Atherosclerosis,
22 (1975) 447-461
447
0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
ASCORBIC ACID AND GLYCOSAMINOGLYCAN BOLISM IN GUINEA PIGS FED NORMAL AND
BALA NAMBISAN
AND
Department of Biochemistry,
AND LIPID ATHEROGENIC
METADIETS
P. A. KURUP University of Kerala, Trivandrum 695001 (India)
(Received January 2nd, 1975) (Revised received January 29th, 1975) (Accepted February
17th, 1975)
SUMMARY
The effect of low and high doses of ascorbic acid on glycosaminoglycan lipid metabolism was studied in guinea pigs fed both normal and atherogenic
and diets.
The high dose of ascorbic acid (25 mg/lOO g body weight/day) decreased the cholesterol level in the liver and aorta but not in the serum in animals fed the normal diet in comparison with those fed the low dose of ascorbic acid (0.1 mg/lOO g body weight/ day). In animals fed the atherogenic diet, cholesterol decreased in the serum and liver, but not in the aorta. Serum triglycerides were not affected by the dose of ascorbic acid in the group on the normal diet, but in the animals receiving the atherogenic diet, the high dose of ascorbic acid caused serum triglycerides to decrease when compared with the low dose. Hepatic and aortic triglycerides decreased in groups on normal and atherogenic diets receiving the high dose of ascorbic acid. Lipoprotein lipase activity was not affected in the aorta by the dose of ascorbic acid either in the normal or atherogenic diet group. It was increased in the liver and heart in both the groups receiving
the low dose of ascorbic
acid but decreased
in the high dose group.
The concentration of all the glycosaminoglycans significantly increased in the aorta of animals on normal diet receiving the high dose of ascorbic acid when compared with the low dose group. In the group on the atherogenic diet, hyaluronic acid was not affected, but all the sulphated glycosaminoglycans increased in the animals receiving the high dose when compared with those receiving the low dose. In the liver all the sulphated glycosaminoglycans increased while hyaluronic acid decreased in both the normal and atherogenic diet groups receiving the high rather than the low dose of ascorbic acid. L-Glutamine :D-fructose-6-phosphate aminotransferase and UDPG dehydrogenase, two key enzymes in the biosynthesis of precursors of glycosaminoglycans, were studied in relation to the dose of ascorbic acid. Hepatic aminotransferase activity was higher both in the normal and atherogenic diet groups when receiving the high rather than the low dose of ascorbic acid. UDPG dehydrogenase
448
B. NAMBISAN,
was not affected by the dose of ascorbic
acid. The activities
P. A. KURUP
of the degrading
enzymes
- hyaluronidase, P-glucuronidase, ,%hexosaminidase and aryl sulphatase - significantly increased both in the normal and atherogenic diet groups when receiving the low rather than the high dose of ascorbic acid. The concentration of PAPS, sulphate activity and sulphotransferase and atherogenic diet groups receiving
activity were all increased in both the normal the high dose of ascorbic acid.
Key words : Aminotransferasc -Aorta - Aryl sulphatase - Ascorbic acid - Cholesterol - Chondroitin sulphates - /I-Glucuronidase - Heparan sulphate - /3-Hexosaminidase - Hyaluronic acid - Hyaluronidase - Liver - PAPS - Phospholipid - Sulphate activating system - Sulphotransferase - Triglyceride UDPG dehydrogenase
INTRODUCTION
There are several reports that ascorbic acid has a cholesterol lowering effect in atherosclerotic animals and man 1- is. There are also a few contradictory reports that it has no such effectll-14. Ginter and Babalai5 found that a high dose of ascorbic acid significantly decreased cholesterol accumulation in the liver of male guinea pigs fed an atherogenic diet, but had no significant effect on serum cholesterol and atheromatous changes in coronary arteries. But Bala Nambisan and Kurupls reported a decrease in the cholesterol level of the serum, liver and aorta in weanling rats fed a normal diet and receiving a high dose of ascorbic acid. Glycosaminoglycans (gg) metabolism has been reported by us to be deranged in atherosclerosisi7.
The effect of ascorbic
acid on the metabolism
of gg does not seem
to have been studied in any detail, although there are some reports on the effect of ascorbic acid deficiency on the total gg in some tissues in guinea pigs. An increase in total gg has been reported by Burlina et al. 18 in the liver of guinea pigs maintained on an ascorbic acid deficient diet, while Gore et al.19 reported a similar increase in the aorta. On the other hand a decrease in the concentration of gg, particularly chondroitin sulphate, was observed by Schultz-Haudt and SigurdzO in the skin of ascorbic acid deficient guinea pigs. Bala Nambisan and Kurupi6 reported an increase in all the gg fractions of the aorta - hyaluronic acid (HA), heparan sulphate (HS), chondroitin sulphate A (Ch S-A), chondroitin sulphate C (Ch S-C), dermatan sulphate (Ch S-B) and heparin (H) - in weanling rats fed a high dose of ascorbic acid. Apart from these observations, no detailed study has been carried out on the role of ascorbic acid in the metabolism of gg. In view of this, the effect of a low and a high dose of ascorbic acid on the metabolism of gg has been studied in guinea pigs. The concentration of the different gg fractions of the aorta and liver, some of the enzymes concerned with the biosynthesis of precursors of gg and degradation of gg as well as biological sulpha-
ASCORBIC
ACID AND ATHEROGENIC
tion have been studied
in guinea
levels and concentration MATERIALS
449
DIET
pigs fed normal
of lipoprotein
and atherogenic
diets. The lipid
lipase have also been investigated.
AND METHODS
Male guinea of 12 animals
pigs (average
initial
weight
-
300 g) were divided
into 6 groups
each and fed as follows:
Normal diet groups 1. Adequate body weight/day.
dose of ascorbic
acid -
basal diet + 1 mg of ascorbic
2. Low (deficient) dose of ascorbic acid 100 g body weight/day. 3. High dose of ascorbic acid - basal body weight/day.
Atherogenic diet groups 4. Adequate dose of ascorbic acid 100 g body weight/day. 5. Low (deficient) dose of ascorbic corbic acid/100 g body weight day. 6. High dose of ascorbic 100 g body weight/day.
acid -
acid/100 g
basal diet + 0.1 mg of ascorbic diet + 25 mg of ascorbic
atherogenic acid -
atherogenic
acid/100 g
diet + 1 mg of ascorbic
atherogenic
acid/
acid/
diet + 0.1 mg of as-
diet + 25 mg of ascorbic
acid/
Ascorbic acid was administered orally to the animals by tube every day. The basal diet contained (g/l00 g diet): crushed wheat - 63, casein - 13, crushed bengal gram - 12, groundnut oil - 4, sodium chloride - 1, calcium carbonate - 3, yeast - 2, and shark liver oil - 2. The atherogenic diet contained (g/l00 g diet): crushed wheat - 51.7, casein - 13, crushed bengal gram - 12, sodium chloride - 1, calcium carbonate - 3, yeast - 2, shark liver oil - 2, cholesterol - 0.3, coconut oil - 15. The animals were fed the respective diets for 4 months and killed at the end of this period.
The tissues were removed
to ice-cold containers
for various
estimations.
1. Estimation of lipids Total cholesterol,
phospholipids
and triglycerides
were estimated
in the serum,
liver and aorta. The tissues were extracted successively at 60” with ethanol:ether (3 :1 v/v, 2 h) followed by chloroform:methanol (1:l v/v, 2 h) as described beforezl. Total cholesterol was estimated by the method of Carr-Drekterz2, phospholipids by the method of Zilversmit and Davis23 and triglycerides by the method of Van Handel and ZilversmiP with the modification that a florisil column was used to remove phospholipids. 2. Estimation of gg in the tissues The tissue (wet weight 500 mg approximately
in the case of liver and 50 mg
450
B. NAMBISAN, P. A. KURUP
in the case of aorta) was defatted with ethanol:ether (3 :I) followed by chloroform: methanol (1 :l v/v) as described before 21. The dry, defatted tissue was subjected to digestion with papain (l/3-1/4 the dry weight of the tissue) for 48 hr at 65-70” in 0.1 M phosphate buffer pH 6.5 containing 0.005 M EDTA and 0.005 M cysteine according to the procedure of Laurent 25. Fresh papain was added at the end of every 16 h. The digest after centrifugation was passed through (10 cm x 1 cm) previously washed with 1% cetylpyridinium
a column chloride
of cellulose* solution, The
different gg fractions 1 = hyaluronic acid (HA), 2 = heparan sulphate (HS), 3 = chondroitin sulphate A (Ch S-A), 4 = chondroitin sulphate C (Ch S-C), 5 = dermatan sulphate (Ch S-B) and 6 = heparin (H) - were eluted according to the procedure of Svejcar and Robertson 26. The individual gg fractions were quantitated by the estimation of uranic acid according to the modified carbazole reaction of Bitter and Muir27. 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 -1 and 5. Further analysis of the fractions by the enzymic method of Murata and Oshimaas, using chondroitinase and chondrosulphatase, has shown in a previous experiment that fraction 2 contains mostly HS, contaminated by small amounts of Ch S-A; fraction 3 has Ch S-A as the major component with traces of Ch S-C; fraction 4 contains Ch S-C with small amounts of Ch S-B. HA and H were found to be mostly uncontaminated in cellulose acetate** electrophoresis. Since this is too tedious for routine analysis of a large number of samples, the analysis was restricted to the chromatography of the CPC-complex over cellulose. The designation of the fractions as HS, Ch S-A and Ch S-C means that these are the major gg in the respective fractions. The identity of the major component in each fraction was confirmed by comparison with standard gg preparations**. 3. Estimation of enzyme activities Lipoprotein lipase activity was estimated in the aorta, liver and heart. Chilled tissue was homogenised with 3 volumes of cold Sorenson’s phosphate buffer (pH 7.38, was centrifuged at 0 “C at 1500 x g for 10 min 6.6 x 1O-2 M). The homogenate and the supernatant was used as the enzyme. Enzyme assay was carried out according to the procedure of Zemplenyi and Grafnetteras. L-Glutamine :D-fructose-6-phosphate aminotransferase (EC 2.6.1.16) activity of the liver and aorta was estimated according to the procedure of Pogell and Gryder30. The tissue immediately after removal from the animal was homogenised under icecold conditions in a solution containing KC1 (0.154 M), EDTA (0.001 M) and glucose-6-phosphate (0.012 M) pH 7.2 (2 ml/g in the case of liver and 2 ml/50 mg in the case of aorta). The homogenate was centrifuged at 1500 x g for 10 min at 0°C and the supernatant was used as the enzyme source. The reaction system containing 0.1 ml of fructose-6-phosphate (Na salt 0.1 M, pH 7.5), 0.15 ml of L-glutamine (0.1
* Microcrystalline, chromatography grade, E. Merck, Germany. ** All obtained from Sigma Chemicals, U.S.A.
ASCORBIC
ACID AND ATHEROGENIC
M), 0.1 ml of reduced
glutathione
7.5) and 0.3 ml of enzyme
451
DIET
(0.1 M), 0.45 ml of 1 M sodium
was incubated
for 1 h at 38°C. The reaction
by the addition of 1 ml of 0.4 N trichloroacetic tent of 1 ml of the supernatant was determined In the controls, the glutamine UDPG
the complete
reaction
was added immediately dehydrogenase (UDP
phosphate
(pH
was stopped
acid (TCA) and the hexosamine conby the method of Pogell and GI yderse.
system minus
glutamine
was incubated
before the addition of TCA. glucose :NAD oxidoreductase,
EC
and
1.1.1.22)
activity was determined according to the procedure of Strominger et ~1.31.Acetonedry powdered liver was extracted at 0°C in 0.1 M glycine buffer pH 8.7 (8 ml per 400 mg of the powder), centrifuged at 1500 x g at 0°C for 10 min and the supernatant was assayed for enzyme activity. The reaction system contained, in addition to the enzyme (0.5 ml), 0.1 pmole UDPG and 0.1 pmole NAD+ in glycine buffer in a total volume of 5 ml. For the estimation
of /I-glucuronidase
(EC 3.2.1.31)
B-N-acetylhexosaminidase
(EC 3.2.1.30), hyaluronidase (EC 3.2.1.35) and aryl sulphatase (EC 3.1.6.1), the tissue (liver and aorta) was homogenised at 0” with 0.1 M acetate buffer, pH 5.0 (4 ml buffer for 1 g of liver and 2 ml for 50 mg of aorta), centrifuged at 1500 x g for 10 min at 0°C and the supernatant was assayed for enzyme activity. B-Glucuronidase and j3-N-acetylhexosaminidase activity were estimated according to the procedure described by Kawai and Annosa using p-nitrophenyl /3-D-glucuronide and p-nitrophenyl #I-IV-acetylglucosaminide respectively as the substrates. 0.1 ml of enzyme was incubated with 0.5 ml of acetate buffer, containing 100 ,ug of p-nitrophenyl p-D-glucuronide in the case of i3-glucuronidase and 100 ,ug of p-nitrophenyl fi-N-acetylhexosaminide activity was assayed according catechol
sulphate
as substrate
in the case of fi-hexosaminidase. Aryl sulphatase to the procedure described by Roy33 using p-nitroand estimating
the nitrocatechol
liberated.
0.1 ml of
the enzyme was incubated with 0.5 ml acetate buffer containing 0.006 M p-nitrocatechol sulphate. Hyaluronidase was assayed as described by Kawai and Annoss, using hyaluronic acid (sodium salt) as the substrate and estimating the reducing sugar liberated by the method of Park and Johnson 34. 0.2 ml of the enzyme was incubated with 0.5 ml acetate buffer containing 150-200 pg HA (sodium salt). 4. Sulphate metabolism The concentration of PAPS (3’-phosphoadenosine-5’-phosphosulphate), sulphate activating activities (ATP sulphurylase, EC 2.7.7.3, and APS kinase, EC 2.7.1.25) and hepatic phenol sulphotransferase (EC 2.8.2.1) activity were estimated by the method of Jansen and Van Kempen3s,ss using methyl umbelliferone. Details of the procedure have been described beforesT. 5. Estimation Protein
of protein was estimated
after TCA precipitation
6. Ascorbic acid content of the tissues This was estimated by the method
by the method
of Roe and Kuetherss.
of Lowry et al.38.
B. NAMBISAN,
452 TABLE ASCORBIC
P. A. KURUP
1 ACID
Group
1 2 3 4 5 6
CONTENT
OF THE TISSUES
Ascorbic acid (,ug/g wet tissue A SEM) adrenals
spleen
liver
430 f 8.6 64.11 & 1.52 1171 * 22.5
127.8 f 2.6 20.5 & 0.42 209 It 4.10
53.6 22.6 177.8
112.6 & 3.2 8.9 5 0.21 156.1 * 3.5
37.85 & 0.61 14.4 f 0.29 104.9 + 2.5
182.4 12.6 716.5
i 3.80 + 0.31 + 20.1
& 1.12 i 0.5 Z!I3.55
Average of the values from the individual tissues of 6 rats in each case rt SEM.
7. Statistical analysis The data in the tables are the average of 6 experiments significance was calculated using Student’s t-test40.
& SEM.
Statistical
RESULTS
1. Ascorbic acid content of the tissues The ascorbic acid content of the liver, spleen and adrenals in the animals of the different groups is given in Table 1. The decrease in the concentration of ascorbic acid in the tissue in the animals fed the atherogenic diet is in agreement with previous reports41. 2. Lipid levels qf the serum, liver and aorta The total cholesterol, phospholipids and triglycerides of the serum, liver and aorta of the animals of the different groups are given in Table 2. In the group on normal diet, serum cholesterol was not significantly affected in the animals fed a low or high dose of ascorbic acid as compared to those fed an adequate dose. The liver cholesterol was significantly higher in the animals receiving the low dose when compared to the adequate- or high dose-group. Aortic cholesterol was significantly lower in the animals fed the high dose of ascorbic acid, as compared to those fed the adequate or low dose. Serum phospholipids were significantly lower in the animals fed the high dose of ascorbic acid, as compared to those fed the low dose. Liver and aortic phospholipids were lower in the low dose than in the adequate- or high-dose groups, while aortic phospholipids were significantly higher in the animals fed the high dose. Serum triglycerides were not significantly altered in the low- or high-dose group when compared with animals fed the adequate dose, while the liver and aortic triglycerides were decreased in the high dose group and increased in the low dose group in comparison with those fed the adequate dose.
190 * 3.9 233.3 h 4.8.’ 167.7 + 3 2%”
serun, (rng/lOO ml 31 SEM)
4
893.5 + 18.5 1400 f 31.1A 631.6 + 13.4n.a
240.8 It 7.3 298.9 -‘- 8.9” 238.6 & 7.2a
liver (mg/IOOg
128.9 j, 2.54 129.3 I 2.62 126.5 i 2.31
110.2 !z 3.3 109 * 3.2 86.8 + 2.6”,”
aorta wet weight & SEM)
AND AORTA
SEM)
333.3 k 6.9 342.1 i 7.2 240.6 A 4.8.Q
140.1 C 2.8 145.9 & 2.8 132.5 +c 2.6”
i
Serum (mg/lOO ml
Phospholipids
3980 i 80.2 3689 i 74.3” 2661 + 54.1A.a
1756 * 34.5 1223 i 24.4” 1842 & 36.ga
liver (mg/lOOg
2268 i 27.2 2250 C 26.1 2386 + 28.6=-
1628 + 33.4 1312 + 28.F 1984 * 39.8-ksa
aorta wet tissue & SEM)
6.31 ??0.12 9.76 _c 0.21” 5.40 & 0.10.‘9”
6.20 i 0.314 6.51 + 0.15 6.05 & 0.13
Serum (mg/lOO ml 41 SEM)
Triglyceride
374 * 7.4 484 * 9.6A 373 5 7.42”
210.7 -c 4.8 243.2 C 5.2” 180.78 & 4 0a.a
liver (mg/lOOg * SEM)
glycerol
1148 & 23.9 1420 h 28.4” 987 i 19.6A+
826.4 + 17.5 962 & 20.1.’ 767.6 _C 15.gBza
aorta wet tissue
Average of the values from individual tissues of 6 animals in each case * SEM. Group 2 and 3 have been compared with group I, and group 5 and 6 with group 4. * P less than 0.01. BP between 0.01 and 0.05. No symbol: greater than 0.05. Group 3 and 6 have also been compared with group 2 and 5 respectively. a P less than 0.01. b P between 0.01 and 0.05.
6
5
3
2
LIVER
Total cholesterol
OF THE SERUM,
79.9 * 2.2 85.6 + 2.5 80.01 i 2.4
LEVELS
2
1
Group
LIPID
TABLE
P
454
B. NAMBISAN, P. A. KURUP
TABLE 3 LIPOPROTEIN
Group
LIPASE
ACTIVITY
Lipoprotein
OF THE TISSUES
lipase activity
(pmole
glycerol/hr/lOO
mg protein
aorta
liver
heart
2 3
72.43 + 1.51 71.51 & 1.5 75.62 zk 1.55
26.39 & 0.59 28.52 i 0.61B 23.63 % 0.52”
20.59 * 0.50 30.91 i 0.71-4 20.78 * 0.51%
4 5 6
55.2 54.6 57.9
15.50 * 0.31 17.82 + 0.36” 13.91 i_ 0.28”+
14.01 & 0.31 21.18 i 0.80” 14.84 * 0.35”
1
.+ 1.2 i 1.15 h 1.28
+ SEM)
Average of the values from individual tissues of 6 animals in each case & SEM. Group 2 and 3 have been compared with group 1, and group 5 and 6 with group 4. A P less than 0.01. * P between 0.01 and 0.05. No symbol: P greater than 0.05. Group 3 and 6 have also been compared with group 2 and 5 respectively. a P less than 0.01. b P between 0.01 and 0.05.
In the atherogenic diet group, serum and liver cholesterol were increased in the low dose group and decreased in the high dose group when compared with those fed the adequate dose, while aortic cholesterol was not significantly affected in both groups. Serum and liver phospholipids were decreased significantly in the animals fed the high dose of ascorbic
acid when compared
with those fed the adequate
or low
dose, while the liver phospholipids were also lower in the low dose group than in the adequate dose group. Aortic phospholipids were increased in the animals of the high dose group. Triglycerides were significantly higher in the serum, liver and aorta of the low dose group, in comparison with the adequate dose group, while they were lower in the serum and aorta in the high dose group. 3. Lipoprotein lipase activity of the aorta, liver and heart The results are given in Table 3. In the normal diet group the aortic enzyme concentration was not affected in the animals fed the low or high dose as compared with those of the adequate group. It was significantly increased in the liver and heart in the low dose group and decreased in the liver in the high dose group as compared with the adequate dose group. In the atherogenic diet group, enzyme activity was not affected in the aorta in the animals fed the low or high doses, was increased in the liver and heart in the low dose group and was decreased in the liver in the high dose group when compared with the adequate dose group. 4. Glycosaminoglycans of the aorta and liver The results are given in Table 4. In the groups on normal diet, HA was significantly decreased in the aorta in the low dose group when compared with the ade-
OF THE
TlSSUES
965 i 21.3 1001 i 25.1 989 * 22.2
1100 5 22.1 990 i 19.8” 1124 + 22.4a
880 + 17.6 695 * 15.3* 956 zt 20.4*+
1180 It 30.5 1036 * 26.2* 1260 i 31.4”+
i_ 25.7 * 21.2 + 29.On 1476 1388 1610 i 29.0”*& + 26.8”*& h 32.1”
1562 i 30.4 1459
i_ 25X* 1491 i 29.1”
1110 + 22.0 1050
1321 i 26.5 1280
1450 + 28.2 1287
Ch S-C Ch S-B
H
926 * 19.3 788
600 & 13.8 520 & 12.4* 675 & 14.3”~~
730 * 15.0 610 * 13.1* 824 & 16.6*.”
+ 16.8” * 15.4“ 956 986 * 19.1*-a * 20.2-43
930 + 18.6 833
272 i 5.4 262 f 5.2 224 k 6.4*la
f 8.2A 298 f 6.8&
305 f 7.5 367
HA
Ch S-A
HA
HS
Liver
Aorta
165 i 4.2 129 + 3.5‘4 167 + 4.4&
& 9.8 412 I_t lO.P+
369 i 9.6 378
HS
112 ??3.3 99.8 & 2.gB 141 & 4.2”~”
zk 5.4 295 * 5.W”
226 * 5.5 221
Ch S-A
126.5 * 3.6 110.8 & 3.1” 139.9 & 4.1-i+
A 7.2 406 + 8.2”+
341 & 7.25 340
Ch S-C
142 * 2.9 112 * 2.4’ 145 I_t 3.0a
IL-6.4 361 & 8.4.‘~a
300 + 1.2 292
Ch S-B
100 + 2.1 98.3 -f- 1.92 110 + 2.3B*”
& 4.4” 310 & 6.3A+
218 4 5.4 224
H
Average of the values from individual tissues of 6 animals in each case & SEM. Group 2 and 3 have been compared with group 1, and group 5 and 6 with group 4. * P less than 0.01. n P between 0.01 and 0.05. No symbol: P greater than 0.05. Group 3 and group 6 have also been compared with group 2 and 5 respectively. &P less than 0.01. b P between 0.01 and 0.05.
6
5
4
3
2
1
LEVELS
uranic acid/g dry defatted tissue + SEM)
Group
(pg
GLYCOSAMINOGLYCAN
TABLE 4
3
5 u
8 2
z %
5
456
9. NAMBISAN, P. A. KURUP
TABLE 5 AMINOTRANSFERASE
AND
UDPG
DEHYDROGENASE
ACTIVITY
OF THE LIVER
Group
Aminotransferase (pmole hexosamine~hrl g protein * SEM)
UDPG dehydrogenase (units/g ofprotein i SEM)
1
24.50 & 0.64 22.63 5 0.62 25.29 f 0.70b
956 f 19.5 960 h 20.1 960 f 20.0
18 * 0.53 13.05 f_ 0.36” 20.15 f 0.61”
820 f 23.4 816 & 23.8 824 & 23.2
2 3 4
5 6
Average of the values from the individual tissues of 6 animals in each case & SEM. Group 2 and 3 have been compared with group 1, and group 5 and 6 with group 4. A P less than 0.01. No symbol: P greater than 0.05. Group 3 and group 6 have also been compared with group 2 and 5 respectively. a P less than 0.01. b P between 0.01 and 0.05.
quate- or high-dose groups. HS, Ch S-A, Ch S-B and H were significantly increased in the high dose group, in comparison with the adequate dose group, while Ch S-C, Ch S-B and H were significantly decreased in the low dose group. All the gg fractions decreased in the low dose group when compared with the high dose group. In the liver, HA increased and H decreased in the low dose group when compared with the adequate dose group. All the sulphated gg increased in the high dose group when compared with the adequate dose group. Compared to the high dose group, all the sulphated
gg fractions
were significantly
reduced in the low dose group.
In the atherogenic diet group, all the sulphated gg in the aorta were significantly lower in the low dose group than in the adequate dose group, while Ch S-A, Ch S-B, Ch S-C and H were higher in the high dose group. All the sulphated gg were higher in the high dose group than in the low dose group. In the liver, HS, Ch S-A, Ch S-C and Ch S-B were significantly lower in the low dose group than in those fed the adequate dose, while Ch S-A, Ch S-C and H were higher in the high dose group. All the sulphated gg fractions were again higher and HA lower in the high dose than in the low dose group. 5. L-Glutamine: D-fructose-6-phosphate aminotransferase ‘and UDPG dehydrogenase activity of the liver The results are given in Table 5. In the group on normal diet increased aminotransferase activity was observed in the animals receiving the high dose of ascorbic acid rather than the low dose. However, enzyme activity was not significantly different when the low or high dose group was compared with the group fed the adequate dose. There was no significant alteration in the UDPG dehydrogenase activity in the low or high dose group when compared with the adequate dose group.
7.54 i 0.18 6.93 i 0.168 5.05 i 0.12.\,a
4 5 6
48.38 & 1.42 51.14 * 1.51 43.82 !C 1.30”
86.0 i 2.51 91.53 i 2.72 81.92 -+ 2.42b
AND LIVER
9.53 i 0.27 11.21 & 0.30” 8.55 & 0.25=”
65.8 + 1.32 74.2 j, 1.51* 66.2 IL 1.35”
77.1 & 1.58 51.48 & 1.10 78.7 * 1.6 53.83 h 1.3 70.79 j, 1.42B,b 46.6 + 0.95BJ
aorta liver (mg nitrocatechol/hr/g protein & SEM)
Aryl sulphatase
22.82 i 0.54 82.16 =I 1.64 31.4 i 0.75* 86.35 & 1.72 21.01 & 0.50nja 80.9 = 1.6”
8.16 i 0.24 9 16 & 0.27B 8.25 + 0.25”
aorta liver (mgp-nitrophenol/hr/g protein & SEM)
16.15 * 0.40 64.33 + 1.92 96.1 + 1.80 17.56 + 0.48 15.85 & 0.41 75.36 C 2.20” 95.8 & 1.75 19.65 & 0.51” 14.4 & 0.38”~~ 54.8 5: 1.62=sa 88.6 & 2.24”~” 16.81 5 0.41%
12.43 h 0.36 13.91 * 0.398 11.90 j, 0.33&
OF THE AORTA
&Hexosaminidase
ACTIVITY
Average of the values from the individual tissues of 6 animals in each case + SEM. Group 2 and 3 have been compared with group 1, and group 5 and 6 with group 4. A P less than 0.01. B P between 0.01 and 0.05. No symbol: P greater than 0.05. Group 3 and group 6 have also been compared with group 2 and 5 respectively. &P less than 0.01. b P between 0.01 and 0.05.
4.93 k 0.14 5.12 * 0.15 4.20 + 0.13*-”
aorta liver (mgp-nitrophenol/hr/g protein 3~ SEM)
SULPHATASE
aorta liver (mg reducing sagar/l6 hr/g protein * SEM)
ARYL
/I-Glucuronidase
/%HEXOSAMINIDASE,
Hyaluronidase
/%GLUClJRONIDASE,
1 2 3
Group
HYALURONIDASE,
TABLE 6
3
a
A
458
B. NAMBISAN, P. A. KURUP
TABLE I LEVEL
OF
PAPS,
SULPHATE
ACTIVATING
SYSTEM
AND PHENOL
SULPHOTRANSFERASE
Group
PAPS levels (pnde
1 2 3
170.6 zk 3.64 166.9 + 3.51 180.27 t_ 3.85nsb
54.2 & 1.10 52.71 + 1.15 62.78 & 1.32”~~
4 5 6
141 138 162
25.32 i 0.52 21.5 + 0.45* 28.55 k 0.57*,”
& 2.8 k 2.6 :k 3.2”~&
ACTIVITY
IN THE LIVER
Sulphotransferase Sulphate activating system methyl umbelliferone sulphatelhrig protein i SEM) 14.11 i 9.28 12.5 i 0.25* 16.11 + 0.32”~” 11.1 & 0.27 10.8 rt 0.26 14.0 * 0.40”*&
Average of the values from the individual tissues of 6 animals in each case i SEM. Group 2 and 3 have been compared with group 1, and group 5 and 6 with group 4. * P less than 0.01. D P between 0.01 and 0.05. No symbol: P greater than 0.05. Group 3 and group 6 have also been compared with group 2 and 5 respectively. a P less than 0.01 b P between 0.01 and 0.05.
In the atherogenic diet group, aminotransferase activity significantly decreased in the low dose group, in comparison with the adequate- or high-dose group. UDPG dehydrogenase
activity
again was not affected in the low or high dose groups.
6. Hyaluronidase, fl-glucuronidase, f+hexosaminidase and aryl sulphatase activity The enzyme activities of the aorta and liver are given in Table 6. In the normal diet group, hyaluronidase, j%glucuronidase, /I-hexosaminidase and aryl sulphatase activity of the liver and aorta were significantly higher in the animals fed the low dose than in those fed the high dose of ascorbic acid. Similar increased values were also observed in the animals on the atherogenic the low dose of ascorbic acid rather than the high dose.
diet when fed
7. Sulphate metabolism The concentration of PAPS, sulphate activating system and phenol sulphotransferase activity of the liver are given in Table 7. In the normal diet group, the concentration of PAPS was significantly higher in the animals fed the high dose of ascorbic acid than in those fed the low dose. Sulphate activating system, which includes ATP sulphurylase and APS kinase, was also considerably higher in the high than in the low dose group. Similar results were also observed in the case of sulphotransferase activity. In animals fed the atherogenic diet, similar increases were seen in the concentration of PAPS, sulphate activating system and sulphotransferase in the animals fed the high dose of ascorbic acid in comparison with those fed the low dose. DISCUSSION
The effect of a high dose of ascorbic
acid on the concentration
of lipids in the
ASCORBIC
ACID
AND ATHEROGENIC
459
DIET
tissues varied in guinea pigs fed the normal emit effect was seen only in the animals
and atherogenic
diets. A hypocholesterol-
fed the atherogenic
diet, while lowering
of
the aortic cholesterol was seen only in animals fed the normal diet and not in the group fed the atherogenic diet. These results on aortic cholesterol in animals fed the atherogenic esterolemic presumably in addition
diet agree with those reported by Ginter and Babalar5, but no hypocholeffect as in our experiments was observed by these workers. This may be due to the difference in the atherogenic diets used, 15 % coconut oil to 0.3 % cholesterol being used in our experiments. Hypotriglyceridemia
again was observed only in the group fed the atherogenic diet, while decrease in aortic and hepatic triglycerides was observed in both groups. However the pattern of change now observed in the concentration of lipoprotein lipase showed no correlation with lipid levels, although previous reports have associated decreased enzyme activity with increased lipid accumulation42 and vice-versa. Administration of a high dose of ascorbic acid to guinea pigs fed normal as atherogenic
diets has been found
to result in an increased
concentration
as well of aortic
and hepatic glycosaminoglycans as compared to those fed a low dose of ascorbic acid. Hyaluronic acid concentration showed a different trend, decreasing in the liver in both the normal and atherogenic diet groups, and increasing only in the aorta in the normal diet group. One possible reason for the decreased concentration of glycosaminoglycans in the animals receiving the low dose of ascorbic acid may be the decreased activity of L-glutamine: u-fructose-6-phosphate aminotransferase, but UDPG dehydrogenase was not appreciably affected. The former is a key enzyme which makes available glucosamine-6-phosphate, the precursor of the hexosamine moiety for synthesis of glycosaminoglycans. Another reason for the low concentration of gg in the low dose group may be increased
degradation
of gg, as is evident
from the
increased activities of hyaluronidase, ,%glucuronidase, /Lhexosaminidase and aryl sulphatase. The decreased sulphate metabolism in the animals receiving the low dose of ascorbic acid may also be another factor. The decrease in the concentration of gg and the activity of biosynthetic enzymes and biological sulphation and increased activity of enzymes concerned with the degradation of gg in the animals fed the atherogenie diet agree with results reported previously43. Ascorbic acid has been reported to activate a number of enzymes, the action being mainly attributed to the protection of -SH groups. It is possible that the decreased activity of L-glutamine:D-fructose-6-phosphate aminotransferase and the enzymes concerned with biological sulphation in the animals of the low ascorbic acid group may be due to this factor. But the increased activity of enzymes concerned with the degradation of gg, cannot be explained on this basis. The report of Mumma and Verlangierr ‘44 that ascorbic acid as ascorbic acid-Z sulphate may play a role in biological sulphation may be pertinent in explaining the increased sulphation observed in the animals on the high dose of ascorbic acid. The results now obtained may indicate a direct involvement of ascorbic acid in the biosynthesis of gg, probably at the level of sulphation and also in the degradation of the gg.
460
B. NAMBISAN, P. A. KURUP
It is tempting
to speculate
mvolved in the development those reported by a number increased
cholesterol
that ascorbic
of atherosclerosis. of others, indicate
in the liver and increased
acid deficiency
in man may be a factor
The results that ascorbic
now obtained as well as acid deficiency results in
triglycerides
in the liver and aorta in
animals fed a normal diet, while an increase in cholesterol and triglycerides is observed in the liver and aorta in the animals fed the atherogenic diet. Ascorbic acid deficiency
has now been shown
with both normal
and atherogenic
to result in a decreased
concentration
diets, and this result is of interest
of aortic
gg
when the reported
decrease in the concentration of sulphated gg17 in atherosclerotic aorta is considered. Thus, ascorbic acid deficiency may contribute to the development of atherosclerosis both by the increased lipid levels and the decreased sulphated gg levels it produces.
REFERENCES 1 KASATKINA, L. V., LOBOVA, N. M. AND SUKASOVA, M. I., In: A. L. MYASNIKOV, Atherosclerosis Moscow, 1967, p. 39. and Thrombosis, Mir Publishers, 2 ZUBOVA, R. F., Effect of vitamin C and B6 level in blood of atherosclerotic patients, Serd-Sosud Sb., (1964) 165. Patol. (Kiev: Zdorovc) 3 INAGAKI, C. AND OIZUMI, K., Effect of ascorbic acid on cholesterol metabolism, Vitamin, 32 (1965) 339. 4 VOLKOVA, K. G., Effect of ascorbic acid on total lipids in the aortic wall in experimental cholesterol atherosclerosis, Ateroskleroz. Sb., (1961) 187. 5 CATELLI, P., BIGNAMI, M., BONATI, Cl. AND GOFRINI, M., Changes in serum cholesterol and lipoproteins in subjects treated with vitamin C, Pathologica, 53 (1961) 267. 6 SMOLENSKII, V. S., EROFEEVA, N. N., PANKRATOVA, N. F., AND ZAPROMETOV, M. N., Influence of vitamin C and vitamin P administration on the development of atherosclerosis, Vir. Resury i ikh Ispoiz Akad. Nauk SSR, Inst. Biokhim Sb., 4 (1959) 158. 7 CORTINOVIS, R., SILENZI, C. AND FARRONI, L., Changes induced by L-ascorbic acid in the blood cholesterol in normal and atherosclerotic subjects, Giorn. Gerontol., 8 (1960) 28. 8 SILENZI, C., CORTINOVIS, A. AND FARRONI, L., Acute changes in lipoproteinograms by administration of ascorbic acid, Giorn. Gerontol., 8 (1960) 32. 9 LEON DE SOLDATI, BALASSANIAN, S. AND STRIZTLER, G., Blood lipid levels in patients with atherosclerosis under treatment with ascorbic acid, Prensa Med. Arg., 47 (1960) 636. 10 GORBACHEV, V. V., Effect of some vitamins on lipid metabolism of atherosclerotic patients, Zdravookhranenic Belorussii, 12 (1959) 22, 11 CHANG, S. C., Effect of mechanical trauma, cortisone and vitamin C upon atherosclerosis in cholesterol-fed rats, New Med. J., 8 (8) (1965) 79. 12 KOSKO, N. K. AND LITVISHKOVA, L. A., Effect of vitamin P and C on the cholesterol level in patients with atherosclerosis, Gipertonich. Bolezni, Atheroscleroz. Koronarn, Nedostatochnost. Sb., (1963) 305. 13 SAMUEL, P. AND SHALCHI, 0. B., Effect of vitamin C on serum cholesterol in patients with hypercholesterolaemia and arteriosclerosis, Circulation, 29 (1964) 24. 14 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. Res., 8 (1968) 891. 15 GINTER, E. AND BABALA, J., The effect of high intraperitoneal doses of vitamin C in guinea pigs fed atherogenic diet, Biologiu, 26 (1971) 449. 16 BALA NAMBISAN AND KURUP, P. A., Effect of massive doses of ascorbic acid and methionine on the levels of glycosaminoglycans in the aorta of weanling rats, Atherosclerosis, 19 (1974) 191. 17 SEETHANATHAN, P. AND KURUP, P. A., Changes in tissue glycosaminoglycans in rats fed a hypercholesterolaemic diet, Atherosclerosis, 14 (1971) 65.
ASCORBIC ACID AND ATHEROGENIC DIET
461
18 BURLINA,A. AND LIPPI, V., Pathologic histology of the liver in relation to ascorbic acid deficiency, Riv. Anat. Patol. Oncol., 16 (1959) XXIV. 19 GORE, l., TANAKA, Y., FUJINAMI,T. AND GOODMAN, M. L., Aortic acid mucopolysaccharides and collagen in scorbutic guinea pigs, 1. Nutr., 87 (3) (1965) 311. 20 SCHULTZ-HAUDT, S. D. AND SIGURD, D., Comparative histochemistry of non-injured skin connective tissue of normal and ascorbic acid deficient guinea pigs, Acta Pathol. Microbial. Stand., 62 (4) (1964) 465. P. AND KURUP, P. A., Changes in tissue glycosaminoglycans in rats fed a hyper21 SEETHANATHAN, 14 (1971) 65. cholesterolaemic diet, Atherosclerosis, 22 CARR, J. J. AND DREKTER, I. J., Estimation of total cholesterol in the serum, C/in. Chem., 2 (1956) 353. of phospholipids, J. Lab. Clin. Med., 23 ZILVERSMIT,D. B. AND DAVIS, A. K., Microdetermination 35 (1950) 155. 24 VAN HANDEL, E. AND ZILVERSMIT,D. B., Micromethod for direct determination of serum triglycerides, J. Lab. Clin. Med., 50 (1957) 152. 25 SCOTT, J. E., Aliphatic ammonium salts in assay of acid polysaccharides from tissues. In: D. GLICK (Ed.), MethodsofBiochemical Analysis, Vol. 8, Interscience, New York, N.Y., 1960, p. 145. 26 SVEJCAR, J. AND ROBERTSON,W. V. B., Micro-separation and determination of mammalian acidic glycosaminoglycans, Anal. Biochem., 18 (1967) 333. 27 BITTER,T. AND MUIR, H. M., A modified uranic acid carbazole reaction, Anal. Biochem., 4 (1962)
330. 28 MURATA, K. AND OSHIMA,Y., Chondroitin sulphate in atherogenic human aorta, Atherosclerosis, 14 (1971) 121. 29 ZEMPLENYI,T. AND GRAFNETTER,D., Species and sex differences in fatty acid release from tissues incubated with lipaemic serum, Brit. J. Exp. Pathol., 39 (1958) 99. in rat liver, 30 POGELL,B. M. AND GRYDER,R. M., Enzymatic synthesis of glucosamine-6-phosphate J. Biol. Chem., 228 (1957) 701. 31 STROMINGER,J. L., MAXWELL, E. S., AXELROD, J. AND KALEKAR, H. M., Enzymatic formation of uridine diphosphoglucuronic acid, J. Biof. Chem., 224 (1957) 79. 32 KAWA~, Y. AND ANNO, K., Mucopolysaccharides degrading enzymes from the liver of the squid, Ommastrephes sloanipacificus, hyaluronidase, Biochim. Biophys. Acta, 242 (1971) 428. 33 ROY, A. B., The sulphatase of ox liver, Biochem. J., 53 (1953) 12. 34 PARK, J. T. AND JOHNSON,M. J., A sub-microdetermination of glucose, J. Biol. Chem., 181 (1949)
149. 35 VAN KEMPEN,G. M. J. AND JANSEN,G. S. I. M., Quantitative assay of 3’-phosphoadenosine5’-phosphosulphate, “active sulphate”, Anal. Biochem, 51 (1973) 324. 36 VAN KEMPEN, G. M. J. AND JANSEN,G. S. 1. M., Quantitative determination of phenolsulphotransferase using 4-methyl umbelliferone, Anal. Biochem., 46 (1972) 438. 37 SUDHAKARAN,P. R. AND KURUP, P. A., Vitamin A and glycosaminoglycans metabolism, J. Nutr., 104 (1974) 871.
38 LOWRY, 0. H., ROSEBROUGH,N. J., FARR, A. L. AND RANDALL, R. J., Protein measurement with the Folin-phenol reagent, J. Biol. Chem., 193 (1952) 265. 39 HAWK, P. B., OSER, B. L. AND SUMMERSON,W. H., In: Practical Physiological Chemistry, 14th edition, McGraw Hill, New York, N.Y., 1965, p. 703. 40 BENNETT,C. A. AND FRANKLIN,N. L., Statistical Analysis in Chemistry and the Chemical Industry, Wiley, New York, N.Y., 1967. 41 RZAKULIEVA,D. M. AND GASANOV, A. S., Variation of ascorbic acid content in the organs and tissues in experimental atherosclerosis, SSR, 22 (3) (1966) 90. 42 SEETHANATHAN, P., VIJAYAGOPAL,P., AUGUSTI, K. T. AND KURUP, P. A., Myocardial lipoprotein lipase in rats fed a hypercholesterolaemic diet, Atherosclerosis, 11 (1970) 333. 43 VIJSYAKUMAR,S. T. AND KURUP, P. A., Metabolism of glycosaminoglycans in atheromatous rats, Atherosclerosis, 21 (2) (1975) 245. 44 MUMMA, R. 0. AND VERLANGIERI,A. J., In vivo sulphation of cholesterol by ascorbic acid-3sulphate as possible explanation for the hypocholesterolaemic effect of ascorbic acid, Fed. Proc., 30 (2) (1971) 370 Abstr.
22 (1975) 447-461
447
0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
ASCORBIC ACID AND GLYCOSAMINOGLYCAN BOLISM IN GUINEA PIGS FED NORMAL AND
BALA NAMBISAN
AND
Department of Biochemistry,
AND LIPID ATHEROGENIC
METADIETS
P. A. KURUP University of Kerala, Trivandrum 695001 (India)
(Received January 2nd, 1975) (Revised received January 29th, 1975) (Accepted February
17th, 1975)
SUMMARY
The effect of low and high doses of ascorbic acid on glycosaminoglycan lipid metabolism was studied in guinea pigs fed both normal and atherogenic
and diets.
The high dose of ascorbic acid (25 mg/lOO g body weight/day) decreased the cholesterol level in the liver and aorta but not in the serum in animals fed the normal diet in comparison with those fed the low dose of ascorbic acid (0.1 mg/lOO g body weight/ day). In animals fed the atherogenic diet, cholesterol decreased in the serum and liver, but not in the aorta. Serum triglycerides were not affected by the dose of ascorbic acid in the group on the normal diet, but in the animals receiving the atherogenic diet, the high dose of ascorbic acid caused serum triglycerides to decrease when compared with the low dose. Hepatic and aortic triglycerides decreased in groups on normal and atherogenic diets receiving the high dose of ascorbic acid. Lipoprotein lipase activity was not affected in the aorta by the dose of ascorbic acid either in the normal or atherogenic diet group. It was increased in the liver and heart in both the groups receiving
the low dose of ascorbic
acid but decreased
in the high dose group.
The concentration of all the glycosaminoglycans significantly increased in the aorta of animals on normal diet receiving the high dose of ascorbic acid when compared with the low dose group. In the group on the atherogenic diet, hyaluronic acid was not affected, but all the sulphated glycosaminoglycans increased in the animals receiving the high dose when compared with those receiving the low dose. In the liver all the sulphated glycosaminoglycans increased while hyaluronic acid decreased in both the normal and atherogenic diet groups receiving the high rather than the low dose of ascorbic acid. L-Glutamine :D-fructose-6-phosphate aminotransferase and UDPG dehydrogenase, two key enzymes in the biosynthesis of precursors of glycosaminoglycans, were studied in relation to the dose of ascorbic acid. Hepatic aminotransferase activity was higher both in the normal and atherogenic diet groups when receiving the high rather than the low dose of ascorbic acid. UDPG dehydrogenase
448
B. NAMBISAN,
was not affected by the dose of ascorbic
acid. The activities
P. A. KURUP
of the degrading
enzymes
- hyaluronidase, P-glucuronidase, ,%hexosaminidase and aryl sulphatase - significantly increased both in the normal and atherogenic diet groups when receiving the low rather than the high dose of ascorbic acid. The concentration of PAPS, sulphate activity and sulphotransferase and atherogenic diet groups receiving
activity were all increased in both the normal the high dose of ascorbic acid.
Key words : Aminotransferasc -Aorta - Aryl sulphatase - Ascorbic acid - Cholesterol - Chondroitin sulphates - /I-Glucuronidase - Heparan sulphate - /3-Hexosaminidase - Hyaluronic acid - Hyaluronidase - Liver - PAPS - Phospholipid - Sulphate activating system - Sulphotransferase - Triglyceride UDPG dehydrogenase
INTRODUCTION
There are several reports that ascorbic acid has a cholesterol lowering effect in atherosclerotic animals and man 1- is. There are also a few contradictory reports that it has no such effectll-14. Ginter and Babalai5 found that a high dose of ascorbic acid significantly decreased cholesterol accumulation in the liver of male guinea pigs fed an atherogenic diet, but had no significant effect on serum cholesterol and atheromatous changes in coronary arteries. But Bala Nambisan and Kurupls reported a decrease in the cholesterol level of the serum, liver and aorta in weanling rats fed a normal diet and receiving a high dose of ascorbic acid. Glycosaminoglycans (gg) metabolism has been reported by us to be deranged in atherosclerosisi7.
The effect of ascorbic
acid on the metabolism
of gg does not seem
to have been studied in any detail, although there are some reports on the effect of ascorbic acid deficiency on the total gg in some tissues in guinea pigs. An increase in total gg has been reported by Burlina et al. 18 in the liver of guinea pigs maintained on an ascorbic acid deficient diet, while Gore et al.19 reported a similar increase in the aorta. On the other hand a decrease in the concentration of gg, particularly chondroitin sulphate, was observed by Schultz-Haudt and SigurdzO in the skin of ascorbic acid deficient guinea pigs. Bala Nambisan and Kurupi6 reported an increase in all the gg fractions of the aorta - hyaluronic acid (HA), heparan sulphate (HS), chondroitin sulphate A (Ch S-A), chondroitin sulphate C (Ch S-C), dermatan sulphate (Ch S-B) and heparin (H) - in weanling rats fed a high dose of ascorbic acid. Apart from these observations, no detailed study has been carried out on the role of ascorbic acid in the metabolism of gg. In view of this, the effect of a low and a high dose of ascorbic acid on the metabolism of gg has been studied in guinea pigs. The concentration of the different gg fractions of the aorta and liver, some of the enzymes concerned with the biosynthesis of precursors of gg and degradation of gg as well as biological sulpha-
ASCORBIC
ACID AND ATHEROGENIC
tion have been studied
in guinea
levels and concentration MATERIALS
449
DIET
pigs fed normal
of lipoprotein
and atherogenic
diets. The lipid
lipase have also been investigated.
AND METHODS
Male guinea of 12 animals
pigs (average
initial
weight
-
300 g) were divided
into 6 groups
each and fed as follows:
Normal diet groups 1. Adequate body weight/day.
dose of ascorbic
acid -
basal diet + 1 mg of ascorbic
2. Low (deficient) dose of ascorbic acid 100 g body weight/day. 3. High dose of ascorbic acid - basal body weight/day.
Atherogenic diet groups 4. Adequate dose of ascorbic acid 100 g body weight/day. 5. Low (deficient) dose of ascorbic corbic acid/100 g body weight day. 6. High dose of ascorbic 100 g body weight/day.
acid -
acid/100 g
basal diet + 0.1 mg of ascorbic diet + 25 mg of ascorbic
atherogenic acid -
atherogenic
acid/100 g
diet + 1 mg of ascorbic
atherogenic
acid/
acid/
diet + 0.1 mg of as-
diet + 25 mg of ascorbic
acid/
Ascorbic acid was administered orally to the animals by tube every day. The basal diet contained (g/l00 g diet): crushed wheat - 63, casein - 13, crushed bengal gram - 12, groundnut oil - 4, sodium chloride - 1, calcium carbonate - 3, yeast - 2, and shark liver oil - 2. The atherogenic diet contained (g/l00 g diet): crushed wheat - 51.7, casein - 13, crushed bengal gram - 12, sodium chloride - 1, calcium carbonate - 3, yeast - 2, shark liver oil - 2, cholesterol - 0.3, coconut oil - 15. The animals were fed the respective diets for 4 months and killed at the end of this period.
The tissues were removed
to ice-cold containers
for various
estimations.
1. Estimation of lipids Total cholesterol,
phospholipids
and triglycerides
were estimated
in the serum,
liver and aorta. The tissues were extracted successively at 60” with ethanol:ether (3 :1 v/v, 2 h) followed by chloroform:methanol (1:l v/v, 2 h) as described beforezl. Total cholesterol was estimated by the method of Carr-Drekterz2, phospholipids by the method of Zilversmit and Davis23 and triglycerides by the method of Van Handel and ZilversmiP with the modification that a florisil column was used to remove phospholipids. 2. Estimation of gg in the tissues The tissue (wet weight 500 mg approximately
in the case of liver and 50 mg
450
B. NAMBISAN, P. A. KURUP
in the case of aorta) was defatted with ethanol:ether (3 :I) followed by chloroform: methanol (1 :l v/v) as described before 21. The dry, defatted tissue was subjected to digestion with papain (l/3-1/4 the dry weight of the tissue) for 48 hr at 65-70” in 0.1 M phosphate buffer pH 6.5 containing 0.005 M EDTA and 0.005 M cysteine according to the procedure of Laurent 25. Fresh papain was added at the end of every 16 h. The digest after centrifugation was passed through (10 cm x 1 cm) previously washed with 1% cetylpyridinium
a column chloride
of cellulose* solution, The
different gg fractions 1 = hyaluronic acid (HA), 2 = heparan sulphate (HS), 3 = chondroitin sulphate A (Ch S-A), 4 = chondroitin sulphate C (Ch S-C), 5 = dermatan sulphate (Ch S-B) and 6 = heparin (H) - were eluted according to the procedure of Svejcar and Robertson 26. The individual gg fractions were quantitated by the estimation of uranic acid according to the modified carbazole reaction of Bitter and Muir27. 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 -1 and 5. Further analysis of the fractions by the enzymic method of Murata and Oshimaas, using chondroitinase and chondrosulphatase, has shown in a previous experiment that fraction 2 contains mostly HS, contaminated by small amounts of Ch S-A; fraction 3 has Ch S-A as the major component with traces of Ch S-C; fraction 4 contains Ch S-C with small amounts of Ch S-B. HA and H were found to be mostly uncontaminated in cellulose acetate** electrophoresis. Since this is too tedious for routine analysis of a large number of samples, the analysis was restricted to the chromatography of the CPC-complex over cellulose. The designation of the fractions as HS, Ch S-A and Ch S-C means that these are the major gg in the respective fractions. The identity of the major component in each fraction was confirmed by comparison with standard gg preparations**. 3. Estimation of enzyme activities Lipoprotein lipase activity was estimated in the aorta, liver and heart. Chilled tissue was homogenised with 3 volumes of cold Sorenson’s phosphate buffer (pH 7.38, was centrifuged at 0 “C at 1500 x g for 10 min 6.6 x 1O-2 M). The homogenate and the supernatant was used as the enzyme. Enzyme assay was carried out according to the procedure of Zemplenyi and Grafnetteras. L-Glutamine :D-fructose-6-phosphate aminotransferase (EC 2.6.1.16) activity of the liver and aorta was estimated according to the procedure of Pogell and Gryder30. The tissue immediately after removal from the animal was homogenised under icecold conditions in a solution containing KC1 (0.154 M), EDTA (0.001 M) and glucose-6-phosphate (0.012 M) pH 7.2 (2 ml/g in the case of liver and 2 ml/50 mg in the case of aorta). The homogenate was centrifuged at 1500 x g for 10 min at 0°C and the supernatant was used as the enzyme source. The reaction system containing 0.1 ml of fructose-6-phosphate (Na salt 0.1 M, pH 7.5), 0.15 ml of L-glutamine (0.1
* Microcrystalline, chromatography grade, E. Merck, Germany. ** All obtained from Sigma Chemicals, U.S.A.
ASCORBIC
ACID AND ATHEROGENIC
M), 0.1 ml of reduced
glutathione
7.5) and 0.3 ml of enzyme
451
DIET
(0.1 M), 0.45 ml of 1 M sodium
was incubated
for 1 h at 38°C. The reaction
by the addition of 1 ml of 0.4 N trichloroacetic tent of 1 ml of the supernatant was determined In the controls, the glutamine UDPG
the complete
reaction
was added immediately dehydrogenase (UDP
phosphate
(pH
was stopped
acid (TCA) and the hexosamine conby the method of Pogell and GI yderse.
system minus
glutamine
was incubated
before the addition of TCA. glucose :NAD oxidoreductase,
EC
and
1.1.1.22)
activity was determined according to the procedure of Strominger et ~1.31.Acetonedry powdered liver was extracted at 0°C in 0.1 M glycine buffer pH 8.7 (8 ml per 400 mg of the powder), centrifuged at 1500 x g at 0°C for 10 min and the supernatant was assayed for enzyme activity. The reaction system contained, in addition to the enzyme (0.5 ml), 0.1 pmole UDPG and 0.1 pmole NAD+ in glycine buffer in a total volume of 5 ml. For the estimation
of /I-glucuronidase
(EC 3.2.1.31)
B-N-acetylhexosaminidase
(EC 3.2.1.30), hyaluronidase (EC 3.2.1.35) and aryl sulphatase (EC 3.1.6.1), the tissue (liver and aorta) was homogenised at 0” with 0.1 M acetate buffer, pH 5.0 (4 ml buffer for 1 g of liver and 2 ml for 50 mg of aorta), centrifuged at 1500 x g for 10 min at 0°C and the supernatant was assayed for enzyme activity. B-Glucuronidase and j3-N-acetylhexosaminidase activity were estimated according to the procedure described by Kawai and Annosa using p-nitrophenyl /3-D-glucuronide and p-nitrophenyl #I-IV-acetylglucosaminide respectively as the substrates. 0.1 ml of enzyme was incubated with 0.5 ml of acetate buffer, containing 100 ,ug of p-nitrophenyl p-D-glucuronide in the case of i3-glucuronidase and 100 ,ug of p-nitrophenyl fi-N-acetylhexosaminide activity was assayed according catechol
sulphate
as substrate
in the case of fi-hexosaminidase. Aryl sulphatase to the procedure described by Roy33 using p-nitroand estimating
the nitrocatechol
liberated.
0.1 ml of
the enzyme was incubated with 0.5 ml acetate buffer containing 0.006 M p-nitrocatechol sulphate. Hyaluronidase was assayed as described by Kawai and Annoss, using hyaluronic acid (sodium salt) as the substrate and estimating the reducing sugar liberated by the method of Park and Johnson 34. 0.2 ml of the enzyme was incubated with 0.5 ml acetate buffer containing 150-200 pg HA (sodium salt). 4. Sulphate metabolism The concentration of PAPS (3’-phosphoadenosine-5’-phosphosulphate), sulphate activating activities (ATP sulphurylase, EC 2.7.7.3, and APS kinase, EC 2.7.1.25) and hepatic phenol sulphotransferase (EC 2.8.2.1) activity were estimated by the method of Jansen and Van Kempen3s,ss using methyl umbelliferone. Details of the procedure have been described beforesT. 5. Estimation Protein
of protein was estimated
after TCA precipitation
6. Ascorbic acid content of the tissues This was estimated by the method
by the method
of Roe and Kuetherss.
of Lowry et al.38.
B. NAMBISAN,
452 TABLE ASCORBIC
P. A. KURUP
1 ACID
Group
1 2 3 4 5 6
CONTENT
OF THE TISSUES
Ascorbic acid (,ug/g wet tissue A SEM) adrenals
spleen
liver
430 f 8.6 64.11 & 1.52 1171 * 22.5
127.8 f 2.6 20.5 & 0.42 209 It 4.10
53.6 22.6 177.8
112.6 & 3.2 8.9 5 0.21 156.1 * 3.5
37.85 & 0.61 14.4 f 0.29 104.9 + 2.5
182.4 12.6 716.5
i 3.80 + 0.31 + 20.1
& 1.12 i 0.5 Z!I3.55
Average of the values from the individual tissues of 6 rats in each case rt SEM.
7. Statistical analysis The data in the tables are the average of 6 experiments significance was calculated using Student’s t-test40.
& SEM.
Statistical
RESULTS
1. Ascorbic acid content of the tissues The ascorbic acid content of the liver, spleen and adrenals in the animals of the different groups is given in Table 1. The decrease in the concentration of ascorbic acid in the tissue in the animals fed the atherogenic diet is in agreement with previous reports41. 2. Lipid levels qf the serum, liver and aorta The total cholesterol, phospholipids and triglycerides of the serum, liver and aorta of the animals of the different groups are given in Table 2. In the group on normal diet, serum cholesterol was not significantly affected in the animals fed a low or high dose of ascorbic acid as compared to those fed an adequate dose. The liver cholesterol was significantly higher in the animals receiving the low dose when compared to the adequate- or high dose-group. Aortic cholesterol was significantly lower in the animals fed the high dose of ascorbic acid, as compared to those fed the adequate or low dose. Serum phospholipids were significantly lower in the animals fed the high dose of ascorbic acid, as compared to those fed the low dose. Liver and aortic phospholipids were lower in the low dose than in the adequate- or high-dose groups, while aortic phospholipids were significantly higher in the animals fed the high dose. Serum triglycerides were not significantly altered in the low- or high-dose group when compared with animals fed the adequate dose, while the liver and aortic triglycerides were decreased in the high dose group and increased in the low dose group in comparison with those fed the adequate dose.
190 * 3.9 233.3 h 4.8.’ 167.7 + 3 2%”
serun, (rng/lOO ml 31 SEM)
4
893.5 + 18.5 1400 f 31.1A 631.6 + 13.4n.a
240.8 It 7.3 298.9 -‘- 8.9” 238.6 & 7.2a
liver (mg/IOOg
128.9 j, 2.54 129.3 I 2.62 126.5 i 2.31
110.2 !z 3.3 109 * 3.2 86.8 + 2.6”,”
aorta wet weight & SEM)
AND AORTA
SEM)
333.3 k 6.9 342.1 i 7.2 240.6 A 4.8.Q
140.1 C 2.8 145.9 & 2.8 132.5 +c 2.6”
i
Serum (mg/lOO ml
Phospholipids
3980 i 80.2 3689 i 74.3” 2661 + 54.1A.a
1756 * 34.5 1223 i 24.4” 1842 & 36.ga
liver (mg/lOOg
2268 i 27.2 2250 C 26.1 2386 + 28.6=-
1628 + 33.4 1312 + 28.F 1984 * 39.8-ksa
aorta wet tissue & SEM)
6.31 ??0.12 9.76 _c 0.21” 5.40 & 0.10.‘9”
6.20 i 0.314 6.51 + 0.15 6.05 & 0.13
Serum (mg/lOO ml 41 SEM)
Triglyceride
374 * 7.4 484 * 9.6A 373 5 7.42”
210.7 -c 4.8 243.2 C 5.2” 180.78 & 4 0a.a
liver (mg/lOOg * SEM)
glycerol
1148 & 23.9 1420 h 28.4” 987 i 19.6A+
826.4 + 17.5 962 & 20.1.’ 767.6 _C 15.gBza
aorta wet tissue
Average of the values from individual tissues of 6 animals in each case * SEM. Group 2 and 3 have been compared with group I, and group 5 and 6 with group 4. * P less than 0.01. BP between 0.01 and 0.05. No symbol: greater than 0.05. Group 3 and 6 have also been compared with group 2 and 5 respectively. a P less than 0.01. b P between 0.01 and 0.05.
6
5
3
2
LIVER
Total cholesterol
OF THE SERUM,
79.9 * 2.2 85.6 + 2.5 80.01 i 2.4
LEVELS
2
1
Group
LIPID
TABLE
P
454
B. NAMBISAN, P. A. KURUP
TABLE 3 LIPOPROTEIN
Group
LIPASE
ACTIVITY
Lipoprotein
OF THE TISSUES
lipase activity
(pmole
glycerol/hr/lOO
mg protein
aorta
liver
heart
2 3
72.43 + 1.51 71.51 & 1.5 75.62 zk 1.55
26.39 & 0.59 28.52 i 0.61B 23.63 % 0.52”
20.59 * 0.50 30.91 i 0.71-4 20.78 * 0.51%
4 5 6
55.2 54.6 57.9
15.50 * 0.31 17.82 + 0.36” 13.91 i_ 0.28”+
14.01 & 0.31 21.18 i 0.80” 14.84 * 0.35”
1
.+ 1.2 i 1.15 h 1.28
+ SEM)
Average of the values from individual tissues of 6 animals in each case & SEM. Group 2 and 3 have been compared with group 1, and group 5 and 6 with group 4. A P less than 0.01. * P between 0.01 and 0.05. No symbol: P greater than 0.05. Group 3 and 6 have also been compared with group 2 and 5 respectively. a P less than 0.01. b P between 0.01 and 0.05.
In the atherogenic diet group, serum and liver cholesterol were increased in the low dose group and decreased in the high dose group when compared with those fed the adequate dose, while aortic cholesterol was not significantly affected in both groups. Serum and liver phospholipids were decreased significantly in the animals fed the high dose of ascorbic
acid when compared
with those fed the adequate
or low
dose, while the liver phospholipids were also lower in the low dose group than in the adequate dose group. Aortic phospholipids were increased in the animals of the high dose group. Triglycerides were significantly higher in the serum, liver and aorta of the low dose group, in comparison with the adequate dose group, while they were lower in the serum and aorta in the high dose group. 3. Lipoprotein lipase activity of the aorta, liver and heart The results are given in Table 3. In the normal diet group the aortic enzyme concentration was not affected in the animals fed the low or high dose as compared with those of the adequate group. It was significantly increased in the liver and heart in the low dose group and decreased in the liver in the high dose group as compared with the adequate dose group. In the atherogenic diet group, enzyme activity was not affected in the aorta in the animals fed the low or high doses, was increased in the liver and heart in the low dose group and was decreased in the liver in the high dose group when compared with the adequate dose group. 4. Glycosaminoglycans of the aorta and liver The results are given in Table 4. In the groups on normal diet, HA was significantly decreased in the aorta in the low dose group when compared with the ade-
OF THE
TlSSUES
965 i 21.3 1001 i 25.1 989 * 22.2
1100 5 22.1 990 i 19.8” 1124 + 22.4a
880 + 17.6 695 * 15.3* 956 zt 20.4*+
1180 It 30.5 1036 * 26.2* 1260 i 31.4”+
i_ 25.7 * 21.2 + 29.On 1476 1388 1610 i 29.0”*& + 26.8”*& h 32.1”
1562 i 30.4 1459
i_ 25X* 1491 i 29.1”
1110 + 22.0 1050
1321 i 26.5 1280
1450 + 28.2 1287
Ch S-C Ch S-B
H
926 * 19.3 788
600 & 13.8 520 & 12.4* 675 & 14.3”~~
730 * 15.0 610 * 13.1* 824 & 16.6*.”
+ 16.8” * 15.4“ 956 986 * 19.1*-a * 20.2-43
930 + 18.6 833
272 i 5.4 262 f 5.2 224 k 6.4*la
f 8.2A 298 f 6.8&
305 f 7.5 367
HA
Ch S-A
HA
HS
Liver
Aorta
165 i 4.2 129 + 3.5‘4 167 + 4.4&
& 9.8 412 I_t lO.P+
369 i 9.6 378
HS
112 ??3.3 99.8 & 2.gB 141 & 4.2”~”
zk 5.4 295 * 5.W”
226 * 5.5 221
Ch S-A
126.5 * 3.6 110.8 & 3.1” 139.9 & 4.1-i+
A 7.2 406 + 8.2”+
341 & 7.25 340
Ch S-C
142 * 2.9 112 * 2.4’ 145 I_t 3.0a
IL-6.4 361 & 8.4.‘~a
300 + 1.2 292
Ch S-B
100 + 2.1 98.3 -f- 1.92 110 + 2.3B*”
& 4.4” 310 & 6.3A+
218 4 5.4 224
H
Average of the values from individual tissues of 6 animals in each case & SEM. Group 2 and 3 have been compared with group 1, and group 5 and 6 with group 4. * P less than 0.01. n P between 0.01 and 0.05. No symbol: P greater than 0.05. Group 3 and group 6 have also been compared with group 2 and 5 respectively. &P less than 0.01. b P between 0.01 and 0.05.
6
5
4
3
2
1
LEVELS
uranic acid/g dry defatted tissue + SEM)
Group
(pg
GLYCOSAMINOGLYCAN
TABLE 4
3
5 u
8 2
z %
5
456
9. NAMBISAN, P. A. KURUP
TABLE 5 AMINOTRANSFERASE
AND
UDPG
DEHYDROGENASE
ACTIVITY
OF THE LIVER
Group
Aminotransferase (pmole hexosamine~hrl g protein * SEM)
UDPG dehydrogenase (units/g ofprotein i SEM)
1
24.50 & 0.64 22.63 5 0.62 25.29 f 0.70b
956 f 19.5 960 h 20.1 960 f 20.0
18 * 0.53 13.05 f_ 0.36” 20.15 f 0.61”
820 f 23.4 816 & 23.8 824 & 23.2
2 3 4
5 6
Average of the values from the individual tissues of 6 animals in each case & SEM. Group 2 and 3 have been compared with group 1, and group 5 and 6 with group 4. A P less than 0.01. No symbol: P greater than 0.05. Group 3 and group 6 have also been compared with group 2 and 5 respectively. a P less than 0.01. b P between 0.01 and 0.05.
quate- or high-dose groups. HS, Ch S-A, Ch S-B and H were significantly increased in the high dose group, in comparison with the adequate dose group, while Ch S-C, Ch S-B and H were significantly decreased in the low dose group. All the gg fractions decreased in the low dose group when compared with the high dose group. In the liver, HA increased and H decreased in the low dose group when compared with the adequate dose group. All the sulphated gg increased in the high dose group when compared with the adequate dose group. Compared to the high dose group, all the sulphated
gg fractions
were significantly
reduced in the low dose group.
In the atherogenic diet group, all the sulphated gg in the aorta were significantly lower in the low dose group than in the adequate dose group, while Ch S-A, Ch S-B, Ch S-C and H were higher in the high dose group. All the sulphated gg were higher in the high dose group than in the low dose group. In the liver, HS, Ch S-A, Ch S-C and Ch S-B were significantly lower in the low dose group than in those fed the adequate dose, while Ch S-A, Ch S-C and H were higher in the high dose group. All the sulphated gg fractions were again higher and HA lower in the high dose than in the low dose group. 5. L-Glutamine: D-fructose-6-phosphate aminotransferase ‘and UDPG dehydrogenase activity of the liver The results are given in Table 5. In the group on normal diet increased aminotransferase activity was observed in the animals receiving the high dose of ascorbic acid rather than the low dose. However, enzyme activity was not significantly different when the low or high dose group was compared with the group fed the adequate dose. There was no significant alteration in the UDPG dehydrogenase activity in the low or high dose group when compared with the adequate dose group.
7.54 i 0.18 6.93 i 0.168 5.05 i 0.12.\,a
4 5 6
48.38 & 1.42 51.14 * 1.51 43.82 !C 1.30”
86.0 i 2.51 91.53 i 2.72 81.92 -+ 2.42b
AND LIVER
9.53 i 0.27 11.21 & 0.30” 8.55 & 0.25=”
65.8 + 1.32 74.2 j, 1.51* 66.2 IL 1.35”
77.1 & 1.58 51.48 & 1.10 78.7 * 1.6 53.83 h 1.3 70.79 j, 1.42B,b 46.6 + 0.95BJ
aorta liver (mg nitrocatechol/hr/g protein & SEM)
Aryl sulphatase
22.82 i 0.54 82.16 =I 1.64 31.4 i 0.75* 86.35 & 1.72 21.01 & 0.50nja 80.9 = 1.6”
8.16 i 0.24 9 16 & 0.27B 8.25 + 0.25”
aorta liver (mgp-nitrophenol/hr/g protein & SEM)
16.15 * 0.40 64.33 + 1.92 96.1 + 1.80 17.56 + 0.48 15.85 & 0.41 75.36 C 2.20” 95.8 & 1.75 19.65 & 0.51” 14.4 & 0.38”~~ 54.8 5: 1.62=sa 88.6 & 2.24”~” 16.81 5 0.41%
12.43 h 0.36 13.91 * 0.398 11.90 j, 0.33&
OF THE AORTA
&Hexosaminidase
ACTIVITY
Average of the values from the individual tissues of 6 animals in each case + SEM. Group 2 and 3 have been compared with group 1, and group 5 and 6 with group 4. A P less than 0.01. B P between 0.01 and 0.05. No symbol: P greater than 0.05. Group 3 and group 6 have also been compared with group 2 and 5 respectively. &P less than 0.01. b P between 0.01 and 0.05.
4.93 k 0.14 5.12 * 0.15 4.20 + 0.13*-”
aorta liver (mgp-nitrophenol/hr/g protein 3~ SEM)
SULPHATASE
aorta liver (mg reducing sagar/l6 hr/g protein * SEM)
ARYL
/I-Glucuronidase
/%HEXOSAMINIDASE,
Hyaluronidase
/%GLUClJRONIDASE,
1 2 3
Group
HYALURONIDASE,
TABLE 6
3
a
A
458
B. NAMBISAN, P. A. KURUP
TABLE I LEVEL
OF
PAPS,
SULPHATE
ACTIVATING
SYSTEM
AND PHENOL
SULPHOTRANSFERASE
Group
PAPS levels (pnde
1 2 3
170.6 zk 3.64 166.9 + 3.51 180.27 t_ 3.85nsb
54.2 & 1.10 52.71 + 1.15 62.78 & 1.32”~~
4 5 6
141 138 162
25.32 i 0.52 21.5 + 0.45* 28.55 k 0.57*,”
& 2.8 k 2.6 :k 3.2”~&
ACTIVITY
IN THE LIVER
Sulphotransferase Sulphate activating system methyl umbelliferone sulphatelhrig protein i SEM) 14.11 i 9.28 12.5 i 0.25* 16.11 + 0.32”~” 11.1 & 0.27 10.8 rt 0.26 14.0 * 0.40”*&
Average of the values from the individual tissues of 6 animals in each case i SEM. Group 2 and 3 have been compared with group 1, and group 5 and 6 with group 4. * P less than 0.01. D P between 0.01 and 0.05. No symbol: P greater than 0.05. Group 3 and group 6 have also been compared with group 2 and 5 respectively. a P less than 0.01 b P between 0.01 and 0.05.
In the atherogenic diet group, aminotransferase activity significantly decreased in the low dose group, in comparison with the adequate- or high-dose group. UDPG dehydrogenase
activity
again was not affected in the low or high dose groups.
6. Hyaluronidase, fl-glucuronidase, f+hexosaminidase and aryl sulphatase activity The enzyme activities of the aorta and liver are given in Table 6. In the normal diet group, hyaluronidase, j%glucuronidase, /I-hexosaminidase and aryl sulphatase activity of the liver and aorta were significantly higher in the animals fed the low dose than in those fed the high dose of ascorbic acid. Similar increased values were also observed in the animals on the atherogenic the low dose of ascorbic acid rather than the high dose.
diet when fed
7. Sulphate metabolism The concentration of PAPS, sulphate activating system and phenol sulphotransferase activity of the liver are given in Table 7. In the normal diet group, the concentration of PAPS was significantly higher in the animals fed the high dose of ascorbic acid than in those fed the low dose. Sulphate activating system, which includes ATP sulphurylase and APS kinase, was also considerably higher in the high than in the low dose group. Similar results were also observed in the case of sulphotransferase activity. In animals fed the atherogenic diet, similar increases were seen in the concentration of PAPS, sulphate activating system and sulphotransferase in the animals fed the high dose of ascorbic acid in comparison with those fed the low dose. DISCUSSION
The effect of a high dose of ascorbic
acid on the concentration
of lipids in the
ASCORBIC
ACID
AND ATHEROGENIC
459
DIET
tissues varied in guinea pigs fed the normal emit effect was seen only in the animals
and atherogenic
diets. A hypocholesterol-
fed the atherogenic
diet, while lowering
of
the aortic cholesterol was seen only in animals fed the normal diet and not in the group fed the atherogenic diet. These results on aortic cholesterol in animals fed the atherogenic esterolemic presumably in addition
diet agree with those reported by Ginter and Babalar5, but no hypocholeffect as in our experiments was observed by these workers. This may be due to the difference in the atherogenic diets used, 15 % coconut oil to 0.3 % cholesterol being used in our experiments. Hypotriglyceridemia
again was observed only in the group fed the atherogenic diet, while decrease in aortic and hepatic triglycerides was observed in both groups. However the pattern of change now observed in the concentration of lipoprotein lipase showed no correlation with lipid levels, although previous reports have associated decreased enzyme activity with increased lipid accumulation42 and vice-versa. Administration of a high dose of ascorbic acid to guinea pigs fed normal as atherogenic
diets has been found
to result in an increased
concentration
as well of aortic
and hepatic glycosaminoglycans as compared to those fed a low dose of ascorbic acid. Hyaluronic acid concentration showed a different trend, decreasing in the liver in both the normal and atherogenic diet groups, and increasing only in the aorta in the normal diet group. One possible reason for the decreased concentration of glycosaminoglycans in the animals receiving the low dose of ascorbic acid may be the decreased activity of L-glutamine: u-fructose-6-phosphate aminotransferase, but UDPG dehydrogenase was not appreciably affected. The former is a key enzyme which makes available glucosamine-6-phosphate, the precursor of the hexosamine moiety for synthesis of glycosaminoglycans. Another reason for the low concentration of gg in the low dose group may be increased
degradation
of gg, as is evident
from the
increased activities of hyaluronidase, ,%glucuronidase, /Lhexosaminidase and aryl sulphatase. The decreased sulphate metabolism in the animals receiving the low dose of ascorbic acid may also be another factor. The decrease in the concentration of gg and the activity of biosynthetic enzymes and biological sulphation and increased activity of enzymes concerned with the degradation of gg in the animals fed the atherogenie diet agree with results reported previously43. Ascorbic acid has been reported to activate a number of enzymes, the action being mainly attributed to the protection of -SH groups. It is possible that the decreased activity of L-glutamine:D-fructose-6-phosphate aminotransferase and the enzymes concerned with biological sulphation in the animals of the low ascorbic acid group may be due to this factor. But the increased activity of enzymes concerned with the degradation of gg, cannot be explained on this basis. The report of Mumma and Verlangierr ‘44 that ascorbic acid as ascorbic acid-Z sulphate may play a role in biological sulphation may be pertinent in explaining the increased sulphation observed in the animals on the high dose of ascorbic acid. The results now obtained may indicate a direct involvement of ascorbic acid in the biosynthesis of gg, probably at the level of sulphation and also in the degradation of the gg.
460
B. NAMBISAN, P. A. KURUP
It is tempting
to speculate
mvolved in the development those reported by a number increased
cholesterol
that ascorbic
of atherosclerosis. of others, indicate
in the liver and increased
acid deficiency
in man may be a factor
The results that ascorbic
now obtained as well as acid deficiency results in
triglycerides
in the liver and aorta in
animals fed a normal diet, while an increase in cholesterol and triglycerides is observed in the liver and aorta in the animals fed the atherogenic diet. Ascorbic acid deficiency
has now been shown
with both normal
and atherogenic
to result in a decreased
concentration
diets, and this result is of interest
of aortic
gg
when the reported
decrease in the concentration of sulphated gg17 in atherosclerotic aorta is considered. Thus, ascorbic acid deficiency may contribute to the development of atherosclerosis both by the increased lipid levels and the decreased sulphated gg levels it produces.
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ASCORBIC ACID AND ATHEROGENIC DIET
461
18 BURLINA,A. AND LIPPI, V., Pathologic histology of the liver in relation to ascorbic acid deficiency, Riv. Anat. Patol. Oncol., 16 (1959) XXIV. 19 GORE, l., TANAKA, Y., FUJINAMI,T. AND GOODMAN, M. L., Aortic acid mucopolysaccharides and collagen in scorbutic guinea pigs, 1. Nutr., 87 (3) (1965) 311. 20 SCHULTZ-HAUDT, S. D. AND SIGURD, D., Comparative histochemistry of non-injured skin connective tissue of normal and ascorbic acid deficient guinea pigs, Acta Pathol. Microbial. Stand., 62 (4) (1964) 465. P. AND KURUP, P. A., Changes in tissue glycosaminoglycans in rats fed a hyper21 SEETHANATHAN, 14 (1971) 65. cholesterolaemic diet, Atherosclerosis, 22 CARR, J. J. AND DREKTER, I. J., Estimation of total cholesterol in the serum, C/in. Chem., 2 (1956) 353. of phospholipids, J. Lab. Clin. Med., 23 ZILVERSMIT,D. B. AND DAVIS, A. K., Microdetermination 35 (1950) 155. 24 VAN HANDEL, E. AND ZILVERSMIT,D. B., Micromethod for direct determination of serum triglycerides, J. Lab. Clin. Med., 50 (1957) 152. 25 SCOTT, J. E., Aliphatic ammonium salts in assay of acid polysaccharides from tissues. In: D. GLICK (Ed.), MethodsofBiochemical Analysis, Vol. 8, Interscience, New York, N.Y., 1960, p. 145. 26 SVEJCAR, J. AND ROBERTSON,W. V. B., Micro-separation and determination of mammalian acidic glycosaminoglycans, Anal. Biochem., 18 (1967) 333. 27 BITTER,T. AND MUIR, H. M., A modified uranic acid carbazole reaction, Anal. Biochem., 4 (1962)
330. 28 MURATA, K. AND OSHIMA,Y., Chondroitin sulphate in atherogenic human aorta, Atherosclerosis, 14 (1971) 121. 29 ZEMPLENYI,T. AND GRAFNETTER,D., Species and sex differences in fatty acid release from tissues incubated with lipaemic serum, Brit. J. Exp. Pathol., 39 (1958) 99. in rat liver, 30 POGELL,B. M. AND GRYDER,R. M., Enzymatic synthesis of glucosamine-6-phosphate J. Biol. Chem., 228 (1957) 701. 31 STROMINGER,J. L., MAXWELL, E. S., AXELROD, J. AND KALEKAR, H. M., Enzymatic formation of uridine diphosphoglucuronic acid, J. Biof. Chem., 224 (1957) 79. 32 KAWA~, Y. AND ANNO, K., Mucopolysaccharides degrading enzymes from the liver of the squid, Ommastrephes sloanipacificus, hyaluronidase, Biochim. Biophys. Acta, 242 (1971) 428. 33 ROY, A. B., The sulphatase of ox liver, Biochem. J., 53 (1953) 12. 34 PARK, J. T. AND JOHNSON,M. J., A sub-microdetermination of glucose, J. Biol. Chem., 181 (1949)
149. 35 VAN KEMPEN,G. M. J. AND JANSEN,G. S. I. M., Quantitative assay of 3’-phosphoadenosine5’-phosphosulphate, “active sulphate”, Anal. Biochem, 51 (1973) 324. 36 VAN KEMPEN, G. M. J. AND JANSEN,G. S. 1. M., Quantitative determination of phenolsulphotransferase using 4-methyl umbelliferone, Anal. Biochem., 46 (1972) 438. 37 SUDHAKARAN,P. R. AND KURUP, P. A., Vitamin A and glycosaminoglycans metabolism, J. Nutr., 104 (1974) 871.
38 LOWRY, 0. H., ROSEBROUGH,N. J., FARR, A. L. AND RANDALL, R. J., Protein measurement with the Folin-phenol reagent, J. Biol. Chem., 193 (1952) 265. 39 HAWK, P. B., OSER, B. L. AND SUMMERSON,W. H., In: Practical Physiological Chemistry, 14th edition, McGraw Hill, New York, N.Y., 1965, p. 703. 40 BENNETT,C. A. AND FRANKLIN,N. L., Statistical Analysis in Chemistry and the Chemical Industry, Wiley, New York, N.Y., 1967. 41 RZAKULIEVA,D. M. AND GASANOV, A. S., Variation of ascorbic acid content in the organs and tissues in experimental atherosclerosis, SSR, 22 (3) (1966) 90. 42 SEETHANATHAN, P., VIJAYAGOPAL,P., AUGUSTI, K. T. AND KURUP, P. A., Myocardial lipoprotein lipase in rats fed a hypercholesterolaemic diet, Atherosclerosis, 11 (1970) 333. 43 VIJSYAKUMAR,S. T. AND KURUP, P. A., Metabolism of glycosaminoglycans in atheromatous rats, Atherosclerosis, 21 (2) (1975) 245. 44 MUMMA, R. 0. AND VERLANGIERI,A. J., In vivo sulphation of cholesterol by ascorbic acid-3sulphate as possible explanation for the hypocholesterolaemic effect of ascorbic acid, Fed. Proc., 30 (2) (1971) 370 Abstr.
