during the development of atherosclerosis, Chem. Abstr., 71 (1969) 286711. KASATIKINA, L. V. AND POZDYUNINA,N. M., States of the principal components in the early stages of experimental atherosclerosis, Arkh. Putol., 31 (1969) 53. SCHMIDT, M., AND DMOCHOWSKI,A., The acid mucopolysaccharides of the normal and atherosclerotic human aorta, Actu Biochem. Polon., 11 (1964) 371. SEETHANATHAN, P. AND KURUP, P. A., Changes in tissue glycosaminoglycans in rats fed a hypercholesterolaemic diet, Atherosclerosis, 14 (1971) 65. LEITES,F. L., Activity of lipoprotein lipase and other lipolytic enzymes in rabbits with experimental atherosclerosis, Put. Fiziol. Eksp. Ter., 9 (1965) 12. ZEMPLBNYI,T. AND GRAFNETTER,D., The lipolytic activity of heart and aorta in experimental atherosclerosis in rabbits, Brit. J. Exp. Pufol., 40 (1959) 312. LEITES,F. L. AND FEDOSEEV,A. N., Changes in the activity of lipolytic enzymes in dogs with experimental atherosclerosis, Arkh. Putol., 26 (1964) 15. ALBERINI,B., LASAGNA, G. C. AND ANGELI, G., Plasma post-heparin clearing in atherosclerosis and diabetes, Actu Geront. (Milan), 11 (1961) 163. SYURIN,A. A. AND BAZAZYAN, G. G., Lipoprotein lipase and blood clotting in patients affected by arteriosclerosis, Vop. Med. Khim., 13 (1967) 42. ZEMPL~NYI,T., The lipolytic and esterolytic activity of blood and tissue and problems of atherosclerosis, Advun. Lipid Res., 2 (1964) 235. SKOREPA,J., FUCIK, M., MARES, P. AND NOVAK, S,, Methyl palmitate as substrate in the estimation of post-heparin lipolytic activity in normal subjects and atherosclerosis, Shorn. Lek., 11 (1969) 316. MEYER, K. D. In: D. NACHMANSOHN(Ed.), Molecular Biology, Elementary Processes of Nerve Conduction and Muscle Contraction, Academic Press, New York, N.Y., 1960. of human arterial tissue, Part 2 (Analysis of KIRK, J. E. AND DYRBYE, M., Mucopolysaccharides total isolated mucopolysaccharide material), J. Gerontol., 12 (1957) 23. KAPLAN, D. AND MEYER, K., Mucopolysaccharides of aorta at various ages, Proc. Sot. Exp. Biol. Med., 105 (1960) 78. SMITH, E. B., The influence of age and atherosclerosis on the chemistry of aortic intima, Part 2 (Collagen and mucopolysaccharides), J. Atheroscler. Res., 5 (1965) 241. BERTKLSEN, S. In: M. SANDLERAND G. H. BOURNE(Eds.), Atherosclerosis and its Origin, Academic Press, New York, N.Y., 1963. VELICAN,C. AND VELICAN,D., Histochemistry of the intima of the human aorta and problems of atherosclerosis, Arch. Puthol., 25 (1963) 16. CARR, J. J. AND DREKTER,I. J., Estimation of total cholesterol in the serum, Clin. Chem., 2 (1956) 353. ZILVERSMIT,D. B. AND DAVIS, A. K., Microdetermination of phospholipids, J. Lab. Clin. Med., 35 (1950) 155. VAN HANDEL, E. AND ZILVERSMIT,D. B., Micromethod for direct determination of serum triglycerides, J. Lab. C/in. Med., 50 (1957) 152. SCOTT, J. E., Aliphatic ammonium salts in the assay of acid polysaccharides from tissues. In: D. GLICK (Ed.), Methods in Biochemical Analysis, Vol. 8, Interscience, New York, N.Y., 1960, P. 145.
14
S. T. VIJAYAKUMAR, S. LEELAMMA, P. A. KURUP
33 SVEJCAR, J. AND ROBERTSON,W. V. B., Microseparation and determination of mammalian acidic glycosaminoglycans, Anal. Biochem., 18 (1967) 333. 34 ANTONOPOULOS, C. A., GARDELL, S., SZIRMAI, J. A. AND DE TYSSONSK,E. R., Determination of glycosaminoglycans from tissues on the microgram scale, Biochim. Bioghys. Actu, 83 (1964) 1. 35 BITTER,T. AND MUIR, H. M., A modified uranic acid carbazole raction, Anal. Biochem., 4 (1962)
330. 36 SEETHANATHAN,B., VIJAYAGOPALAN,P., AUGUSTI, K. T. AND KURUP, P. A., Myocardial lipoprotein lipase in rats fed a hypercholesterolaemic diet, Atherosclerosis, 11 (1970) 333. 37 NIKOLOVA,M. P., MIKHAILOVA,I. Yu., AND STOYANOV,D. P., Comparative study of experimental atherosclerosis production in white rats, Fed. Proc., 25 (3) (1966) T 458. 38 GORE, I., TANAKA, Y. AND KIMOTO,E., Changes in the acid mucopolysaccharides of human aortae with age and atherosclerosis, Circulution, 30, Suppl. III (1964) 10. 39 KUMAR, V., BERENSON,G. S., Rurz, H., DALFERES,JR., E. R. AND STRONG,J. P., Acid mucopolysaccharides of human aorta, Part 1 (Variations with maturation), J. Atheroscler. Res., 7 (1967) 573. 40 HALL, D. A., The production of plasma clearing factor in vitro, Biochem. J., 10 (1958) 5.
Company, Amsterdam
- Printed in The Netherlands
CHANGES IN AORTIC GLYCOSAMINOGLYCANS AND LIPOPROTEIN LIPASE ACTIVITY IN RATS WITH AGE AND ATHEROMA
S. T. VIJAYAKUMAR, Department
S. LEELAMMA
of Biochemistry,
AND
P. A. KURUP
University of Kerala, Trivandrum-695001
(India)
(Received August 17th, 1973) (Revised received April 18th and August 27th, 1974) (Accepted August 28th, 1974)
SUMMARY
The changes in the individual glycosaminoglycans of the aorta and in lipoprotein lipase activity of the aorta, liver and heart have been studied at various stages in the development of mild atheroma in the rat. Three responses were seen: (a) Hyaluronic acid initially decreased, then increased; (b) Heparan sulphate and chondroitin sulphates A and C initially increased, then decreased. (c) Chondroitin sulphate-B and heparin increased with progressing lipid infiltration and decreased markedly only in the later stages. Ageing changes were also investigated in the rat aorta: total cholesterol, phospholipids and triglycerides increased progressively from weaning to 9 months of age. Hyaluronic acid decreased after weaning, reached a minimum at 6 months and then increased thereafter. Heparan sulphate and chondroitin sulphate-C reached a maximum at 6 months and then decreased thereafter. Chondroitin sulphates A and B showed a similar but less marked pattern of change with age. Heparin progressively increased with age. Aortic lipoprotein lipase activity increased in the early stages of atheroma and then decreased as the lipid infiltration became more severe. The ageing study showed that enzyme activity was quite high at weaning, decreased considerably at 3 months, but thereafter fell only slightly.
Key words : Aorta - Atherogenic diet - Cholesterol - Chondroitin sulphates - Heparan sulphate - Heparin - Hyaluronic acid - Lipoprotein lipase - Phospholipid Rat
2
S. T. VIJAYAKUMAR,
S. LEELAMMA,
P. A. KURUP
INTRODUCTION
The changes in the concentration of different glycosaminoglycans (gg) in the aorta with atherosclerosis have been the subject of many investigations. The early observations based on histochemical methods, generally suggested an increase in the ggi?s, but these methods were mainly used qualitatively. Conflicting results have been obtained in more recent studies based on biochemical separation and estimation of different ggs-10. The spectrum of reported results comprises decrease/no change in hyaluronic acid (HA) and decrease/increase in the chondroitin sulphates, dermatan sulphate and heparan sulphate. Discrepancies between various reports probably arise from the different treatments that the tissues were subjected to, the different ways in which the results were expressed, the species of animals used and also because of the difficulties inherent in the complete extraction and separation of gg fractions. These differences may also be related to variations in the severity of the atherosclerosis produced in the experimental animals used, depending on the nature of the atherogenic diet and the period during which the animals were maintained on the diet. Kumar et al.11, as a result of their studies in the aorta of Negro females, reported that an increase of acid mucopolysaccharides was characteristic of fatty streaks and a decrease characteristic of fibrous plaques, and of complicated and calcified lesions. Other reports on the changes in the gg with development of atheroscletosis include the observation of Baev et aZ.12on decreased HA and increased chondroitin sulphates in the human aorta with the development of atherosclerosis; that of Kasatikina and Pozdyuninals on the early increase of chondroitin sulphate-B (Ch S-B) and later increase of chondroitin sulphate-C (Ch S-C) in atherosclerotic rabbit aorta; that of Schmidt and Dmochowskii4 on the increase of dermatan sulphate and heparan sulphate and the decrease in HA and chondroitin sulphates in the human aorta in advanced atherosclerosis; and the observation of Seethanathan and Kurupis on the increase in HA and decrease in the sulphated gg in the aorta of rats fed a high-fat-cholesterol diet for 6 months. Because these conflicting reports may be due to variations in the severity of atherosclerosis in the experimental animals or subjects studied, the aortic gg levels have here been studied in rats maintained for different periods on an atherogenic diet. The role of lipoprotein lipase in atherosclerosis has also received considerable attention, but there are conflicting reports on the level of this enzyme. Increaseis-is, decreaselp-21 and no change 22 have all been reported. Here again the discrepancies between the various reports may be related to variations in the severity of atherosclerosis. Although there has been considerable interest in the changes of the gg in atherosclerosis, the changes in the gg with respect to age have been little studied. Meyers3 observed increase in the concentration of gg in rapidly dividing and growing tissues and decrease in older tissues, while others 24-2s failed to find any significant change in the gg with age. Bertelsen27 reviewing the results obtained by histologists, observed increased gg in both intima and media in the earlier decades, but the trend in later life was not clear. Velican and Velicanss reported an increase in the sulphated gg with age.
GLYCOSAMINOGLYCANS
AND
LIPOPROTEIN
LIPASE
3
In view of the apparent contradiction in these reports, it seemed appropriate to study in our animals the changes in gg with age. As it was difficult to get human material for controlled studies, this work has been confined to rats. MATERIALS
AND
METHODS
(a) Changes in gg and lipoprotein lipase in rats fed the atherogenic diet for diferent periods
Young male albino rats (average weight 80 g) were divided into 3 groups of 30 rats each. They were fed as follows: Group I: Normal laboratory diet (Hind Lever rat feed) supplied by M/s. Hindustan Lever Limited. Its ingredients include cereals, oilcakes, bran and other milk solids and contain 24 % protein and 4 % ether extractables. Group II: High-fat-cholesterol diet of the following composition: sucrose 57.5%; casein - 16%; hydrogenated groundnut oil - 15%; cholesterol - 5%; sodium cholate - 0.5 %; salt mixture (Hubbel, Mendel and Wakeman) - 2 %; yeast tablets - 2 %; and sharkliver oil - 2 %. Group III: High-fat-cholesterol diet (same as above) + 25 mg of potassium perchlorate/lOO g of body weight/day. Ten rats in each group were killed at the end of the lst, 3rd and 6th month of feeding. Serum, liver, aorta and heart were collected for various estimations. (b)
Changes in gg and lipoprotein lipase with age
Male albino rats used for the experiments were maintained on Hind Lever rat feed, described above. Weanling, 3, 6 and 9 month-old rats were used. The animals were fasted overnight before sacrifice and the aortas were removed for the various estimations. Only those aortas that fulfilled the criteria of having no grossly observable atherosclerosis were used for the estimations. Estimation of lipids. Total cholesterol, phospholipids and triglycerides were estimated in the serum, liver and aorra. Total cholesterol was estimated by the method of Carr and Drekteraa, phospholipids by the method of Zilversmit and Davis30 and triglycerides by the method of Van Handel and Zilversmitsi, with the modification that a Florisil column was used to remove the phospholipids. Estimation of difirent gg in the aorta. The aorta (pooled sample) was defatted by successive extractions at 60 “C with ethanol-ether (3 : 1, v/v) followed by chloroform-methanol (1 :I, v/v), each for 2 h. The dry defatted tissue was subjected to papain digestion at 65-70°C in 0.1 M phosphate buffer (pH 6.5), containing 0.005 M EDTA and 0.005 M cysteine hydrochloride according to the procedure of Laurent32. The digestion was carried out for 48 h with addition of fresh papain at the end of every 16 h. The digest was centrifuged and the clear supernatant was passed through a column of cellulose. The cellulose column (10 cm x 1 cm; microcrystalline chromatography grade, E. Merck, Germany) was washed first with distilled water and then with
4
S. T. VIJAYAKUMAR, S. LEELAMMA, P. A. KURUP
15 ml of 1% cetylpyridinium chloride (CPC). The digest was then passed through the column and washed again with 15 ml of 1% CPC. Elution was carried out successively with 15 ml of each of the following solvents in the order given below, as described by Svejcar and Robertson3s. Each eluate of 15 ml was collected separately. 1. 0.3 M NaCl in 0.05 % CPC 2. 0.28 M MgCls in 0.05 % CPC 3. Wash with 15 ml of 0.05 % CPC 4. n-Propanol-methanol-glacial acetic acid-water (40 :20: 15 :38.5, by vol.) containing 0.4 % CPC 5. Wash with 15 ml of 0.05 % CPC 6. 0.75 M MgCla in 0.1 A4 acetic acid containing 0.05 % CPC 7. 0.75 M MgCle in 0.05 % CPC 8. Wash with 15 ml of 0.05 % CPC 9. 1.25 M MgCle in 0.05 % CPC These fractions have been shown to contain the following as the major gg component@: (1) hyaluronic acid (HA); (2) heparan sulphate (HS); (4) chondroitin sulphate-A (Ch S-A); (6) chondroitin sulphate-C (Ch S-C); (7) chondroitin sulphate-B (Ch S-B); (9) heparin (H). The identity of the major component in each fraction was confirmed by comparison with standard gg*. To each fraction (15 ml), was added 10 ml of 1% CPC, diluted with 45 ml water; the mixture was allowed to stand at room temperature for 24 h and then centrifuged for 1 h at 3000 x g. The precipitate was washed by centrifugation three times for 1 h with 95 % ethanol saturated with sodium chloride. The residue was dissolved in 1.5 ml of 0.01 M NaOH and used to determine uranic acid by the modified carbazole reaction of Bitter and Muirss. Estimation of lipoprotein lipase. Lipoprotein lipase activity was estimated in the aorta, liver and heart. Chilled tissue was homogenized with 3 vol. of cold Sorenson’s phosphate buffer (pH 7.38,6.6 x 10-2M). The homogenate was centrifuged at 0°C at 2000 x g for 10 min; the supernatant was used as the enzyme source. Enzyme assay was carried out by the method of Korn, as described beforeas. Enzyme activity was expressed as micromoles of glycerol liberated/h/g of wet tissue and also, in the case of aorta, per 10 mg of protein. RESULTS I. Lipid levels of the serum, liver and aorta in rats fed the atherogenic
diet for difSerent
periods
The results are given in Tables 1 and 1A. As can be seen, the total cholesterol and triglycerides in the serum, liver and aorta, expressed on a wet weight basis, increase progressively with the duration of the atherogenic diet. In the case of aorta, a similar increase in these lipids is also observed when the results are referred to tissue * Standards of hyaluronic acid, heparan sulphate, chondroitin sulphate-A, chondroitin sulphateB, chondroitin sulphate-C and heparin were obtained from Sigma Chemicals, U.S.A.
AND LIVER
64.9 228.8 268.4
After 6 months Group I Group II Group III
+ 2.4 f 3.6 & 3.8
88.3 -+ 2.15 168.5 * 3.1 190.4 i 4.2
81.1 120.6 137.5
85.29 * 2.6 74.8 I_t 3.1 71.22 f 1.9
(mg/lOO ml i
S.E.)
FED ATHEROGENIC
phospholipids
IN RATS
7.11 i 0.89 15.83 & 1.12 18.36 & 1.56
6.9 i 0.65 12.50 & 0.85 14.60 & 1.06
6.42 & 0.64 8.49 & 1.01 9.1 & 0.93
as
FOR DIFFERENT
triglycerides glycerol
DIETS
& 4.8 & 6.4 zt 7.1
* 3.2 * 4.1 + 3.6
537.4 f 4.6 1286 I_t 7.4 1860 + 6.9
442 865 996
435 544 703 i 6.8 i 7.2 + 5.9
2841 3719 4280
i 6.2 * 7.01 & 7.6
2807.2 i 6.9 3206 i 7.2 3562 & 7.5
2715 2012 2003
phospholipids
559 i 4.8 1240 * 6.9 1460 & 7.8
523 & 5.2 868 16.4 1096 & 6.6
508 i 3.8 611 i 4.1 833 + 5.6
triglycerides glycerol (tng/lOO g of wet tissue * S.E.)
total cholesterol
Liver
PERIODS
Average of the values from 6 rats in each group. Comparison between Groups I and II, and I and III in each case. In all cases P < 0.01.
f 2.3 + 4.3 i- 3.9
60.5 * 1.98 158.28 + 2.64 179.6 xt 3.52
After 3 months Group I Group II Group III
total cholesterol
Serum
OF THE SERUM
61.25 * 2.50 93.52 + 2.25 104.5 & 3.30
LEVELS
1
After 1 month Group I Group II Group III
Period
LIPID
TABLE
as
171 * 2.05 296 & 3.1 334 * 4.3
After 6 months Group I Group II Group III
22.19 & 0.24 41.46 & 1.64 50.32 & 1.82
22.26 & 0.21 29.1 * 0.18 36.2 _c 0.24
1018 * 4.5 1640 + 6.4 1926 + 7.2
992 i 3.8 1420 * 6.8 1680 j, 7.1
983 i 4.3 989 * 5.1 b 991 + 5.4&
136 232 298
136 195 231 i 3.2 + 4.1 + 5.2
+ 2.5 & 3.2 & 3.4
139.4 It 1.4 139.6 & 1.6b 138 & 2b
mgJg of tissue protein h S.E.
2529 i 6.6 4116 YF 7.1 5210 + 8.2
2503 & 5.5 3329 zk 7.4 3706 zt 6.7
2412 + 7.6 2703 + 6.4 2967 I!Z6.8
3.6 4.2 4.6
339 f 3.8 585 + 4.6 809 zk 4.2
345 & 4.2 459 * 5.1 510 + 6.4
342 i 381 i 414 i
mgjg of tissue protein i S.E.
as glycerol
mgJlO0 g of wet tissue X!ZS.E.
Triglycerides
The aorta from 2 rats have been pooled and the value given is the average of 5 experiments from 10 rats. Comaprison between Groups I and II and I and III in each case. No symbol: P < 0.01 ; a P between 0.01 and 0.05; b P bteween 0.05 and 0.1.
166 * 1.4 212 i_ 2.2 264 * 2.6
22.41 i 0.26 22.72 * 0.31% 22.85 i 0.32
mgj100 g of wet tissue + S.E.
mg/g of tissue protein & S.E.
PERIODS
mgj100 g of wet tissue * S.E.
FOR DIFFERENT
Phospholipids
DIETS
Total cholesterol
FED ATHEROGENIC
After 3 months Group I Group II Group III
IN RATS
158 & 1.3 161 & 2.01” 162 * 1.9
LIPIDS
After 1 month Group I Group II Group III
Period
AORTIC
TABLE 1A
GLYCOSAMINOGLYCANS
AND LIPOPROTEIN
LIPASE
Fig. 1. Aorta from rats fed for 6 months on (left) atherogenic diet plus potassium perchlorate, (right) high-fat-cholesterol diet plus cholate. Frozen, Sudan black, x 40.
and
protein. Phospholipids of the serum and liver, on the other hand, decrease at the end of the 1st month as compared with the rats fed the normal diet, but progressively increase after the 3rd month. Either on a wet weight or tissue protein basis, the aortic phospholipid level is not appreciably affected at the end of the 1st month except in the case of rats of Group III (wet weight), but subsequently it increases progressively. In the rats fed the high-fat-cholesterol diet + potassium perchlorate, the increase in the lipids is much greater. The aorta in these rats shows more atheroma than in those fed the high-fat-cholesterol diet alone (Fig. 1). 2. Changes in the lipid levels of the aorta with age The results are given in Table 2. The total cholesterol, phospholipids and triglycerides show a progressive increase from weaning to 9 months of age. 3. Changes in the gg levels of the aorta in rats fed the atherogenic
diet for diferent
periods
The results are given in Table 3. HA shows a decrease in the aorta at the end of TABLE 2 CHANGES
IN AORTIC
LIPIDS
WITH
Weight
AGE IN THE RAT
AORTA
Total cholesterol
(g 3.z S.E.)
(1) (2) (3) (4)
Weanling 3 months 6 months 9 months
30 f 110 + 155 f 205 i
2.5 5.2 4.5 5.8
Phospholipids
Triglyceride
glycerol
(mg/lOO g wet tissue 3= S.E.)
142.8 + 1.58 i 166 f 171 *
1.86 2.01 2.24 2.60
852 + 983 jz 992 i1018 %
3.01 3.60 3.45 4.15
2054 2412 2503 2529
f i & +
6.05 6.46 5.80 6.71
The aorta from 2 rats have been pooled and the values given are the average of 5 experiments from 10 rats. Comparison between groups 1 and 2, 1 and 3 and 1 and 4. In all cases P < 0.01.
GLYCOSAMINOGLYCANS
IN RATS
FED ATHEROGENIC
DIETS
349 + 3.6 341 k 3.2 335 zt 2.9
325 * 1.8 337 zt 2.6 352 If 2.8
361 & 3.01 411 + 4.2 463 i 2.8
After 1 month Group I Group 11 Group III
After 3 months Group I Group II Group III
After 6 months Group I Group II Group III
1618 + 4.3 1544 i_ 4.1 1501 & 3.6
1663 + 4.8 1641 & 3.2 1611 + 3.8
1522 + 5.6 1556 % 6.1 1513 i4.9a
HS
405 + 3.9 381 * 4.1 360 i 4.0
412 k 3.8 437 & 4.2 461 i 4.6
395 * 4.5 408 i 6.1 417 * 4.9
Ch S-A
S.E.)
FOR DIFFERENT
PERIODS
1487 * 5.01 1403 i 4.0 1372 & 5.6
1509 & 5.2 1483 * 4.1 1427 ??4.6
1413 ??3.6 1425 i 4.6 1403 * 5.1
Ch S-C
346 i 2.9 295 & 3.1 237 i_ 2.6
352 * 3.6 387 zt 4.8 419 ??5.2
333 * 2.8 346 * 3.6 354 * 4.1
Ch S-B
415 * 3.0 410 f 4.6b 385 i 4.2
391 ??4.0 430 & 2.9 467 & 3.2
388 i 2.9 401 * 3.1 422 f 3.6
H
IN AORTIC
398 349 323 411
HA
& i_ + +
GG WITH
1.25 1.65 2.02 2.44
AGE IN RATS
1017 1522 1663 1618
HS
i_ & A *
2.10 2.38 3.62 3.15
Ch S-C
383 395 412 405
i_ i i i
1.05 1.02 2.11 1.87
795 & 1413 + 1509 & 1487 i
from 10 rats.
309 333 352 346
& * C +
Ch S-B tissue 31 S.E.
2.00 2.64 3.05 3.44
pg of uranic acid/g of dry defatted
Ch S-A
The aorta from 2 rats have been pooled and the values given are the average of 5 experiments Comparison between Groups 1 and 2, 1 and 3 and 1 and 4. In all cases P < 0.01.
(1) Weanling (2) 3 months (3) 6 months (4) 9 months
CHANGES
TABLE 4
2.31 2.10 1.86 1.34
319 388 391 415
H
i + & &
1.89 1.6 1.90 2.06
The aorta from 2 rats have been pooled and the values given are the average of 5 experiments from 10 rats. Comparison between Groups I and II and I and III in each case. No symbol: P < 0.01; aP between 0.01 and 0.05; b P between 0.05 and 0.1
HA
Period
(Expressed as pg of uranic acid per g of dry defatted tissue i
AORTIC
TABLE 3
GLYCOSAMINOGLYCANS
AND LIPOPROTEIN LIPASE
9
the 1st month, which is greater in the animals of Group III receiving potassium perchlorate. It increases at the end of the 3rd and 6th months, the animals of the perchlorate group showing higher values. Thus after the initial decrease, HA shows a progressive increase. In the case of HS, there is an initial increase at the end of the 1st month, followed by a decrease after the 3rd month in the rats fed high-fat-cholesterol diet. In the animals of group III receiving potassium perchlorate, however, HS decreases throughout. Ch S-A increases at the end of the 1st month; this becomes more marked after the 3rd month, but thereafter decreases at the end of the 6th month in the case of rats fed the high-fat-cholesterol diet. These changes in Ch S-A are more marked in the case of the rats receiving potassium perchlorate. In the case of Ch S-C, there is an initial increase after the 1st month, but a decrease after the 3rd and 6th months in the rats fed the high-fat-cholesterol diet. Animals of Group III receiving potassium perchlorate show a more marked decrease. Ch S-B shows a progressive increase after the 1st and 3rd months, but decreases considerably after the 6th month in the case of animals of both Groups I and III, the changes being more marked in the animals of Group III. Heparin shows a similar increase after the 1st and 3rd months in the animals of Group II. H increases more in the rats of Group III. After the 6th month there is no significant difference in the level of H in the animals of Group II compared with the controls, while there is a decrease in the animals of Group III. 4. Changes in the glycosanzinoglycans of the aorta with age The results are given in Table 4. HA concentration is high in the weanling rat aorta and decreases progressively, reaching a minimum in the 6 month-old rats and then increases afterwards. The concentration in the 9 month-old rats is higher than that in the weanling rats. HS is lowest in the weanling rat aorta and increases progressively; it reaches a maximum in the 6 month-old rats and then decreases in the 9 month-old rats. Ch S-A shows a similar pattern of change, but its extent is considerably less. Ch S-C is lowest in the weanling rat aorta, but shows a considerable increase in the 3 month-old rat. It again increases in the 6 month-old rats, but decreases in the 9 month-old rats. The change in the Ch S-B is similar to that in the case of Ch S-A. Heparin (H) on the other hand shows a progressive increase throughout. 5. Lipoprotein lipase activity of the aorta, liver and heart in rats fed the atherogenic diet for different periods
The results are given in Table 5. As can be seen, the lipoprotein lipase activity of the aorta on a wet weight basis is not appreciably affected in the animals of Groups II and III at the end of the 1st month, but increases at the end of the 3rd month. It decreases at the end of the 6th month, the changes being more marked in the animals of Group III. On a tissue protein basis, enzyme activity increases at the end of the 1st and 3rd months in animals of both groups and decreases at the end of the 6th month. In the liver, enzyme activity increases after the 1st month. At 3 months it remains more or less unaffected in Group II, but decreases in Group III. After the 6th month,
S. T. VIJAYAKUMAR, S. LEELAMMA, P. A. KURUP
10 TABLE 5 LIPOPROTEIN DIFFERENT
LIPASE
ACTIVITY
OF THE
AORTA,
LIVER
AND
HEART
IN RATS
FED
ATHEROGENIC
DIETS
FOR
PERIODS
Period
Aorta _umoIeof glycerol liberated per h per
Liver
Heart
pmole of glycerol liberated per h per g of wet tissue & S.E.
10 mg of protein f S.E.
g of wet tissue * S.E.
After 1 month Group I Group II Group III
2.06 f 0.02 2.21 i. 0.04& 2.22 f 0.02
14.54 & 1.02 15.64 i 1.60c 15.87 i 0.98b
13.53 f 1.06 16.16 xt 1.24 16.82 & 1.08
11.34 f 0.87 11.40 f l.OOC 11.41 f 1.2oc
After 3 months Group I Group II Group III
1.99 * 0.06 2.18 k 0.04 2.20 * 0.06
14.40 zt 0.96 15.81 f 1.01 15.91 * 0.99
13.52 * 0.86 13.23 h l.Olc 12.10 i 0.91a
11.26 * 0.86 11.25 5 0.92c 11.15 i 0.91c
After 6 months Group I Group II Group III
1.95 * 0.02 1.73 i 0.03 1.69 i 0.06
14.34 xt 0.92 12.16 zt 0.84 10.92 & 1.01
13.43 i 0.88 10.47 + 1.01 8.64 zt 0.96
11.16 & 0.87 8.95 + 1.01 7.68 + 0.90
Average of the value from 6 rats in each group. Comparison between Groups 1 and II and I and III in each case. No symbol: P < 0.01; aP between 0.01 and 0.05; b P between 0.05 and 0.1; c P > 0.2.
it decreases in both groups, the decrease being more marked in Group III. In the heart, there is no appreciable change in the enzyme level after the 1st and the 3rd months, but a perceptible both groups.
decrease after the 6th month
is observed
in the animals
of
6. Changes in lipoprotein lipase activity of the aorta with age The results are given in Table 6. As can be seen, the enzyme activity is quite high in the weanling rat aorta. It decreases considerably in the aorta of the 3 month-old rat, but afterwards
shows only a small tendency
to decrease.
DISCUSSION
Rats are relatively resistant to induced atherosclerosis and feeding a high-fatcholesterol diet containing sodium cholate has been observed to result only in mild lipid infiltration in the aorta 37, the extent of which increases with the duration of feeding. Administration of an antithyroid substance, such as potassium perchlorate, together with a high-fat-cholesterol-cholic acid diet for long periods has been found to result in more severe lipid infiltration in the aorta in agreement with previous reports37.
GLYCOSAMINOGLYCANS AND LIPOPROTEIN LIPASE
11
TABLE 6 CHANGES
IN AORTIC
LIPOPROTEIN
LIPASE
WITH
AGE IN RATS
Enzyme activity (pmoles of glycerol liberated/g of wet tissue per h * S. E.) (1) (2) (3) (4)
Weanling 3 months 6 months 9 months
25.84 14.54 14.40 14.34
+ f f +
2.25 0.46 0.38 0.26
Average of the values from 6 rats in each group. Comparison between Groups 1 and 2, 1 and 3 and 1 and 4. In all cases P < 0.01.
The pattern of change in the different gg fractions of the aorta varies with increased lipid infiltration. The changes in the gg fraction are similar in the animals of Group II, receiving only high fat-cholesterol diet and Group III, receiving in addition potassium perchlorate, the extent of the change being greater in the animals of the latter group. HA decreased in the early stage, but increased as the lipid infiltration became greater. HS increasedin the early stages and then decreased. Ch S-A and ChS-C increased in the early stage and then decreased. Ch S-B and heparin (H) continued to rise with increased lipid infiltration and decreased markedly only in the later stage. The nature of the changes in the individual gg now obtained is in general similar to that reported by Kumar et al.11 in the human aorta with increasing severity of atherosclerosis, with the exception of the increase in HA that we found. Kumar et al. found an initial increase for sulphated gg, but a decline in fibrous and calcified lesions. One reason for the many divergent previous results (see INTRODUCTION) might be that animals may have been used in different stages in the development of atherosclerosis. Closer examination of these results reveals that increased sulphated gg has been mostly reported in animals maintained for a short period on the atherogenic diet and also in the fatty streak of the human aorta. This increase in the early stages might represent a reaction of the arterial tissue to injury. As the lesions develop and become advanced, the capacity of the arterial tissue to synthesise gg may be affected or the activity of enzymes degrading gg may increase or both these factors may operate. The initial slight decrease and subsequent increase in HA that we observed is, however, difficult to explain. The pattern of ageing changes now observed in the rat aorta varies among the different gg. HA decreases with age, reaches a minimum in the 6 month-old rat and then increases in the 9 month-old animals. Almost all reports so far published indicate that HA slowly decreases with age in manssJs. Kumar et al.39 observed that HA tended to reach peak levels at an early age and then only gradually decreased in the female human aorta. The pattern of change - initial decrease and then increase - now reported in the rat differs from the previous reports in the human aorta.
12
S. T. VIJAYAKUMAR,
S. LEELAMMA, P. A. KURUP
HS has now been found to increase reaching a peak value in the 6 month-old rat and subsequently shows a decrease in the 9 month-old rat; this agrees with other reports on ageing changes for HS 25,38,3g.HS is widely distributed in tissues but its actual physiological function is not clear. Ch S-A and Ch S-B increased with age, reaching a peak in the 6 month-old rat, and subsequently decreased in the 9 month-old rat. Kaplan and Meyer25 reported a relative increase in Ch S-B with age. Kumar et al. 39 found that Ch S-B increased in the intima and then decreased, while Ch S-A progressively increased in the human aorta. Aortic Ch S-C in the current studies considerably increased in the 3 month-old rat when compared with the weanling, reached a peak value in the 6-month-old rat and then decreased. Kaplan and Meyer25 reported a decrease in Ch S-C with age, while the results reported by Kumar et ~1.~9 indicate an initial increase followed by a decrease, similar to our results. Heparin in our experiments continues to show an increase with age; but no other reports are available on this gg during ageing. Lipoprotein lipase activity increased in the early stages and then decreased as lipid infiltration became more severe. Increased lipolytic activity in the early stages has been reported by several workersts-1s. Seethanathan et al.36 observed considerably decreased myocardial lipoprotein lipase activity in rats fed a high-fat-cholesterol diet for 6 months. It is possible that the physiological role of this enzyme may be to prevent fat accumulation in tissues. Results obtained in several laboratories are consistent with this view. The increased lipolytic activity now reported in the early stages of lipid infiltration may represent an adaptation (enzyme induction) to increased penetration of lipids into the tissues. However the continuous influx of lipids into the tissues may promote degenerative changes; degenerating cells can no longer produce sufficient lipolytic enzyme. The pattern of change in lipoprotein lipase with progressive lipid infiltration almost parallels that in the sulphated gg. There is some evidence that lipoprotein lipase is a mucoprotein, containing a heparin-like mucopolysaccharide as an integral part of the enzyme. Heparin is known to stimulate in vivo release of this enzyme and the role of an acid mucopolysaccharide in the production of lipoprotein lipase has been clarified40. Change in this enzyme activity, parallel with that in gg concentration, is therefore understandable.
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14
S. T. VIJAYAKUMAR, S. LEELAMMA, P. A. KURUP
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