Metabolism of glycosaminoglycans in atheromatous rats

2 I (1975) 245.-258

Athrrosclerosis,

(3 Elsevier Scientific Publishing Company, Amsterdam

METABOLISM RATS

OF

GLYCOSAMINOGLYCANS

ENZYMES CONCERNED GLYCOSAMINOGLYCANS

S. T. VIJAYAKUMAR Drpartmwt

of Biochemistry,

WITH SYNTHESIS,

AND

24.5

~ Printed in The Netherlands

IN

ATHEROMATOUS

DEGRADATION

AND SULPHATION

OF

P. A. KURUP

UtCversity

of’ Kerala,

Tvivandrurn

695001 (India)

(Received August 27th, 1974) (Accepted November 5th, 1974)

SUMMARY

Some important enzymes concerned with the biosynthesis of the precursors of glycosaminoglycans (gg), degradation of gg and biological sulphation have been studied

in rats fed an atherogenic

diet. L-Glutamine-D-fructose-6-phosphate

transferase and glucosamine-6-phosphate-N-acetylase biosynthesis of hexosamine precursors of gg atherogenic diet. UDPG pyrophosphorylase, glucuronic

acid-5’-epimerase,

precursors

of gg, also decreased

which are concerned

amino-

- 2 enzymes concerned with the decreased in the liver in rats fed the UDPG dehydrogenase and U DPG with the biosynthesis

of the uranic

in the liver in the diet-fed rats. The activities

of some

of the enzymes concerned with degradation of gg - hyaluronidase, /+glucuronidase, /+hexosaminidase, cathepsin and aryl sulphatase - increased both in the liver and aorta. The hepatic concentration of PAPS significantly decreased in the diet-fed rats. The sulphate-activating system, which includes ATP sulphurylase, APS kinase and sulphotransferase, also decreased. Thus the overall picture is one of decreased synthesis of gg and their increased

Key words:

Aryl sulphatase

degradation

- Cathepsin

in the atheromatous

~ Cholesterol

Glucosamine-6-phosphate-N-acetylase D-Fructose6phosphate fl- Hexosaminidase

lipids - Sulphate-activating UDPG dehydrogenase

~ Chondroitin

- P-Glucuronidase

aminotransferase

~ Hyaluronic

rats.

sulphates

- L-Glutamine-m

- Heparan sulphate - Heparin --

acid - Hvaluronidase

system - Sulphotransferase

- UDPG S-epimerase

- PAPS - Phospho- Triglycerides

- UDPG pyrophosphorylase

--

S.

246

T. VIJAYAKUMAR,

P. A. KURUP

INTRODUCTION

No detailed

investigation

seems to have been carried

out so far on the metabol-

ism of glycosaminoglycans (gg) in atherosclerosis. The only data at present available relate to the conflicting reports on the concentration of gg fractions in the atherosclerotic

aortic tissue’-ll.

enzymes

concerned

include

increase

There are also some reports

with the degradation in the

activity

on the activity

of some of the

of gg in the aorta in atherosclerosis.

of ,!I-glucuronidaseta-19,

fl-N-acetyl

These

glucosamini-

and decrease in the activity of aryl dasel?*t6, cathepsins20Jl and hyaluronidase14Ja sulphatase on a wet-tissue basis, but no change on a tissue-N basisas. Regarding the enzymes concerned with the synthesis of precursors of gg, L-glutamine-D-fructose-6phosphate aminotransferase has been reported to show no significant change in atherosclerotic

aortic

tissuea”,

while UDPG

pyrophosphorylase

has been found

increase with age and atherosclerosisa5. Apart from this, no information the other enzymes concerned with the metabolism of gg or of sulphate atherosclerosis.

In view ofthis,

detailed investigations

were undertaken

is available metabolism

to on in

on the enzymes

of gg metabolism. These include degrading enzymes (/?-glucuronidase, B-hexosaminidase, cathepsin, aryl sulphatase and hyaluronidase); biosyntheticenzymes(L-glutamineD-fructose-6-phosphate aminotransferase, UDPG pyrophosphorylase, UDPG dehydrogenase, glucosamine-6-phosphate-N-acetylase and UDP glucuronic acid-5’epimerase); the concentration of 3’-phosphoadenosine 5’-phosphosulphate (PAPS, the biological sulphating agent); the sulphate-activating system (which includes ATP sulphurylase and APS kinase) and sulphotransferase. Since feeding a high-fat cholesterol-sodium cholate diet to rats has been found to result only in mild lipid infiltration in the aorta, the work has also been carried out in rats fed the high-fat cholesterolsodium cholate diet along with an antithyroid substance like potassium perchlorate. More severe lipid infiltration has been found to develop in the aorta in the latter casetr. MATERIALS

AND METHODS

Young male albino

rats (average

weight 80 g) were divided

into 3 groups

of I5

rats each. They were fed as follows: Group I. Normal laboratory diet (Hind Lever rat feed) supplied by Hindustan Lever Limited, which contains 24% protein and 4”/ ether-extractable material. The ingredients include cereals, oil-cakes, bran and milk solids. Group II. High-fat cholesterol diet: 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 (as above) + 25 mg potassium perchlorate/ 100 g body weight/day. The rats were maintained on their respective diets for 6 months, at the end of which they were killed. The serum, liver and aorta were collected for various estimations.

GLYCOSAMINOGLYCANS

IN ATHEROMA

Total cholesterol, phospholipids Extraction

247

and triglycerides

of the serum, liver and aorta

was carried out at 60°C with ethanol (1 : 1, v/v) as described previously2’j. -ether (3:1), followed by cloroform-methanol Total cholesterol was estimated by the method of Carr-Drektera7, phospholipids by the method of Zilversmit-Davis28 and triglycerides by the method of Van Handel and Zilversmi Estimation

of the tissues for lipid estimation

t2s modified

by use of a florisil column

to remove phospholipids.

of gg of the liver and aorta

The dry, defatted tissue obtained digestion according to the procedure

after lipid extraction was subjected to papain of Laurent at 65570°C in 0.1 M phosphate

buffer (pH 6.5) containing 0.005 M EDTA and 0.005 M cysteine for 48 hr3(‘. After centrifugation. the digest was passed through a column of cellulose” (10 cm x I cm), previously fractions,

washed in I “/, cetyl pyridinium chloride (CPC) solution. The different gg I -hyaluronic acid (HA), 2-heparan sulphate (HS), 3-chondroitin sulphate

A (Ch S-A), 4-chondroitin

sulphate C (Ch S-C), 5-chondroitin sulphate B (Ch S-B) and 6-heparin (H) were eluted according to the procedure of Svejcar and Robertson”‘. Complete resolution of the gg fractions is not achieved by any of the methods available at present: in this method, some degree of overlapping occurred between fractions 2 and 3,3 and 4 and also 4 and 5. In a preliminary analysis of these fractions, using the and chondrosulphatase, it has enzymic method of Murata et a1.32 with chondroitinase been shown that fraction 2 contains mostly HS, contaminated by small quantities of Ch S-A: fraction 3 has Ch S-A as the major fraction with traces of Ch S-C, while fraction 4 contains Ch S-C with small quantities of Ch S-B. HA and H were found to be mostly uncontaminated. Since this complete analysis is too time-consuming for routine analysis of a large number of samples, the procedure has been restricted to the cellulose

chromatography

of the CPC complexes.

The designation

of the fractions

as

HS, Ch S-A, Ch S-C and Ch S-B only means that these are the major gg in the respective fractions. The identity of the major component in each fraction was confirmed by gg fractions were comparison with standard gg preparations* j’. The individual quantitated by the estimation of uranic acid using the modified carbazole reaction of Bitter and Muir33. Estimation

of enzyme activities

The validity and reproducibility of the enzyme assays have been established previously3”,3j. L-Glutamine-D-fructose-6-phosphate aminotransferase activity of the liver and aorta was estimated according to the procedure of Pogell and Gryder”“. UDPG dehydrogenase activity was determined according to the procedure of Strominger rt a1.37 and UDPG pyrophosphorylase activity according to the procedure of Villar Palasi and Larnera*. UDP glucuronic acid 5’-epimerase activity was determined

* Microcrystalline chromatography grade, E. Merck, ** All obtained from Sigma Chemicals. U.S.A.

Germany.

S. T. VIJAYAKUMAR,

248 according

to the procedure

N-acetylase 40. For the estimation dase, cathepsin

of Davidson3g,

of I-glucuronidase,

and aryl sulphatase,

P. A. KURUP

as was that of glucosamine-6-phosphate /I-N-acetyl

hexosaminidase,

the tissue (liver and aorta)

0 “C with 0.1 A4 acetate buffer (pH 5.0), and the supernatant

hyaluroni-

was homogenized

P-Glucuronidase and /I-N-acetyl hexosaminidase activity were estimated according the procedure described by Kawai and Anno41, using p-nitrophenyl-/3-D-glucuronide and

p-nitrophenyl-/3-N-acetyl

glucosaminide

respectively

at

used as the enzyme source.

as the

substrates.

to Aryl

sulphatase activity was assayed according to the procedure described by Roy4”, using 4-nitro-catechol sulphate as the substrate. Hyaluronidase activity was assayed as described by Kawai and Anno41 using hyaluronic acid (Na salt) as the substrate and estimating the N-acetyl hexosamines liberated by the method of Reissig et ~1.~~. Cathepsin activity was determined 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”‘. Sulphate metabolism 3’-Phosphoadenosine-5’-phosphosulphate was estimated according to the procedure of Jansen and Van Kempen45. The validity and reproducibility of the method were established by using standard solutions of PAPS in the range 0.5-3 ,uM (final concentration). A 20 ‘A (wt/vol) homogenate was prepared under ice-cold conditions in isotonic KC1 solution, and centrifuged at 0°C at 2000 x g for 10 min. The supernatant was kept in a boiling water bath for 60 set to inactivate enzymes. The PAPS present in the supernatsnt (A) was estimated by using the sulphotransferase system (B) from normal rat liver (supernatant from 20% homogenate in isotonic KC1 solution) and methyl umbelliferone. The reaction system containing 1.5 ml of heat-treated supernatant (A) + 1 ml of Tris-HCI

buffer (pH 7.4, 0.4 M) containing

0.5 mM EDTA,

1.5 mM methyl umbelli-

ferone + 0.2 mA4 KHzP04 (pH 7.4) + 0.5 ml sulphotransferase system (B) was incubated at 37°C for 15 min. The reaction was terminated by immersion in a boiling water bath for 60 sec. (The necessary control for the PAPS present in (B) was carried out by replacing (A) with same volume of buffer in the reaction system. An aliquot of the supernatant after centrifugation was passed through Dowex 50 (H+ form) and the methyl umbelliferone sulphate Kempen and Jansen46.

eluted, hydrolysed

and estimated

as described

by Van

Sulphate-activating system in the liver The supernatant from the 20% homogenate (A) (without heat inactivation) was used as the source of the sulphate-activating system and PAPS was generated by incubating it with ATP and inorganic sulphate. The PAPS formed was estimated as before. The reaction system containing 1.5 ml of (A) + 4 ml of the Tris-HCI buffer (0.4 M, pH 7.4), containing 2.2 mM of MgCl2, 50 mM KzS04 and 4.4 mM ATP, was incubated at 37°C for 1 hr. The reaction was arrested by immersion in a boiling water

IA

LIPID

AORTIC

LIVER

0.56

248.6 :

2.32

216.8 1 I .86

61.5

II

III

Group

Group

B P O.l-0.2. In all other

I

Group

Group

0.206

0.18

:m 0.0017

0.0016

cases P ‘-. 0.01.

340 I3.20

292 mt 2.60 1~4.80”

52.18

2012 ~_ 16.40

1598 112.8

-1 4.10

8.6

1026 -’

21.68 = 2.4 42.62

il~lg!Ioo g wet tissue _ S.E.M.)

img/g tissue protein t S.E.M.)

(r?lg/loo g wet tissue = S.E.M.)

168 :t 1.71

Phospholipid.7

Total cholesterol

from

0.072 -:: 0.0005

given are the average

198.7 ~_ I .80

172.6 _ I .61

98.5 _ 0.72

trigl~eride ~nlrllol/I00 HII = S.E.M.)

Groups

= 16.8

10.5

5.10

132 11.28

302 7 2.60

246 2. 2.20

34.6

25.6

4169 _ 40.21

3642 :

2768 :

-~ 0.13

z 0.06

Group I;

52.17

0.05 8.67 = 0.70

35.43 i 0.31 0.35

3.32 zz 0.034 5.54 f

24.08 1 0.202

-______ iumol/g tissw protein i S.E.M.)

with

16.108 z 0.14

12.93

5.65

ililmol~Ioo g WC1 tissue z S.E.M.)

Triglyceride

II. In all cases

triglyceride in1~??01/100 g wet tissue i S.E.M.)

with Group

II and Ill have been compared

img:g tissue protein J S. E. M.)

12 rats.

1920

1196

520.6 z

totul cholesterol phospholipid;, ltug/IOO g rrvt tissue 1 S.E.M.)

111 has been compared

phospholipicl;, total chokstcrol lrngj100 ml -~ S.E.M.)

1, and Group

Liw

with Group

.%tW~l

II and III have been compared

The aortas from 2 rats have been pooled and the values Group III has also been compared with Group II.

IS

TABLE

LEVELS

cholesterol

Group III High-fat

- potassium perchlorate

cholesterol

High-fat

Group II

Normal

Group I

AND

from 6 rats. Groups

OF THE SERUM

Average of the values P i 0.01.

LEVELS

TABLE

LIPID

LEVELS

IN THE

AORTA

AND

LIVER

1538 + 14.89

1496 1 14.56h

Group 11 424 i 4.35

Group 111 473 * 4.82

il P 0.1-0.2. b P 0.05-0.1. C P 0.1-0.2.

1620 1 16.10

354 & 3.24

392 z 3.46

420 + 5.8 402 + 3.6” 380 * 3.4

298 & 3.10

1380 = 12.60 1352 241 + 13.10~~ i 2.20

410 13.8

360 zk 3.2

1460 i 12.8

148 = 1.32

119 ~ 1.02

59 I 0.61

HA (/tg wonic

HA Ch S-C Ch S-B HS Ch S-A Ipg urorlic acid/g dry defatted tissue = S.E.M.)

H

Liver

Aorta

382 C 3.04

Group 1

Group

81 = 0.79

85 = 0.75

68 = 0.60

112 T 1.01 141 zz 1.24 133 z 1.28

43 * 0.36 92 I 0.81 103 z 0.99

126 7 0.96

92.8 + 0.81

57 _+ 0.4 1

Ch S-C Ch S-A Ch S-B HS acid,‘g dry defa!ted tisstre + S.E.M.)

52.3 * 0.51

95.5 -c 0.96

t 0.68

77

H

The aorta from 2 rats was pooled and the results given are the average from 12 rats. The liver values are the average from 6 rats. Groups I1 and III have been compared with Group I, and Group III has been compared with Group 11.

GLYCOSAMINOGLYCAN

TABLE 2

0

e

GLYCOSAMINOGLYCANS

1N ATHEROMA

251

bath for 60 sec. The necessary enzyme blank was provided by using heat-inactivated (A). The following were added in each case to I .5 ml of the supernatant: 0.5 ml EDTA (250 mM, pH 7.4) in Tris-buffer 0.5 ml of methyl umbelliferone transferase

and 0.5 ml KHaP04 (final concentration

system (B). The system was incubated

(final concentration 0.2 mM) and 1.5 mM) and 0.5 ml of sulpho-

for 30 min at 37°C and the reaction

was arrested by heating in a water bath for 60 sec. A blank was also carried out using heat-inactivated B. The methyl umbelliferone sulphate formed was estimated as before. Phenol sulphotransferase

actiCt!x

PAPS was generated

as above

by using a normal

rat liver homogenate

(B) in

place of (A) in the first step of the sulphate-activating system. In the second step, the supernatant from the experimental rat liver homogenate (A) was used as the source of sulphotransferase and the methyl umbelliferone sulphate formed was estimated. Statistical

analysis

The data given in the tables are the average of 6 experiments cal significance

was calculated

using Student’s

+ S.E.M.

Statisti-

t-test4T.

RESULTS

Histological examination of the aorta revealed a more severe form of lipid infiltration in the animals of the perchlorate group than that seen in the cholesterol-cholate Lipid

group,

as reported

in the case of the previous

levels of the serum, lirer

series”.

and aorta

The results are given in Table 1. The total cholesterol, phospholipid and triglyceride were significantly higher in the serum, liver and aorta in the animals of the perchlorate

group

when

compared

with the corresponding

cholesterol group (except in the case of total cholesterol tein basis). Glycosaminoglycan

lerels

qf the

values

in the high-fat

of the aorta on a tissue-pro-

aorta and liver

The results are given in Table 2. As has previously been reported”, the sulphated gg decreased, while HA increased in the aorta in the animals of Groups II and 111 when compared with Group I (except in the case of H in the animals of Group II). Comparison of Group III with Group II showed significantly higher values for HA and lower values for the sulphated gg except HS and Ch S-C. However, the gg fractions in the liver of Groups II and III were increased when compared with Group I. Comparison of Group III with Group II showed significantly higher values of HA. Ch S-A and Ch S-B, but lower values of HS, Ch S-C and H. Enzymes in\,oh>ed in the synthesis of the precursors

The levels of r_-glutamine-o-fructose-6-phosphate

of gg

aminotransferase

(E C2.6.1.16)

OF ENZYMES

CONCERNED

WITH

THE SYNTHESIS

OF PRECURSORS

OF gg

76.3 = 1.2 (15.26 j, 0.24)

71.05 i 1.42% (14.21 x 0.28)a

Group II

Group 111

a P 0.01-0.05. b P 0.1-0.2. In all other cases P < 0.01.

83.84 * 0.88 (16.76 i 0.18)

27.82 = 0.56 ( 5.54 e 0.11)

43.1 & 0.65 ( 8.62 i 0.13)

56.28 & 0.62 (11.25 * 0.12)

581 + 8.62” (116.2 * 1.72)”

602 = 9.03 (120.4 I 1.81)

649 = 6.8 (129.8 1 1.36)

53 (10.6

= 0.82 = 0.16)

70.05 _c 0.90 (14.01 i 0.18)

86.0 z 0.88 (17.2 = 0.18)

(units/g protein = S.E.M.)

lunits/g protein & S.E.M.)

protein

liver I mg glucosamine 4 S.E.M.)

aorta formedjhrjg

Hepatic UDGP pyrophosphorylase

Hepatic UDPG dehydrogenase

L-Glutamine-D-fructose-&phosphate aminotransferase activity

Group I

Group

98.95 * 1.98” ( 19.97 + 0.39)h

103.6 I_t 1.56) (20.72 = 0.31)

118.3 E 1.2 (23.66 = 0.24)

(mg uranic acid formedjhrjg protein i S.E.M.)

Hepatic UDP glucuronic acid-Sepimerase

83.4 + 1.67 (16.68 = 0.33)

110.94 & 1.67 (22.12 * 0.33)

122.3 * 1.25 (24.46 i 0.25)

(mg N-acetyl glucosamine formed/hrjg protein + S.E.M.)

Hepatic glucosamine-6-phosphate N-acetylase

The values given in brackets are those on tissue weight basis (per g wet tissue). The values given are the average from 6 rats. Groups II and 111 have been compared with Group I and Group III has been compared with Group II.

LEVELS

TABLE 3

OF gg-DEGRADING

4

EhZYMES

II

III

Group

Group

_

56.91 i 0.62 (1 1.38 = 60.4 0.12) ! 0.74 (12.08 -- 0.15) 98.7 I 1.20 (19.74 z 0.24)

43.05 = 0.44 ( 8.61 71.80.09) -A:0.85 (14.36 - 0.1) -82.6 = 1.01 (16.52 = 0.20)

0.01.

liver

._

aorta

~___

irng N-acetyl hexosamine liheratedjhrlg protein S.E.M.)

Hyaluvorzidase

In all cases P i

I

Group

Group _

91.2 _ 1.14 (19.44 z 0.23)

0.20)

19.2 10.84 (15.84 - 0.17) -88.29 -~ I .02 (17.25

66.85 :m0.68 (13.37 i 0.13) 80.9 IO.94 (16.18 + 0.19) 105 1.52 (20.5 _- 0.30)

liver

aorta

l:-Glucurotlirlrrse ~~~ irng p-nitrophenol .fornted/hr/g tissue protein L S.E.M.)

)

1 3.01 (52.28 0.60)

i 2.65 (45.4 261.4 - 0.53)

194.3 = 2.01 (38.86 2270.40)

h- 3.81 (67.8 z 0.76)

I+ 3.61 (61 .Ol 3390.72)

_

II and

242.8 : 2.84 (48.56 305 L 0.57)

I52 z 1.68 (30.4 0.34) 217 = 2.6 (43.4 IK 0.52) 311 : 3.82 (62.2 0.76)

79.32 z 0.83 (15.84 0.17) ‘-87.51 ‘- 1.31 (17.50 I 0.26) 119.6 z 1.34 (23.8 2 0.2)

94.8 z 0.98 (19.12 : 0.19) 103.4 = 1.55 (20.72 IO.31 148.7 11.82 (29.76 .m0.36)

107 z 1.09 (21.4 z 0.21) 122 ~’ 1.48 (24.4 mu0.29) 169 z 2.24 (33.8 II 0.45)

live,

live,

uorta-

i mg p-nitrocatechol liberated, hr,g protein -* S.E.M.) ~ ~~~~ liver aorta

Groups

ittig tyrosine liherated;‘hr,‘g protein - S.E.M.)

6 rats.

Aryl sulphatase

from

Cathepsins

given are the average

il-Hesosa,llini~la.re -~ ln~g p-nitrophenol formed/hr/g tissue protein 7 S. E.M.)

The values given in brackets are those expressed on a tissue weight basis (per g wet tissue). The values III have been compared with Group 1, and Group III has been compared with Group 11.

LEVELS

TABLE

S. T. VIJAYAKUMAR,

254 UDPG

dehydrogenase

(UDP

glucose-NAD

phosphate-N-acetyl

transferase,

the rats fed atherogenic As compared aminotransferase

EC 1.I. I .22), UDPG

oxidoreductase,

pyrophosphorylase (UTP-glucose-l-phosphate glucosamine-6-phosphate-N-acetylase (Acetyl

P. A. KURUP

uridyl transferase, EC 2.7.7.9) COA-2-amino-2-deoxy-D-glucose-6-

EC 2.3. I .4) and UDP glucuronic

acid 5’-epimerase

in

diet are given in Table 3.

to Group

I, the activity

was significant

decreased

of L-glutamine-D-fructose-6-phosphate in the liver and aorta

in the animals

of

Groups 11 and III, both on tissue-protein and tissue weight basis. Group III compared to Group II showed significantly lower values on both tissue protein and tissue weight basis. Similar decreases in UDPG pyrophosphorylase, UDPG dehydrogenase, UDP glucuronic acid-5’-epimerase and glucosamine-6-phosphate-N-acetylase were also observed in the liver on tissue-protein or tissue weight basis in the animals of Groups II and III as compared

to Group

I. Group

cantly lower values of aminotransferase, 6-phosphate-N-acetylase

III compared

UDPG

on both tissue-protein

Enzymes concerned with the degradation

to Group

pyrophosphorylase

II showed

signifi-

and glucosamine

-

and tissue weight basis.

of gg

The levels of hyaluronidase (EC 3.2.1.35), fl-glucuronidase (EC 3.2.1.31) {jhexosaminidase (EC 3.2. I .30), cathepsin (EC 3.4.4.23) and aryl sulphatase (EC 3.1.6. I ) are given in Table 4. When compared with Group I, all the degrading enzymes showed significantly increased activity in both the liver and aorta in the animals of Groups II and III, both on tissue protein and tissue weight basis. Group significantly higher values in all cases.

III compared

to Group

II showed

TABLE 5 LEVELS

OF HEPATIC

PAPS,

SULPHATE

ACTlVATlNG

SYSTEM

AND

PHENOL

SULPHOTRANSFERASE

ACTIVITY

The values given in brackets are those on tissue weight basis (per g wet tissue). Average of the values from 6 rats. Groups II and III have been compared with group I, and Group 111has been compared with Group II. Gr0llp

PAPS

(mg methyl

Z&hate system umbrllif~~rone wlphate

Sulphotransjtirase

activating

formed/hr/g

protein

activity

+ S. E.M.) _

Group I

157.28 1; 1.61 (3 I .45 * 0.32)

22.9 + 0.23 (4.58 + 0.05)

31.5 Ito. (6.31 t 0.06)

Group II

141.3 & 1.51 (28.26 i 0.30)

17.84 i 0. I9 (3.57 i 0.04)

28.02 + 0.30 (5.61 i 0.06)

Group 111

120.6 Itr 1.24 (24.12 i 0.25)

II.98 i 0.16 (2.39 i 0.03)

20.9 i 0.24 (4.18 i 0.05)

In all cases P < 0.01.

GLYCOSAMINOGLYCANS

255

IN ATHEROMA

bchondroltln Cho”drolt,“-4-s”lphate

Fig.

1. Metabolic

pathway

4 for

---I

4

PAPS

Cho”dTOlt,“-6-S”lphale

0’

biosynthesis

of glycosaminoglycans.

Sulpliatt~ nwtaholisnl

The levels of PAPS,

the sulphate-activating

system

ferase activity of the liver are given in Table 5. When compared with Group I, PAPS decreased

and phenol

sulphotrans-

in the liver in the animals

of

Groups II and 111 both on tissue-protein and tissue weight basis. The sulphate-activating system, which includes ATP sulphurylase (EC 2.7.7.3) and APS kinase (EC 2.7.1.25) decreased considerably. showed a similar decrease. Group

The phenol sulphotransferase (EC 2.8.2. I ) also III in comparison with Group II showed significant-

ly lower values in all cases. DlSCUSSIOh

The general

route for the synthesis

of gg from glucose is reasonably

well under-

stood (Fig. 1). Some of the important enzymes in this biosynthetic pathway decreased in the liver of rats fed the atherogenic diet. t_-Glutamine-o-fructose-6-phosphate aminotransferase is a key enzyme since it provides glucosamine-6-phosphate, the precursor of the hexosamine moiety in the gg. The enzyme decreased significantly both in the liver and aorta; this is in contrast to a previous report, stating that the aminotransferase activity of atherosclerotic aortic tissue is not significantly different to normal tissue”J. Glucosamine-6-phosphate-N-acetylase also decreased in the liver. The decrease in these 2 enzymes would result in decreased availability of the hexosamine precursors-UDP glucosamine, UDP N-acetyl glucosamine and UDPG Nacetyl galactosamine for gg synthesis. Likewise, 3 important enzymes in the biosynthetic pathway of the uranic acid precursor for gg synthesis also decreased. UDPG pyrophosphorylase forms UDPG from glucose-l-phosphate and UTP. UDPG dehydrogenase provides UDP-glucuronic acid from UDPG, while UDP glucuronic acid S’epimerase converts UDP glucuronic acid to UDP iduronic acid. The decreased activity of these 3 enzymes would result in decreased availability of uranic acid precursors-

S. T.

256 UDP glucuronic

acid and UDP iduronic

acid-required

VIJAYAKUMAR,

P. A. KURUP

for gg synthesis.

Thus syn-

thesis of gg would be expected to decrease in the animals fed the atherogenic diet. In this connection, UDPG pyrophosphorylase has been previously reported to increase in the aorta in atherosclerosisz5.

This enzyme was not studied

review, but it was found to decrease in the liver. All the enzymes studied that degrade gg increased and aorta. These included

hyaluronidase,

P-glucuronidase,

in aorta in the present

in activity

in both the liver

/$hexosaminidase,

cathep-

sin and aryl sulphatase. The increased activity of these enzymes ~ in agreement with previous reports - can result in increased degradation of gg. Aryl sulphatase has been previously

reported

to show no significant

change in the aorta,

but the present

results

indicate that this enzyme activity was considerably increased in the rats fed the atherogenie diet. Little attention has so far been paid to sulphation in atherosclerosis, except for the incorporation of “304 into gg fractions. Sulphation was significantly decreased in the liver in the diet-fed rats. PAPS, the biological sulphate-donor, was considerably decreased. ATP sulphurylase first forms APS from ATP and sulphate; APS is then phosphorylated

to PAPS by ATP in the presence of APS kinase. The sulphate-activat-

ing system studied activity

here includes

of the sulphate-activating

both ATP sulphurylase

and APS kinase.

system can result in decreased

Decreased

PAPS formation.

Sulphotransferase activity also decreased in the diet-fed rats. The magnitude of the changes was generally much greater in the perchlorate group than in the simple highfat-high-cholesterol group. Thus the overall picture is one of decreased synthesis and increased degradation of gg in the animals on the atherogenic diet. This may explain the decreased amounts of sulphated gg in the aorta observed in both atheromatous groups. The increase in HA in the aorta is difficult to explain as is also the increase of gg in the liver-even though the biosynthetic enzymes decreased and the degrading enzymes increased. Nevertheless, the concentration aorta, is very slight.

of gg in the liver, when compared

with that in the

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(;LYCOSAMINOC;LYCANS

IN ATHEROMA

251

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