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Hyperlipidemia in guinea-pigs induced by ascorbic acid deficiency The effects of cholesterol, dl -ethionine and aflatoxin
249
Atherosclerosis, 38 (1981) 249-254 @Elsevier/North-Holland Scientific Publishers, Ltd.
HYPERLIPIDEMIA IN GUINEA-PIGS INDUCED BY ASCORBIC ACID DEFICIENCY The Effects of Cholesterol, DL-Ethionine and Aflatoxin FUMI YOKOTA,
YUKO IGARASHI and RYOKUERO
The National Institute of Nutrition, 1, Toyama-cho,
SUZUE
Shinjuku-ku,
Tokyo 162 (Japan)
(Received 6 June, 1980) (Revised, received 30 July, 1980) (Accepted 30 July, 1980)
Summary A study was made of hyperlipidemia caused by ascorbic acid deficiency and of the effects of cholesterol, DGethionine and aflatoxin on plasma lipoprotein fractions of normal and scorbutic guinea pigs. The plasma lipoprotein fractions of scorbutic animals showed a significantly higher level of pre-&lipoprotein and a lower level of a-lipoprotein. By adding DL-ethionine to the control group, the pre$-lipoprotein fraction was remarkably elevated and by adding cholesterol, the ol-lipoprotein level was greatly reduced and the P-lipoprotein level was increased. Addition of aflatoxin to the control diet resulted in a rather high concentration of cu-lipoprotein and a low pre_P-lipoprotein level. High concentrations of triglyceride and phospholipid were seen in the plasma of scorbutic guinea pigs. The probable cause of hyperlipidemia induced by ascorbic acid deficiency is partly retarded degradation of cholesterol resulting from impaired ‘la-hydroxylation, and partly that ascorbic acid deficiency may affect other enzyme systems that control triglyceride of phospholipid metabqhsm, such as lipoprotein lipase activity, or synthesis or breakdown of these enzymes. Key words:
Aflcrtoxin - Ascorbic acid - Cholesterol - DL-Ethionine Lipoprotein fmctions
- Hyperlipidemia
-
Introduction Recent research on hyperlipidemia has shown a consistent association between relatively low plasma concentrations of high density lipoprotein (HDL) cholesterol and increased risk of coronary heart disease [ 1,2].
250
Some investigators have recently reported on hyperlipidemia caused by deficiency of ascorbate [ 3,4], and Bordia has described the effect of vitamin C on blood lipids in coronary artery disease [5]. Guinea pigs with ascorbic acid deficiency have significantly higher serum and liver cholesterol levels than control animals; the reason for this has been sought in the biosynthesis as well as the catabolism of cholesterol [ 41. Since ascorbic acid is an important cofactor in the hydroxylation of many substances, it is possible that it also stimulates the hydroxylation of cholesterol, causing transformation of cholesterol to bile acids in the liver, Ginter et al. [6] noted that biosynthesis of bile acids is reduced in scorbutic guinea pigs, and Guchhait et al. [ 71 found a reduction in cholesterol catabolism in scorbuticguinea pigs. If ascorbic acid affects the overall conversion of cholesterol into bile acids, it is probable that the effect is related to cholesterol 7a-hydroxylase. There are 2 possibilities regarding the effect of ascorbic acid on cholesterol 7a-hydroxylation. Ascorbic acid might be an essential factor for enzyme activity or it might affect enzyme synthesis or breakdown. Bjorkhem et al. [3] have recently observed that 7cu-hydroxylation is .markedly reduced in scorbutic animals compared with those treated with ascorbate. It has also been suggested that ascorbate affects the synthesis or breakdown of the 7a-hydroxylating system, in particular the cytochrome P-450 component. Thus, the mechanisms of hypercholesteremia caused by ascorbic acid deficiency are gradually being clarified. On the other hand, the mechanisms of non-cholesterol hyperlipidemia caused by deficiency of ascorbic acid have not been thoroughly elucidated. This experiment was designed to study electrophoretically the metabolic alteration in lipoprotein fractions of the blood plasma in normal and scorbutic guinea pigs, when fed a cholesterol-rich diet or treated with the hepatotoxic substances, DGethionine and aflatoxin. Materials and Methods Materials Forty-eight guinea pigs of the Hartley strain and a powdered diet free of ascorbic acid were obtained from Clea Japan Inc. Supplements of ascorbic acid were purchased from Iwaki Seiyaku Co., L. of Japan and supplements aflatoxin from Makor Chemicals of Jerusalem, Israel. Reagents used for the determination of cholesterol were obtained from Wako Pure Chemical Industries, L. of Japan and DGethionine and cholesterol from Sigma Chemical Company. All the chemicals and the equipment used in the lipoprotein electrophoresis were procured from Gelman Instrument Company. Animals As shown in Table 1, the animals were divided into 8 groups, 6 in each group, and were maintained on diets as described below for 3 weeks. The control group was fed the powdered diet free of ascorbic acid supplemented with 0.5% Gascorbic acid and the scorbutic group the ascorbic acid-free diet. Supplements of 0.5% cholesterol and 0.2% gall powder were added to the cholesterol-supplemented groups and 50 pg of aflatoxin B1 dissolved in dimethylsulfoxide was given orally to the animals of the aflatoxin groups every day for
251 TABLE
1
Control
-AsA -AsA
+ Cholesterol groups
diet + AsA 0.5% diet + cholesterol 0.5% + gall powder 0.290 + cholesterol 0.5% diet { + gsU powder 0.2%
Control { -AsA
+ Aflatoxin groups
Control + AF B 1 50 @g/day -AsA + AF B, 50 pg/day
+ Ethionine groups
Control + DL-ethionine 0.5% -AsA diet + DL-ethionine 0.5%
Abbreviations:
AsA = Ascorbic acid: AF = Aflatoxin.
3 weeks. In the ethionine groups, 0.5% DGethionine supplements were given for 4 days after 2 weeks of feeding on the control or ascorbic acid-deficient diet. During the experimental period, food and water were given ad libitum. Animals were kept in individual cages at constant temperature and humidity and were weighed daily. After 3 weeks, animals were killed by decapitation, and plasma obtained. Plasma cholesterol and P-lipoprotein determination Plasma total cholesterol was determined by the method &lipoprotein by the method of Fried et al. [9].
of Zak et al. [ 81 and
Plasma lipoprotein fractions obtained by electrophoresis Separated plasma was stored at l-4” C until use. Lipoprotein fractions were separated in accordance with Technical Bulletin No. 28 of the Gelman Instrument Company. Cellulose acetate membrane (Separaphore 111) was used as the sample carrier, and Tris-Barbital-Sodium Barbital buffer, pH 8.8, was used for electrophoresis. Lipoprotein complexes were separated by electrophoresis into chylomicrons, &lipoproteins, pre-/3-lipoproteins, ar-lipoproteins an4 free fatty acids. Chylomicrons remained at the application point (toward the negative pole), while free fatty acids migrated fastest and were found closest to the anode (positive pole). Other fractions were located between chylomicrons and free fatty acids in the order stated. A normal sample usually contained /3-, pre-fland a-fractions only. Electrophoresis was carried out at a constant voltage of 200 V (current 4.5-6.0 mA) for 25 min. After electrophoretic separation, the fractions were visualized by staining with Oil Red 0. The individual spots were then quantitated with a densitometer at 525 nm by a Gelman computing densitometer (DCD-16). Results As shown in Fig. 1, the body weight of ascorbic acid-deficient animals gradually decreased after 2 weeks of feeding on the respective diets, except in the cholesterol-supplemented groups whose decrement of body weight was not so severe as the other groups even at the end of 3 weeks.
252
A
4
“I
9
I
B
500
0
0
1
2
I
3
I
0
1
Week
2
3
W.?YZk
,I+
,-::-i:-:-:_:i
m
+ ethkmine
8
4 days
2OO-
2ca
0
1
2
3
Week
0
1
2
3
We&C
Fig. 1. Body weight. A: control groups: 0~ positive control, o----o -AsA control; B: plus cholesterol groups: ?? control + cholesterol + gall powder, o----o -AsA + cholesterol + gall powder; C: Plus AF groups: .control + AF. O-AsA + AF; D: plus ethionine groups: ?? _ control, O----o -AsA. Each point represents mean f SE for 6 animals in each group.
Plasma total cholesterol values are shown in Table 2. By the addition of aflatoxin or ethionine to the control diet, no significant increment could be observed in the plasma total cholesterol levels, but the addition of cholesterol to the control diet brought about a significant increase. In the scorbutic groups, TABLE
2
Plasma total cholesterol (mg/dl) c&ntrol -AsA
24.6 * 1.0 *la 65.0 f 2.3 b
Control + cholesterol -AsA + cholesterol
42.6 f 1.9 b 87.5.k 8.2 c
Control + AF -AsA + AF
31’5 f 1.4 a 46.9 i 3.0 a.b
Control + ethionine -AsA + ethionine
27.9 i 0.9 a 51.5 i 1.8b
a&c
*Mean*SE(n=6). Mew hahg same superscripts (vbaP) are not significantly different. whereas those with different superscripta (tia.a.b) are significantly different (P < 0.01).
253 TABLE 3 Plasma triglyceride and phospholipid (mg/dl) Group
Triglyceride
Phospholipid
Control -AsA
26.8 f 3.4 *,a 91.4 f 12.6 b
49.8 f 4.8 * 133.8 f 8.5 b
Notations as for Table 2.
TABLE 4 Electrophoretic
fractions of plasma lipoproteins (96)
Groups
Alpha
Pm-beta
Beta
Beta/alpha (ratio)
Control -AsA
21.4 f 6.9 *& 6.3 -k0.8 a
5.9 r 2.9 b 31.9 + 6.3 *
58.2 f 4.2 aqb 54.5 f 5.3 a.b
2.5 f. 0.7 a 13.9 i 1.5 b
1.4 f 2.4 a 5.0 f 1.0 a
6.4 k 1.2b 22.9* 1.9a
72.9 f 2.2 a 68.4 * 3.5 a
12.0 i 4.0 b 19.3 f 4.2 c
Control + cholesterol -AsA + cholesterol Control + AF -AsA + AF
34.9 2 1.5d 12.4 +_1.8 a.b
4.8 r 0.3 b 16.1 f 6.0 a.b
59.8 f 2.3 a,h 59.2 + 8.3 a.b
1.9 f 0.1 a 6.6 f 1.2 a
Control + ethionine -AsA + ethionine
20.1 i 0.9 b 12.3 + 1.0 *
38.3 + 1.7 a 24.5 * 2.5 a
33.9 + 1.5 b 56.8 f 1.7 a.b
3.5 f 0.2 a 6.7 + 0.7 b
Notations as for Tables 1 and 2.
all plasma cholesterol levels increased in comparison with the corresponding control groups. As shown in Table 3, plasma triglyceride and phospholipid levels in the scorbutic groups were very much higher than the control group. Triglyceride in the control group was 26.8 mg/dl and in the scorbutic group 91.4 mg/dl. Phospholipid in the control group was 49.8 mg/dl and in the scorbutic group 133.8 mg/dl. Electrophoretic separations of plasma lipoproteins are shown in Table 4. A significantly higher &lipoprotein to e-lipoprotein ratio was observed in the scorbutic group, i.e. 13.9 versus 2.5 in the control group. In the cholesterolsupplemented groups, an elevated level of /3-lipoprotein was noted in both the control-plus-cholesterol group and scorbutic-plus-cholesterol group. With aflatoxin supplements in the control group, a high level of o-lipoprotein and a low level of prep-lipoprotein were observed. Following 2 weeks on the control or ascorbic acid-deficient diet; 0.5% DL-ethionine was added to each diet which was then fed for 4 days. A significant increase in the pre-/l-lipoprotein level was observed in the control-plusethionine group. Discussion In this experiment, high concentrations of triglyceride and phospholipid were observed in the plasma of ascorbic acid-deficient guinea pigs. In electrophoretic fractions of plasma lipoproteins, a significantly higher level of pre_P-
254
lipoprotein and a lower level of cu-lipoprotein were noted in the plasma of ascorbic acid-deficient animals. Since preQ-lipoprotein contained a considerably higher level of triglyceride, the elevation of the pre-o-fraction must be due to the higher concentration of triglyceride in the plasma of ascorbic acid-deficient guinea pigs. The cause of hyperlipidemia in the ascorbic acid-deficient animals could partly be retarded degradation of cholesterol by impaired ‘la-hy droxylation induced by ascorbic acid deficiency. Ascorbic acid deficiency may also affect other enzymes that control triglyceride or phospholipid metabolism, such as lipoprotein lipase activity, or synthesis or breakdown of these enzymes. In order to confirm these observations, further studies on enzyme activities are in progress in our laboratory. After addition of cholesterol, the a-lipoprotein level was greatly reduced, while the &lipoprotein level was increased. This may be due to the higher concentration of cholesterol in /3-lipoprotein. By adding aflatoxin to the control diet, a rather high concentration of a-lipoprotein and a low level of preQ-lipoprotein were observed, but by adding DL-ethionine to the control group, the prep-lipoprotein fraction was remarkably elevated. Thus, by the addition of cholesterol, aflatoxin or Dcethionine, characteristic changes were noted in the electrophoretic fractions of plasma lipoprotein, but the explanation for the mechanisms of these metabolic changes in individual cases must await further investigations. References 1 Rhoads. G.G., Gulbrandsen, CL. and Kagan. A., Serum lipoprotein and coronary heart disease in a population study of Hawaii Japanese men, N. En& J. Med., 294 (1976) 293. 2 Gordon, T.. CasteRi, W.P., Hjdrtland. M.C.. Kannel. W.B. and Dawber, T.R., High density lipoprotein as a protective factor against coronary heart disease - The Framingham Study, Amer. J. Med., 62 (1977) 707. 3 Bjorkhem, I. and Kallner. A., Hepatic 7 o-hydroxylation of cholesterol in aacorbatedeficient and ascorbate+upplemented guinea pigs, J. Lipid Res., 17 (1976) 360. 4 Gmter. E.. Cholesterol-Vitamin C controls its transformation to bile acids, Science, 179 (1973) 702. 6 Bordia. A.K., The effect of vitamin C on blood lipids. fibrinolytic activity and platelet adhesiveness in patients with coronary artery disease,Atheroscleroais, 36 (1980) 181. 6 Ginter, E.. Nemec. R., Cerven. J. and Mfkus, L.. Quantification of lowered cholesterol oxidation in guinea pigs with latent vitamin C deficiency, Lipids, 8 (1973) 135. 7 Guchhait. R.. Guha. B.C. and Gsnguli. N.C.. Metabolic studies on rcorbutic guinea pigs - Catabolism of 4-14C-cholesterol in viva and in vitro, Biochem, J.. 86 (1963) 193. 8 Zak. B.. Luz. D.A. and Fisher, M.. Determination of serum cholesterol, Amer. J. Med. Technol.. 23 (1967) 283. 9 Fried, R. and Hoeflmayr, T., Eine ehrfache mdirekte Bestimmungrmethode der 6-Lipoproteide, Kim. Wschr., 41 (1963) 246.
Atherosclerosis, 38 (1981) 249-254 @Elsevier/North-Holland Scientific Publishers, Ltd.
HYPERLIPIDEMIA IN GUINEA-PIGS INDUCED BY ASCORBIC ACID DEFICIENCY The Effects of Cholesterol, DL-Ethionine and Aflatoxin FUMI YOKOTA,
YUKO IGARASHI and RYOKUERO
The National Institute of Nutrition, 1, Toyama-cho,
SUZUE
Shinjuku-ku,
Tokyo 162 (Japan)
(Received 6 June, 1980) (Revised, received 30 July, 1980) (Accepted 30 July, 1980)
Summary A study was made of hyperlipidemia caused by ascorbic acid deficiency and of the effects of cholesterol, DGethionine and aflatoxin on plasma lipoprotein fractions of normal and scorbutic guinea pigs. The plasma lipoprotein fractions of scorbutic animals showed a significantly higher level of pre-&lipoprotein and a lower level of a-lipoprotein. By adding DL-ethionine to the control group, the pre$-lipoprotein fraction was remarkably elevated and by adding cholesterol, the ol-lipoprotein level was greatly reduced and the P-lipoprotein level was increased. Addition of aflatoxin to the control diet resulted in a rather high concentration of cu-lipoprotein and a low pre_P-lipoprotein level. High concentrations of triglyceride and phospholipid were seen in the plasma of scorbutic guinea pigs. The probable cause of hyperlipidemia induced by ascorbic acid deficiency is partly retarded degradation of cholesterol resulting from impaired ‘la-hydroxylation, and partly that ascorbic acid deficiency may affect other enzyme systems that control triglyceride of phospholipid metabqhsm, such as lipoprotein lipase activity, or synthesis or breakdown of these enzymes. Key words:
Aflcrtoxin - Ascorbic acid - Cholesterol - DL-Ethionine Lipoprotein fmctions
- Hyperlipidemia
-
Introduction Recent research on hyperlipidemia has shown a consistent association between relatively low plasma concentrations of high density lipoprotein (HDL) cholesterol and increased risk of coronary heart disease [ 1,2].
250
Some investigators have recently reported on hyperlipidemia caused by deficiency of ascorbate [ 3,4], and Bordia has described the effect of vitamin C on blood lipids in coronary artery disease [5]. Guinea pigs with ascorbic acid deficiency have significantly higher serum and liver cholesterol levels than control animals; the reason for this has been sought in the biosynthesis as well as the catabolism of cholesterol [ 41. Since ascorbic acid is an important cofactor in the hydroxylation of many substances, it is possible that it also stimulates the hydroxylation of cholesterol, causing transformation of cholesterol to bile acids in the liver, Ginter et al. [6] noted that biosynthesis of bile acids is reduced in scorbutic guinea pigs, and Guchhait et al. [ 71 found a reduction in cholesterol catabolism in scorbuticguinea pigs. If ascorbic acid affects the overall conversion of cholesterol into bile acids, it is probable that the effect is related to cholesterol 7a-hydroxylase. There are 2 possibilities regarding the effect of ascorbic acid on cholesterol 7a-hydroxylation. Ascorbic acid might be an essential factor for enzyme activity or it might affect enzyme synthesis or breakdown. Bjorkhem et al. [3] have recently observed that 7cu-hydroxylation is .markedly reduced in scorbutic animals compared with those treated with ascorbate. It has also been suggested that ascorbate affects the synthesis or breakdown of the 7a-hydroxylating system, in particular the cytochrome P-450 component. Thus, the mechanisms of hypercholesteremia caused by ascorbic acid deficiency are gradually being clarified. On the other hand, the mechanisms of non-cholesterol hyperlipidemia caused by deficiency of ascorbic acid have not been thoroughly elucidated. This experiment was designed to study electrophoretically the metabolic alteration in lipoprotein fractions of the blood plasma in normal and scorbutic guinea pigs, when fed a cholesterol-rich diet or treated with the hepatotoxic substances, DGethionine and aflatoxin. Materials and Methods Materials Forty-eight guinea pigs of the Hartley strain and a powdered diet free of ascorbic acid were obtained from Clea Japan Inc. Supplements of ascorbic acid were purchased from Iwaki Seiyaku Co., L. of Japan and supplements aflatoxin from Makor Chemicals of Jerusalem, Israel. Reagents used for the determination of cholesterol were obtained from Wako Pure Chemical Industries, L. of Japan and DGethionine and cholesterol from Sigma Chemical Company. All the chemicals and the equipment used in the lipoprotein electrophoresis were procured from Gelman Instrument Company. Animals As shown in Table 1, the animals were divided into 8 groups, 6 in each group, and were maintained on diets as described below for 3 weeks. The control group was fed the powdered diet free of ascorbic acid supplemented with 0.5% Gascorbic acid and the scorbutic group the ascorbic acid-free diet. Supplements of 0.5% cholesterol and 0.2% gall powder were added to the cholesterol-supplemented groups and 50 pg of aflatoxin B1 dissolved in dimethylsulfoxide was given orally to the animals of the aflatoxin groups every day for
251 TABLE
1
Control
-AsA -AsA
+ Cholesterol groups
diet + AsA 0.5% diet + cholesterol 0.5% + gall powder 0.290 + cholesterol 0.5% diet { + gsU powder 0.2%
Control { -AsA
+ Aflatoxin groups
Control + AF B 1 50 @g/day -AsA + AF B, 50 pg/day
+ Ethionine groups
Control + DL-ethionine 0.5% -AsA diet + DL-ethionine 0.5%
Abbreviations:
AsA = Ascorbic acid: AF = Aflatoxin.
3 weeks. In the ethionine groups, 0.5% DGethionine supplements were given for 4 days after 2 weeks of feeding on the control or ascorbic acid-deficient diet. During the experimental period, food and water were given ad libitum. Animals were kept in individual cages at constant temperature and humidity and were weighed daily. After 3 weeks, animals were killed by decapitation, and plasma obtained. Plasma cholesterol and P-lipoprotein determination Plasma total cholesterol was determined by the method &lipoprotein by the method of Fried et al. [9].
of Zak et al. [ 81 and
Plasma lipoprotein fractions obtained by electrophoresis Separated plasma was stored at l-4” C until use. Lipoprotein fractions were separated in accordance with Technical Bulletin No. 28 of the Gelman Instrument Company. Cellulose acetate membrane (Separaphore 111) was used as the sample carrier, and Tris-Barbital-Sodium Barbital buffer, pH 8.8, was used for electrophoresis. Lipoprotein complexes were separated by electrophoresis into chylomicrons, &lipoproteins, pre-/3-lipoproteins, ar-lipoproteins an4 free fatty acids. Chylomicrons remained at the application point (toward the negative pole), while free fatty acids migrated fastest and were found closest to the anode (positive pole). Other fractions were located between chylomicrons and free fatty acids in the order stated. A normal sample usually contained /3-, pre-fland a-fractions only. Electrophoresis was carried out at a constant voltage of 200 V (current 4.5-6.0 mA) for 25 min. After electrophoretic separation, the fractions were visualized by staining with Oil Red 0. The individual spots were then quantitated with a densitometer at 525 nm by a Gelman computing densitometer (DCD-16). Results As shown in Fig. 1, the body weight of ascorbic acid-deficient animals gradually decreased after 2 weeks of feeding on the respective diets, except in the cholesterol-supplemented groups whose decrement of body weight was not so severe as the other groups even at the end of 3 weeks.
252
A
4
“I
9
I
B
500
0
0
1
2
I
3
I
0
1
Week
2
3
W.?YZk
,I+
,-::-i:-:-:_:i
m
+ ethkmine
8
4 days
2OO-
2ca
0
1
2
3
Week
0
1
2
3
We&C
Fig. 1. Body weight. A: control groups: 0~ positive control, o----o -AsA control; B: plus cholesterol groups: ?? control + cholesterol + gall powder, o----o -AsA + cholesterol + gall powder; C: Plus AF groups: .control + AF. O-AsA + AF; D: plus ethionine groups: ?? _ control, O----o -AsA. Each point represents mean f SE for 6 animals in each group.
Plasma total cholesterol values are shown in Table 2. By the addition of aflatoxin or ethionine to the control diet, no significant increment could be observed in the plasma total cholesterol levels, but the addition of cholesterol to the control diet brought about a significant increase. In the scorbutic groups, TABLE
2
Plasma total cholesterol (mg/dl) c&ntrol -AsA
24.6 * 1.0 *la 65.0 f 2.3 b
Control + cholesterol -AsA + cholesterol
42.6 f 1.9 b 87.5.k 8.2 c
Control + AF -AsA + AF
31’5 f 1.4 a 46.9 i 3.0 a.b
Control + ethionine -AsA + ethionine
27.9 i 0.9 a 51.5 i 1.8b
a&c
*Mean*SE(n=6). Mew hahg same superscripts (vbaP) are not significantly different. whereas those with different superscripta (tia.a.b) are significantly different (P < 0.01).
253 TABLE 3 Plasma triglyceride and phospholipid (mg/dl) Group
Triglyceride
Phospholipid
Control -AsA
26.8 f 3.4 *,a 91.4 f 12.6 b
49.8 f 4.8 * 133.8 f 8.5 b
Notations as for Table 2.
TABLE 4 Electrophoretic
fractions of plasma lipoproteins (96)
Groups
Alpha
Pm-beta
Beta
Beta/alpha (ratio)
Control -AsA
21.4 f 6.9 *& 6.3 -k0.8 a
5.9 r 2.9 b 31.9 + 6.3 *
58.2 f 4.2 aqb 54.5 f 5.3 a.b
2.5 f. 0.7 a 13.9 i 1.5 b
1.4 f 2.4 a 5.0 f 1.0 a
6.4 k 1.2b 22.9* 1.9a
72.9 f 2.2 a 68.4 * 3.5 a
12.0 i 4.0 b 19.3 f 4.2 c
Control + cholesterol -AsA + cholesterol Control + AF -AsA + AF
34.9 2 1.5d 12.4 +_1.8 a.b
4.8 r 0.3 b 16.1 f 6.0 a.b
59.8 f 2.3 a,h 59.2 + 8.3 a.b
1.9 f 0.1 a 6.6 f 1.2 a
Control + ethionine -AsA + ethionine
20.1 i 0.9 b 12.3 + 1.0 *
38.3 + 1.7 a 24.5 * 2.5 a
33.9 + 1.5 b 56.8 f 1.7 a.b
3.5 f 0.2 a 6.7 + 0.7 b
Notations as for Tables 1 and 2.
all plasma cholesterol levels increased in comparison with the corresponding control groups. As shown in Table 3, plasma triglyceride and phospholipid levels in the scorbutic groups were very much higher than the control group. Triglyceride in the control group was 26.8 mg/dl and in the scorbutic group 91.4 mg/dl. Phospholipid in the control group was 49.8 mg/dl and in the scorbutic group 133.8 mg/dl. Electrophoretic separations of plasma lipoproteins are shown in Table 4. A significantly higher &lipoprotein to e-lipoprotein ratio was observed in the scorbutic group, i.e. 13.9 versus 2.5 in the control group. In the cholesterolsupplemented groups, an elevated level of /3-lipoprotein was noted in both the control-plus-cholesterol group and scorbutic-plus-cholesterol group. With aflatoxin supplements in the control group, a high level of o-lipoprotein and a low level of prep-lipoprotein were observed. Following 2 weeks on the control or ascorbic acid-deficient diet; 0.5% DL-ethionine was added to each diet which was then fed for 4 days. A significant increase in the pre-/l-lipoprotein level was observed in the control-plusethionine group. Discussion In this experiment, high concentrations of triglyceride and phospholipid were observed in the plasma of ascorbic acid-deficient guinea pigs. In electrophoretic fractions of plasma lipoproteins, a significantly higher level of pre_P-
254
lipoprotein and a lower level of cu-lipoprotein were noted in the plasma of ascorbic acid-deficient animals. Since preQ-lipoprotein contained a considerably higher level of triglyceride, the elevation of the pre-o-fraction must be due to the higher concentration of triglyceride in the plasma of ascorbic acid-deficient guinea pigs. The cause of hyperlipidemia in the ascorbic acid-deficient animals could partly be retarded degradation of cholesterol by impaired ‘la-hy droxylation induced by ascorbic acid deficiency. Ascorbic acid deficiency may also affect other enzymes that control triglyceride or phospholipid metabolism, such as lipoprotein lipase activity, or synthesis or breakdown of these enzymes. In order to confirm these observations, further studies on enzyme activities are in progress in our laboratory. After addition of cholesterol, the a-lipoprotein level was greatly reduced, while the &lipoprotein level was increased. This may be due to the higher concentration of cholesterol in /3-lipoprotein. By adding aflatoxin to the control diet, a rather high concentration of a-lipoprotein and a low level of preQ-lipoprotein were observed, but by adding DL-ethionine to the control group, the prep-lipoprotein fraction was remarkably elevated. Thus, by the addition of cholesterol, aflatoxin or Dcethionine, characteristic changes were noted in the electrophoretic fractions of plasma lipoprotein, but the explanation for the mechanisms of these metabolic changes in individual cases must await further investigations. References 1 Rhoads. G.G., Gulbrandsen, CL. and Kagan. A., Serum lipoprotein and coronary heart disease in a population study of Hawaii Japanese men, N. En& J. Med., 294 (1976) 293. 2 Gordon, T.. CasteRi, W.P., Hjdrtland. M.C.. Kannel. W.B. and Dawber, T.R., High density lipoprotein as a protective factor against coronary heart disease - The Framingham Study, Amer. J. Med., 62 (1977) 707. 3 Bjorkhem, I. and Kallner. A., Hepatic 7 o-hydroxylation of cholesterol in aacorbatedeficient and ascorbate+upplemented guinea pigs, J. Lipid Res., 17 (1976) 360. 4 Gmter. E.. Cholesterol-Vitamin C controls its transformation to bile acids, Science, 179 (1973) 702. 6 Bordia. A.K., The effect of vitamin C on blood lipids. fibrinolytic activity and platelet adhesiveness in patients with coronary artery disease,Atheroscleroais, 36 (1980) 181. 6 Ginter, E.. Nemec. R., Cerven. J. and Mfkus, L.. Quantification of lowered cholesterol oxidation in guinea pigs with latent vitamin C deficiency, Lipids, 8 (1973) 135. 7 Guchhait. R.. Guha. B.C. and Gsnguli. N.C.. Metabolic studies on rcorbutic guinea pigs - Catabolism of 4-14C-cholesterol in viva and in vitro, Biochem, J.. 86 (1963) 193. 8 Zak. B.. Luz. D.A. and Fisher, M.. Determination of serum cholesterol, Amer. J. Med. Technol.. 23 (1967) 283. 9 Fried, R. and Hoeflmayr, T., Eine ehrfache mdirekte Bestimmungrmethode der 6-Lipoproteide, Kim. Wschr., 41 (1963) 246.