Nutrient Requirements
and Interactions
HIROSHI K1MÃœRA, YÃœKIO YAMADA, YASÃœHIKO MORITA, HITOSH1 IKEDA* AND TAKAO MATSÃœO* Technical Research and Development Department, Vitamin and Food Division, and *Biological Research Laboratories, Research and Development Division, Takeda Chemical Ind., Ltd., Jusohonmachi 2-17-85, Yodogawa-ku, Osaka, 532, Japan With the exception of humans, other primates and guinea pigs, most mammals are able to synthesize the amount of ascorbic acid that they require and therefore cannot be used in experiments studying ascorbic acid deficiency. In 1980, a specific rat strain incapable of synthesizing ascorbic acid was developed from the Wistar rat strain. This characteristic in the ODS (Osteogenic Disorder-Shionogi) rats is controlled by a single autosomal recessive gene (Makino and Katagiri 1980). Like humans, they lack Lgulonolactone oxidase activity, which is necessary for ascorbic acid biosynthesis (Mizushima et al. 1984). Therefore, the ODS rat is a useful experimental model for investigations of ascorbic acid deficiency. We used ODS rats to assess dietary ascorbic acid requirements as related to plasma and lipoprotein lipid peroxidation.
ABSTRACT The effects of dietary ascorbic acid on plasma lipoprotein and liver lipid peroxide concentrations were examined using ODS od/od rats with a genetic defect in the ability to synthesize ascorbic acid. ODS od/ od rats were fed purified diets supplemented with 0 to 300 mg ascorbic acid/kg diet for 21 d. An ascorbic acid-free diet induced body weight loss, depleted ascorbic acid in the plasma and increased thiobarbituric acid-reactive substances in the plasma and liver as compared with rats fed ascorbic acid supplemented diets and with normal ODS +/+ rats fed the ascorbic acid-free diet. Increasing ascorbic acid concentration in the diet inhibited the development of these ascorbic acid deficiency symptoms in a dose-dependent manner. The dietary requirement of ascorbic acid to maintain normal body weight gain and plasma lipid peroxide concentra tions was -150 mg ascorbic acid/kg diet. On the other hand, even 300 mg ascorbic add/kg diet was insuffi cient to maintain a hepatic concentration of ascorbic acid comparable to that in the liver of ODS +/+ rats. The lipid peroxide concentration in plasma LDLand liver was significantly elevated in ODS od/od rats fed the ascorbic acid-free diet. Supplementing the diet with 300 mg ascorbic acid/kg kept those concentrations within the normal ranges seen in the ODS +/+ rats. J. Nutr. 122: 1904-1909, 1992.
MATERIALS AND METHODS Animals and diets. A notice of the Prime Minister's Office of Japan (No. 6 of 27 March 1980) for
INDEXING KEY WORDS:
the care and use of laboratory animals was followed. Six-week-old male ODS od/od (od/od) and ODS +/+ (+/+) rats purchased from CLEA Japan (Tokyo, Japan) were fed a standard nonpurified rat diet (CE-2, CLEA Japan) supplemented with 280 mg ascorbic acid/kg diet for 1 wk. Rats were then fed a purified diet (Table 1) prepared in accordance with Horio et al. (1985). Male ODS +/+ rats that are able to synthesize ascorbic acid were used as a control. The animals were housed individually in a room with a temperature of 23 ±1°C,humidity of 55 ±5%,
•ascorbic acid •lipid peroxides •ODS rats •lipoproteins
Ascorbic acid is a cofactor for enzymes such as prolyl hydroxylase and lysyl hydroxylase and plays an important role in the synthesis of the collagen in tissues and bone (Mclennan et al. 1988, Pinnell et al. 1987, Tsunenari et al. 1990). Recent studies have asso ciated lipid peroxidation in vivo with arterial lesions,thus considerable interest has been focused on the antioxidative effect of ascorbic acid (Fujinami 1985, Fujinami et al. 1980, Yoshikawa 1985).
and a 12-h light:dark cycle (light: 0800 to 2000 h). The animals were allowed free access to the diet and tap water. Their body weights and food intakes were measured between 0800 and 0830 h every few days.
0022-3166/92 $3.00 ©1992 American Institute of Nutrition. Received 4 February 1992. Accepted 12 May 1992. 1904
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Dietary Ascorbic Acid Depresses Plasma and Low Density Lipoprotein Lipid Peroxidation in Genetically Scorbutic Rats
ASCORBIC ACID AND LIPID PEROXIDATION
TABLE 1 Composition of the basal diet
mixture2Vitamin mixture3Choline
Analysis of lipoproteins. kept at -80°C was thawed
Plasma that had been and then centrifuged to
permit fractionation of lipoproteins according to the method of Bronzert and Brewer (1977). Two 175-uL plasma samples were placed into tubes and KBr added to one to adjust the density to 1.063 kg/L; nothing g30.05.01.00.25.04.054.20.01 was added to the other. After mixing by pipetting the samples were centrifuged at a density of 1.063 kg/L, or plasma density of 1.006 kg/L, at 222,000 x g for 2.5 h at 10°Cusing an Lp-42 Ti rotor (Beckman Instru
chlorideCottonseed oilCellulose powder4Comstarch:sucrose |2:1)Sodium seleniteAmountg/100 Casein, Dairy Board Co., Ltd., New Zealand. 2Harper mineral mixture. The basal diet contains the following (mg/kg diet): Ca, 5898; P, 3946; K, 4929; Na, 4927; Cl, 7609; Fe, 53; Mg, 492; S, 666; Zn, 5; Cu, 22; I, 0.2; Mo, 0.1. 3Harper vitamin mixture. The basal diet contains the following jmg/kg diet): retinyl acetate, 9.3; cholecalciferol, 0.06; all-rac-atocopherol acetate, 120; menadione, 0.6; thiamin HC1, 5.9; riboflavin, 5.9; pyridoxine-HCl, 2.9; vitamin B-12, 0.02; D-biotin, 0.1; folie acid, 0.2; Ca-pantothenate, 23.5; nicotinic acid, 29.4; inositol, 117.6; ascorbic acid was not included. 4Cellulose powder, type E, Toyo Roshi, Ltd. (Tokyo, Japan).
Experiment 1. ODS od/od rats were divided into four groups of five according to body weight, so that each group had approximately the same average weight (144-160 g when the experiment started). An ascorbic acid-free purified diet was given to the control group of ODS +/+ rats for 21 d. The four experimental groups were fed the purified diet supple mented with 0 (group AAO), 50 (group AA50), 150 (group AA150) or 300 (group AA300) mg ascorbic acid/kg over the same period. On the final day of the feeding period, the rats were anesthetized with diethyl ether and killed by collecting the blood from the abdominal aorta using a syringe containing sodium heparin as an anticoagulant (Rosenberg 1977). Experiment 2. ODS od/od rats were divided into two groups of five having approximately the same mean body weight. The ascorbic acid-free purified diet was fed to one group (group AAO), and the pu rified diet supplemented with 300 mg ascorbic acid/ kg was fed to the other group (group AA300) for 19 d. ODS +/+ rats fed the ascorbic acid-free purified diet were the control group. On the final day of the feeding period, blood samples were collected from the abdominal aorta under diethyl ether anesthesia. Ethylenediamine tetraacetic acid as an anticoagulant, glutathione as an antioxidant and chloramphenicol were added to the samples (Avogaro et al. 1988). The plasma obtained by centrifugation was analyzed, and a portion of the plasma was frozen at -80°C under a nitrogen at mosphere. After rats were killed by bleeding, livers were removed immediately and stored at -20°C until assayed.
1905
ments, Fullerton, CA). Plasma centrifuged at a density of 1.006 kg/L contains chylomicra and VLDL in the upper layer and LDL and HDL in the lower layer. Plasma centrifuged at a density of 1.063 kg/L contains chylomicra, VLDL and LDL in the top layer and HDL in the bottom. After removing the top frac tions in each tube with an aspirator, cholesterol, tri glycérideand thiobarbituric acid-reactive substance (TBARS) concentrations in the bottom fractions were measured. At the same time, plasma concentrations of total cholesterol, triglycéride, TBARS and ascorbic acid were assayed. The concentration of each lipoprotein fraction was calculated as follows from experimental results: CM + VLDL (density < 1.006) = X - Y LDL (1.006 < density < 1.063) = Y - Z HDLfdensity > 1.063) = Z where X = total lipoprotein (plasma) concentration; Y = LDL + HDL (bottom fraction at a density of 1.006 kg/L); and Z = HDL (bottom fraction at a density of 1.063 kg/L). Measurement of thiobarbituric acid-reactive sub stances. Levels of TBARS in plasma lipoproteins were measured according to the method of Yagi (1976). Livers that had been stored at -20°C were homoge nized with 10 volumes of an ice-cold 11.5 g/L aqueous solution of potassium chloride. The lipoperoxide level in the liver homogenate was measured by the thiobar bituric acid method of Ohkawa et al. (1979). The protein concentration of liver homogenate was deter mined by the method of Lowry et al. (1951). Measurement of ascorbic acid. Livers that had been stored at -20°C (Horio et al. 1987) were homoge nized with 20 volumes of an ice-cold 50 g/L aqueous solution of trichloroacetic acid. Plasma frozen at -80°C was thawed and analyzed immediately. The ascorbic acid concentrations in the liver and the plasma were determined by the cc,a'dipyridyl method (Zannoni et al. 1974). Measurement of cholesterol and triglycéride. Total plasma and lipoprotein concentrations of cholesterol (Allain et al. 1974) and triglycéride(Spayd et al. 1978) were measured by enzymatic colorimetrie methods (cholesterol C-test, COD-POD-4-AA method, Wako Pure Chemicals, Osaka, Japan; Cleantech TG-S, GK-GPO method, Diaiatron, Tokyo, Japan).
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ComponentCasein1Mineral
IN ODS RATS
1906
KIMURA ET AL.
579
12 DURATION
14
16
19
21
(days)
FIGURE 1 Effect of various dietary ascorbic acid levels on body weight gain in ODS rats (Experiment 1). ODS od/od rats were fed purified diets containing 0 (AAO, •), 50 (AA50, O), 150 (AA150, a) or 300 (AA300, A) mg ascorbic acid/kg diet for 3 wk. ODS +/+ rats (control, •) were fed the purified diet with no added ascorbic acid. Data are means of five rats except at 21 d. Means ±SEM not sharing a common super script letter are significantly different at P < 0.05 on d 21.
Statistical analysis. Data were evaluated by ANOVA, and subsequently by Duncan's multiplerange test (Duncan 1957). Values significantly different at P < 0.05.
were considered
RESULTS Experiment 1. In the AAO group of od/od rats, body weight began to decrease after d 9 and declined rapidly thereafter. Body weight began to decrease after d 12 in the AA50 group (Fig. 1). Body weight gain for 21 d in the AA150 and AA300 groups were equivalent and were comparable to the weight gain in the control group. Food intake in the AAO group was significantly lower than in all other groups (Table 2).
TABLE 2 Effect of dietary ascorbic acid on food intake of ODS rats fed graded levels of ascorbic acid1 Diet group2
Experiment Experiment
1 2Control19.4
±0.3a ±0.5d ±0.4CAA15017.8 19.5 ±1.1aAAO14.5 14.1 ±0.6bAA50già 16.3
±0.6bAA30019.8 ±0.7a 20.2 ±0.9a
'Values are means ±SEM(n - S}. Values with different superscript letters in a row are significantly different at P < 0.05. 2Control group was composed of ODS +/+ rats fed a purified diet with no ascorbic acid. Experimental groups were ODS od/od rats fed a purified diet with (mg ascorbic acid/kg diet): AAO, 0; AA50, 50¡AA150, 150; AA 300, 300.
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O
The plasma ascorbic acid concentration in the AAO group was not detectable. The plasma ascorbic acid concentrations in the AA50, AA150 and AA300 groups increased as the dietary ascorbic acid concen tration increased (Fig. 2, upper panel}. The plasma ascorbic acid concentration in the AA300 group was not significantly different from that in the control rats. Lipid peroxide concentrations in the plasma, as measured by TEARS concentration, in the AAO group was significantly greater than in control rats. The plasma TBARS concentrations observed in the od/od groups receiving ascorbic acid-supplemented diets decreased as the amount of ascorbic acid added to the diet increased, and that in the AA300 group was the same as that found in +/+ rats (Fig. 2, lower panel}. Experiment 2. Food intake and body weight gain in the AAO group were significantly lower than in the AA300 and control groups (Table 2, Fig. 3). The concentration of LDL-TBARS in the AAO group was significantly higher than that in the control group and the AA300 group (Table 3). The total plasma cholesterol concentration in the AAO group tended to be lower than that in the AA300 and control groups (Table 3). The cholesterol concen trations of chylomicra + VLDL in the AAO group and the AA300 group were significantly lower than in the control group (Table 3). On the other hand, the LDLcholesterol concentrations in the AAO and AA300 groups were significantly higher than that in the control. The HDL cholesterol concentration in the AAO group was significantly lower than that in the other two groups. The plasma total triglycéride concentration in the AAO group was less than one third of the control value, and, in the AA300 group, was about two thirds of the control value (Table 3). The triglycéride con centration of chylomicra + VLDL in the AAO group was less than one fourth of the control value, and, in the AA300 group, was about two thirds of the control value. The liver ascorbic acid concentration in the AAO group was very low (Fig. 4). It was higher in the AA300 group but was still lower than the concen tration found in the control group. The liver TBARS
ASCORBIC ACID AND LIPID PEROXIDATION
concentration in the AAO group was significantly higher than that in the control group and was inter mediate in the AA300 group.
Due to their hereditary lack of the L-gulonolactone oxidase activity essential to the final stage of ascorbic od/od
T
10
O ASCORBIC
O
50
150
ACID CONCENTRATION
300
(mg/kg diet)
1907
acid biosynthesis, ODS od/od rats cannot synthesize ascorbic acid (Horio et al. 1985, Makino and Katagiri 1980, Mizushima et al. 1984). Therefore, like humans they need dietary ascorbic acid for normal growth. The experiments reported here showed that the body weight of ODS od/od rats increases at nearly the same rate as that in the control rats if they are given a diet containing a minimum level of between 50 and 150 mg ascorbic acid/kg diet. However, only in ODS od/od rats fed 300 mg ascorbic acid/kg diet was the concentration of plasma ascorbic acid comparable to that in the control rats. Lipid peroxidation products in plasma, as measured by TEARS, were significantly greater in the AAO group compared with the AA150 and AA300 groups, but this increase was inhibited in a dose-dependent manner with ascorbic acid supplementation. The ad dition of 50 mg ascorbic acid/kg diet was insufficient; addition of 150-300 mg ascorbic acid/kg diet was found to be necessary to keep the peroxide concentra tions as low as those seen in the control rats in Experiment 1. Fujinami et al. (1980) recently suggested that lipid peroxides may disturb the endothelium in the blood vessels and trigger atherogenesis. The role of atherogenesis as a critical factor in arteriosclerosis has at tracted attention. Because ascorbic acid is an antioxidant, it likely suppresses lipid peroxidation and may have a suppressive action on the disturbance of the endothelium in the blood vessels (Fujinami 1985). In
od/od
< m
*2
i i a
O ASCORBIC
O
50
150
ACID CONCENTRATION
300 (mg/kg diet)
FIGURE 2 Effect of various dietary ascorbic acid levels on plasma ascorbic acid concentration (upper panel] and thiobarbituric acid-reactive substance (TEARS) concen tration (lower panel) in ODS od/od and ODS +/+ rats (Ex periment 1). Means ±SEM (n = 5) not sharing a common superscript letter are significantly different at P < 0.05. ODS od/od rats were fed a purified diet with 0, 50, 150 or 300 mg ascorbic acid/kg diet for 3 wk. ODS +/+ rats, control, were fed the purified diet with no ascorbic acid for the same period.
0
6
10
12
14
17
19
DURATION (days) FIGURE 3 Effect of dietary ascorbic acid on body weight gain in ODS rats (Experiment 2). ODS od/od rats were fed purified diets containing 0 (AAO, o) or 300 (AA300, o) mg ascorbic acid/kg diet for 19 d. ODS +/+ rats (control, •) were fed the purified diet with no ascorbic acid for the same period. Data are means of five rats except at 19 d. Means ± SEM not sharing a common superscript letter are signifi cantly different at P < 0.05 on d 19.
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DISCUSSION
IN ODS RATS
KIMURA ET AL.
1908
TABLE 3 substance (TBARS), cholesterol
and
groups2AAO2.43
\imol/LTotalChylomicra+VLDLLDLHDLCholesterol, TEARS, 0.080.772 ± 0.088a0.230 ± 0.066b1.65 ± 0.08a1122 ±
0.090.214 ± 0.063b0.869 ± 0.090a1.35 ± 0.04b807 ±
0.060.732 ± 0.089a0.374 ± 0.064b1.51 ± 0.08ab1040±
67517 49a292 75b313 18a1931
155128 61b546 109a133 56b521
93146 63b641 49a251 32a1412
\unollLTotalChylomicra+VLDLLDLHDLTriglycéride,3 ± ± ± ±
± ± ± ±
± ± ± ±
\imol/LTotalChylomicra+VLDLLDLHDLControl2.65 124a1818 ± 113*33.4± 18.176.1± ± 16.4Diet
38C391 ± 38C65.1 ±
113b1299 ± 102b40.0± 26.264.6± 14.774.8± ± 12.1AA3002.62 ± 4.1
'Values are means ±SEMfor five rats per group. Values in a row not sharing a common superscript letter are significantly
different at P
and Interactions
HIROSHI K1MÃœRA, YÃœKIO YAMADA, YASÃœHIKO MORITA, HITOSH1 IKEDA* AND TAKAO MATSÃœO* Technical Research and Development Department, Vitamin and Food Division, and *Biological Research Laboratories, Research and Development Division, Takeda Chemical Ind., Ltd., Jusohonmachi 2-17-85, Yodogawa-ku, Osaka, 532, Japan With the exception of humans, other primates and guinea pigs, most mammals are able to synthesize the amount of ascorbic acid that they require and therefore cannot be used in experiments studying ascorbic acid deficiency. In 1980, a specific rat strain incapable of synthesizing ascorbic acid was developed from the Wistar rat strain. This characteristic in the ODS (Osteogenic Disorder-Shionogi) rats is controlled by a single autosomal recessive gene (Makino and Katagiri 1980). Like humans, they lack Lgulonolactone oxidase activity, which is necessary for ascorbic acid biosynthesis (Mizushima et al. 1984). Therefore, the ODS rat is a useful experimental model for investigations of ascorbic acid deficiency. We used ODS rats to assess dietary ascorbic acid requirements as related to plasma and lipoprotein lipid peroxidation.
ABSTRACT The effects of dietary ascorbic acid on plasma lipoprotein and liver lipid peroxide concentrations were examined using ODS od/od rats with a genetic defect in the ability to synthesize ascorbic acid. ODS od/ od rats were fed purified diets supplemented with 0 to 300 mg ascorbic acid/kg diet for 21 d. An ascorbic acid-free diet induced body weight loss, depleted ascorbic acid in the plasma and increased thiobarbituric acid-reactive substances in the plasma and liver as compared with rats fed ascorbic acid supplemented diets and with normal ODS +/+ rats fed the ascorbic acid-free diet. Increasing ascorbic acid concentration in the diet inhibited the development of these ascorbic acid deficiency symptoms in a dose-dependent manner. The dietary requirement of ascorbic acid to maintain normal body weight gain and plasma lipid peroxide concentra tions was -150 mg ascorbic acid/kg diet. On the other hand, even 300 mg ascorbic add/kg diet was insuffi cient to maintain a hepatic concentration of ascorbic acid comparable to that in the liver of ODS +/+ rats. The lipid peroxide concentration in plasma LDLand liver was significantly elevated in ODS od/od rats fed the ascorbic acid-free diet. Supplementing the diet with 300 mg ascorbic acid/kg kept those concentrations within the normal ranges seen in the ODS +/+ rats. J. Nutr. 122: 1904-1909, 1992.
MATERIALS AND METHODS Animals and diets. A notice of the Prime Minister's Office of Japan (No. 6 of 27 March 1980) for
INDEXING KEY WORDS:
the care and use of laboratory animals was followed. Six-week-old male ODS od/od (od/od) and ODS +/+ (+/+) rats purchased from CLEA Japan (Tokyo, Japan) were fed a standard nonpurified rat diet (CE-2, CLEA Japan) supplemented with 280 mg ascorbic acid/kg diet for 1 wk. Rats were then fed a purified diet (Table 1) prepared in accordance with Horio et al. (1985). Male ODS +/+ rats that are able to synthesize ascorbic acid were used as a control. The animals were housed individually in a room with a temperature of 23 ±1°C,humidity of 55 ±5%,
•ascorbic acid •lipid peroxides •ODS rats •lipoproteins
Ascorbic acid is a cofactor for enzymes such as prolyl hydroxylase and lysyl hydroxylase and plays an important role in the synthesis of the collagen in tissues and bone (Mclennan et al. 1988, Pinnell et al. 1987, Tsunenari et al. 1990). Recent studies have asso ciated lipid peroxidation in vivo with arterial lesions,thus considerable interest has been focused on the antioxidative effect of ascorbic acid (Fujinami 1985, Fujinami et al. 1980, Yoshikawa 1985).
and a 12-h light:dark cycle (light: 0800 to 2000 h). The animals were allowed free access to the diet and tap water. Their body weights and food intakes were measured between 0800 and 0830 h every few days.
0022-3166/92 $3.00 ©1992 American Institute of Nutrition. Received 4 February 1992. Accepted 12 May 1992. 1904
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Dietary Ascorbic Acid Depresses Plasma and Low Density Lipoprotein Lipid Peroxidation in Genetically Scorbutic Rats
ASCORBIC ACID AND LIPID PEROXIDATION
TABLE 1 Composition of the basal diet
mixture2Vitamin mixture3Choline
Analysis of lipoproteins. kept at -80°C was thawed
Plasma that had been and then centrifuged to
permit fractionation of lipoproteins according to the method of Bronzert and Brewer (1977). Two 175-uL plasma samples were placed into tubes and KBr added to one to adjust the density to 1.063 kg/L; nothing g30.05.01.00.25.04.054.20.01 was added to the other. After mixing by pipetting the samples were centrifuged at a density of 1.063 kg/L, or plasma density of 1.006 kg/L, at 222,000 x g for 2.5 h at 10°Cusing an Lp-42 Ti rotor (Beckman Instru
chlorideCottonseed oilCellulose powder4Comstarch:sucrose |2:1)Sodium seleniteAmountg/100 Casein, Dairy Board Co., Ltd., New Zealand. 2Harper mineral mixture. The basal diet contains the following (mg/kg diet): Ca, 5898; P, 3946; K, 4929; Na, 4927; Cl, 7609; Fe, 53; Mg, 492; S, 666; Zn, 5; Cu, 22; I, 0.2; Mo, 0.1. 3Harper vitamin mixture. The basal diet contains the following jmg/kg diet): retinyl acetate, 9.3; cholecalciferol, 0.06; all-rac-atocopherol acetate, 120; menadione, 0.6; thiamin HC1, 5.9; riboflavin, 5.9; pyridoxine-HCl, 2.9; vitamin B-12, 0.02; D-biotin, 0.1; folie acid, 0.2; Ca-pantothenate, 23.5; nicotinic acid, 29.4; inositol, 117.6; ascorbic acid was not included. 4Cellulose powder, type E, Toyo Roshi, Ltd. (Tokyo, Japan).
Experiment 1. ODS od/od rats were divided into four groups of five according to body weight, so that each group had approximately the same average weight (144-160 g when the experiment started). An ascorbic acid-free purified diet was given to the control group of ODS +/+ rats for 21 d. The four experimental groups were fed the purified diet supple mented with 0 (group AAO), 50 (group AA50), 150 (group AA150) or 300 (group AA300) mg ascorbic acid/kg over the same period. On the final day of the feeding period, the rats were anesthetized with diethyl ether and killed by collecting the blood from the abdominal aorta using a syringe containing sodium heparin as an anticoagulant (Rosenberg 1977). Experiment 2. ODS od/od rats were divided into two groups of five having approximately the same mean body weight. The ascorbic acid-free purified diet was fed to one group (group AAO), and the pu rified diet supplemented with 300 mg ascorbic acid/ kg was fed to the other group (group AA300) for 19 d. ODS +/+ rats fed the ascorbic acid-free purified diet were the control group. On the final day of the feeding period, blood samples were collected from the abdominal aorta under diethyl ether anesthesia. Ethylenediamine tetraacetic acid as an anticoagulant, glutathione as an antioxidant and chloramphenicol were added to the samples (Avogaro et al. 1988). The plasma obtained by centrifugation was analyzed, and a portion of the plasma was frozen at -80°C under a nitrogen at mosphere. After rats were killed by bleeding, livers were removed immediately and stored at -20°C until assayed.
1905
ments, Fullerton, CA). Plasma centrifuged at a density of 1.006 kg/L contains chylomicra and VLDL in the upper layer and LDL and HDL in the lower layer. Plasma centrifuged at a density of 1.063 kg/L contains chylomicra, VLDL and LDL in the top layer and HDL in the bottom. After removing the top frac tions in each tube with an aspirator, cholesterol, tri glycérideand thiobarbituric acid-reactive substance (TBARS) concentrations in the bottom fractions were measured. At the same time, plasma concentrations of total cholesterol, triglycéride, TBARS and ascorbic acid were assayed. The concentration of each lipoprotein fraction was calculated as follows from experimental results: CM + VLDL (density < 1.006) = X - Y LDL (1.006 < density < 1.063) = Y - Z HDLfdensity > 1.063) = Z where X = total lipoprotein (plasma) concentration; Y = LDL + HDL (bottom fraction at a density of 1.006 kg/L); and Z = HDL (bottom fraction at a density of 1.063 kg/L). Measurement of thiobarbituric acid-reactive sub stances. Levels of TBARS in plasma lipoproteins were measured according to the method of Yagi (1976). Livers that had been stored at -20°C were homoge nized with 10 volumes of an ice-cold 11.5 g/L aqueous solution of potassium chloride. The lipoperoxide level in the liver homogenate was measured by the thiobar bituric acid method of Ohkawa et al. (1979). The protein concentration of liver homogenate was deter mined by the method of Lowry et al. (1951). Measurement of ascorbic acid. Livers that had been stored at -20°C (Horio et al. 1987) were homoge nized with 20 volumes of an ice-cold 50 g/L aqueous solution of trichloroacetic acid. Plasma frozen at -80°C was thawed and analyzed immediately. The ascorbic acid concentrations in the liver and the plasma were determined by the cc,a'dipyridyl method (Zannoni et al. 1974). Measurement of cholesterol and triglycéride. Total plasma and lipoprotein concentrations of cholesterol (Allain et al. 1974) and triglycéride(Spayd et al. 1978) were measured by enzymatic colorimetrie methods (cholesterol C-test, COD-POD-4-AA method, Wako Pure Chemicals, Osaka, Japan; Cleantech TG-S, GK-GPO method, Diaiatron, Tokyo, Japan).
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ComponentCasein1Mineral
IN ODS RATS
1906
KIMURA ET AL.
579
12 DURATION
14
16
19
21
(days)
FIGURE 1 Effect of various dietary ascorbic acid levels on body weight gain in ODS rats (Experiment 1). ODS od/od rats were fed purified diets containing 0 (AAO, •), 50 (AA50, O), 150 (AA150, a) or 300 (AA300, A) mg ascorbic acid/kg diet for 3 wk. ODS +/+ rats (control, •) were fed the purified diet with no added ascorbic acid. Data are means of five rats except at 21 d. Means ±SEM not sharing a common super script letter are significantly different at P < 0.05 on d 21.
Statistical analysis. Data were evaluated by ANOVA, and subsequently by Duncan's multiplerange test (Duncan 1957). Values significantly different at P < 0.05.
were considered
RESULTS Experiment 1. In the AAO group of od/od rats, body weight began to decrease after d 9 and declined rapidly thereafter. Body weight began to decrease after d 12 in the AA50 group (Fig. 1). Body weight gain for 21 d in the AA150 and AA300 groups were equivalent and were comparable to the weight gain in the control group. Food intake in the AAO group was significantly lower than in all other groups (Table 2).
TABLE 2 Effect of dietary ascorbic acid on food intake of ODS rats fed graded levels of ascorbic acid1 Diet group2
Experiment Experiment
1 2Control19.4
±0.3a ±0.5d ±0.4CAA15017.8 19.5 ±1.1aAAO14.5 14.1 ±0.6bAA50già 16.3
±0.6bAA30019.8 ±0.7a 20.2 ±0.9a
'Values are means ±SEM(n - S}. Values with different superscript letters in a row are significantly different at P < 0.05. 2Control group was composed of ODS +/+ rats fed a purified diet with no ascorbic acid. Experimental groups were ODS od/od rats fed a purified diet with (mg ascorbic acid/kg diet): AAO, 0; AA50, 50¡AA150, 150; AA 300, 300.
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O
The plasma ascorbic acid concentration in the AAO group was not detectable. The plasma ascorbic acid concentrations in the AA50, AA150 and AA300 groups increased as the dietary ascorbic acid concen tration increased (Fig. 2, upper panel}. The plasma ascorbic acid concentration in the AA300 group was not significantly different from that in the control rats. Lipid peroxide concentrations in the plasma, as measured by TEARS concentration, in the AAO group was significantly greater than in control rats. The plasma TBARS concentrations observed in the od/od groups receiving ascorbic acid-supplemented diets decreased as the amount of ascorbic acid added to the diet increased, and that in the AA300 group was the same as that found in +/+ rats (Fig. 2, lower panel}. Experiment 2. Food intake and body weight gain in the AAO group were significantly lower than in the AA300 and control groups (Table 2, Fig. 3). The concentration of LDL-TBARS in the AAO group was significantly higher than that in the control group and the AA300 group (Table 3). The total plasma cholesterol concentration in the AAO group tended to be lower than that in the AA300 and control groups (Table 3). The cholesterol concen trations of chylomicra + VLDL in the AAO group and the AA300 group were significantly lower than in the control group (Table 3). On the other hand, the LDLcholesterol concentrations in the AAO and AA300 groups were significantly higher than that in the control. The HDL cholesterol concentration in the AAO group was significantly lower than that in the other two groups. The plasma total triglycéride concentration in the AAO group was less than one third of the control value, and, in the AA300 group, was about two thirds of the control value (Table 3). The triglycéride con centration of chylomicra + VLDL in the AAO group was less than one fourth of the control value, and, in the AA300 group, was about two thirds of the control value. The liver ascorbic acid concentration in the AAO group was very low (Fig. 4). It was higher in the AA300 group but was still lower than the concen tration found in the control group. The liver TBARS
ASCORBIC ACID AND LIPID PEROXIDATION
concentration in the AAO group was significantly higher than that in the control group and was inter mediate in the AA300 group.
Due to their hereditary lack of the L-gulonolactone oxidase activity essential to the final stage of ascorbic od/od
T
10
O ASCORBIC
O
50
150
ACID CONCENTRATION
300
(mg/kg diet)
1907
acid biosynthesis, ODS od/od rats cannot synthesize ascorbic acid (Horio et al. 1985, Makino and Katagiri 1980, Mizushima et al. 1984). Therefore, like humans they need dietary ascorbic acid for normal growth. The experiments reported here showed that the body weight of ODS od/od rats increases at nearly the same rate as that in the control rats if they are given a diet containing a minimum level of between 50 and 150 mg ascorbic acid/kg diet. However, only in ODS od/od rats fed 300 mg ascorbic acid/kg diet was the concentration of plasma ascorbic acid comparable to that in the control rats. Lipid peroxidation products in plasma, as measured by TEARS, were significantly greater in the AAO group compared with the AA150 and AA300 groups, but this increase was inhibited in a dose-dependent manner with ascorbic acid supplementation. The ad dition of 50 mg ascorbic acid/kg diet was insufficient; addition of 150-300 mg ascorbic acid/kg diet was found to be necessary to keep the peroxide concentra tions as low as those seen in the control rats in Experiment 1. Fujinami et al. (1980) recently suggested that lipid peroxides may disturb the endothelium in the blood vessels and trigger atherogenesis. The role of atherogenesis as a critical factor in arteriosclerosis has at tracted attention. Because ascorbic acid is an antioxidant, it likely suppresses lipid peroxidation and may have a suppressive action on the disturbance of the endothelium in the blood vessels (Fujinami 1985). In
od/od
< m
*2
i i a
O ASCORBIC
O
50
150
ACID CONCENTRATION
300 (mg/kg diet)
FIGURE 2 Effect of various dietary ascorbic acid levels on plasma ascorbic acid concentration (upper panel] and thiobarbituric acid-reactive substance (TEARS) concen tration (lower panel) in ODS od/od and ODS +/+ rats (Ex periment 1). Means ±SEM (n = 5) not sharing a common superscript letter are significantly different at P < 0.05. ODS od/od rats were fed a purified diet with 0, 50, 150 or 300 mg ascorbic acid/kg diet for 3 wk. ODS +/+ rats, control, were fed the purified diet with no ascorbic acid for the same period.
0
6
10
12
14
17
19
DURATION (days) FIGURE 3 Effect of dietary ascorbic acid on body weight gain in ODS rats (Experiment 2). ODS od/od rats were fed purified diets containing 0 (AAO, o) or 300 (AA300, o) mg ascorbic acid/kg diet for 19 d. ODS +/+ rats (control, •) were fed the purified diet with no ascorbic acid for the same period. Data are means of five rats except at 19 d. Means ± SEM not sharing a common superscript letter are signifi cantly different at P < 0.05 on d 19.
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DISCUSSION
IN ODS RATS
KIMURA ET AL.
1908
TABLE 3 substance (TBARS), cholesterol
and
groups2AAO2.43
\imol/LTotalChylomicra+VLDLLDLHDLCholesterol, TEARS, 0.080.772 ± 0.088a0.230 ± 0.066b1.65 ± 0.08a1122 ±
0.090.214 ± 0.063b0.869 ± 0.090a1.35 ± 0.04b807 ±
0.060.732 ± 0.089a0.374 ± 0.064b1.51 ± 0.08ab1040±
67517 49a292 75b313 18a1931
155128 61b546 109a133 56b521
93146 63b641 49a251 32a1412
\unollLTotalChylomicra+VLDLLDLHDLTriglycéride,3 ± ± ± ±
± ± ± ±
± ± ± ±
\imol/LTotalChylomicra+VLDLLDLHDLControl2.65 124a1818 ± 113*33.4± 18.176.1± ± 16.4Diet
38C391 ± 38C65.1 ±
113b1299 ± 102b40.0± 26.264.6± 14.774.8± ± 12.1AA3002.62 ± 4.1
'Values are means ±SEMfor five rats per group. Values in a row not sharing a common superscript letter are significantly
different at P
