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Influence of peanut oil of different origins on lipid metabolism in rats
NUTRITION RESEARCH, Vol. 10, pp. 997-1004,1990 0271:5317/90 $3.00 + .00 Printed in the USA. Copyright (c) 1990 Pergamon Press plc. All rights reserved.
INFLUENCE OF PEANUT OIL OF DIFFERENT ORIGINS ON LIPID METABOLISM IN RATS David Kritchevsky, PhD 1., Shirley A. Tepper 1, Elaina Ryder, MS 1, Larry M. Davidson 1, Josephine Miller, PhD 2, R. E. Worthington, PhD 2, and David M. Klurfeld, PhD 1 1Wistar Institute 'of Anatomy and Biology, Philadelphia, PA 19104 2Department of Food Science, University of Georgia, Agriculture Experiment Station, Experiment, GA 30212
ABSTRACT Mechanism(s) underlying the unexpected atherogenicity of peanut oil are obscure. Triglyceride structure a n d / o r composition may contribute to atherogenicity. The studies reported here were designed to explore further effects on lipid metabolism. We have compared in rats the effects on lipid metabolism of: corn oil (CO), commercial peanut oil (PNO), randomized PNO (PNOR), and oils prepared from two specific cultivars of peanut, India White (IW) and Jenkins Jumbo (JJ). The oils (15%) were fed for 3 weeks in a semipurified diet. There were no differences in weight gain, liver weight, or serum lipids. Liver cholesterol levels were not significantly different, but liver triglycerides were lower in rats fed IW or JJ. Feces were collected at 10 and 20 days. Fecal fat (%) was similar in all groups. All rats excreted increasing amounts of 16:0 and 18:0, but less 18:1 and 18:2, suggesting conservation of the more unsaturated fatty acids. Fecal excretion of neutral steroids was similar in the five dietary groups, but acidic steroid excretion was lower in rats fed CO and PNO than in the other groups. Triglyceride content of the intestinal mucosa differed significantly, being (mg/g): CO - 10.8 + 1.7; PNO - 19.15 + 1.6; PNOR - 10.5 + 1.8; IW - 17.6 + 1.8; and JJ 14.6 + 0.9. In every group, mucosal 18:1/18:2 was higher than that of CO fed. Retention of triglycerides in the mucosa may be related to atherogenic effects. KEY WORDS: Fatty acids, gut mucosal lipids, liver lipids, oleic/linoleic acid ratio, peanut oil, serum lipids.
* To whom correspondence and reprint requests should be addressed: David Kritchevsky, Wistar Institute of Anatomy and Biology, 3601 Spruce St., Philadelphia, PA, USA. 997
998
D. KRITCHEVSKY et al.
INTRODUCTION Peanut oil (PNO) has been shown to be unexpectedly atherogenic in rats (1), rabbits (2) and monkeys (3). Comparison of the atherogenic effects of peanut oils from North America, South America and Africa showed the South American oil (which had the lowest 18:1/18:2 ratio) to be the most atherogenic for rabbits fed cholesterol (4). In an effort to investigate further the possibility that the 18:1/18:2 ratio may affect lipid metabolism, we compared the effects on lipid metabolism in rats of corn oil (CO) (18:1/18:2 = 0.45), PNO (18:1/18:2 = 1.55), and oils prepared from two special cultivars of peanut. These oils are designated as India White (IW) (18:1/18:2 = 1.02) and Jenkins Jumbo (JJ) (18:1/18:2 = 3.06). Randomized peanut oil (PNOR), which has the same fatty acid composition as PNO but different triglyceride structure, has been shown not to be atherogenic for rabbits (5). This oil was also fed to a group of rats to determine if its effects differed from those of native peanut oil. Our findings are the basis of this report. METHODS AND MATERIALS Male Wistar rats were randomized into groups of six rats each. The average starting weight of the groups was 182 + 0.3 g. Rats were kept in individual cages in an air-conditioned (21~ room maintained on a 12-hour light-dark cycle. The rats were fed a semipurified diet containing 15% fat (Table 1). The diet provided 411 kcal/100 g. The fats used were CO, PNO, PNOR, IW and JJ. Food and water were provided ad libitum. The diets were mixed to our specifications and pelleted by Dyets, Inc. (Bethlehem, PA). The fatty acid spectra of the fats used are detailed in Table 2. TABLE 1 Composition of Diet Ingredient*
%
% of Calories
Fat Casein Sucrose Cellulose Mineral Mix (AIN-76) Vitamin Mix (AIN-76A)
15 20 49 10 5 1
32.8 19.5 47.7
*Diet provides 411 kcal/100 g. Feces from each rat were collected after 10 and 20 days on the diets. Each individual sample was lyophilized, powdered and extracted with acidified chloroform-methanol (2:1). The extracted fat was dried over anhydrous sodium sulfate, transesterified and the methyl esters subjected to gas chromatography. The fatty acid methyl esters were analyzed using a temperature programmed (175-2050C) Hewlett-Packard Model 5830A gas chromatograph with a six-foot column of 10% SP2300 on 80/100 Supelcoport (Supelco, Inc., Bellefont, PA). Fatty acids were identified and quantitated by comparison with authentic standards (Nu Chek Prep, Inc., Elysian, MN).
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TABLE 2 Fatty Acid Composition of Oils Fatty Acid 14:0 16:0 16:1 18:0 18:1 18:2 18:3 20:0 20:1 20:4 22:0 24:0 Other
CO 10.5
26.8 59.6 1.0 0.4 0.2 0.1 0.3 1.1
PNO
PNO-IW
PNO-JJ
0.1 10.8 0.2 2.3 46.8 30.2
11.4 0.1 4.3 37.6 37.0
7.8 0.1 4.2 60.9 19.9
2.0 1.7
2.4 1.0
2.0 1.0
3.3 1.9 0.7
4.2 1.5 0.5
2.6 1.1 0.4
*CO, corn oil; PNO, peanut oil; PNOR, randomized peanut oil; IW, India White; JJ, Jenkins Jumbo. Aliquots (200 mg) of pooled feces from each group (1200 mg total) were saponified for 3 hours at 70~ in 10 ml of 1N KOH in 50% ethyl alcohol. The neutral steroids were extracted with petroleum ether (4 x 10 ml), and the extracts were dried over anh.-Na2SO4 and taken to dryness. The weighed residue was designated as neutral steroid. The aqueous residue from the petroleum ether extraction was acidified to pH 3 with conc. HC1. The acidified solution was extracted with ethyl ether (4 x 10 ml), and the residue remaining after evaporation of the ether was designated acidic steroid. At 21 days the rats were fasted for 18 hours, then killed by barbiturate injection. Serum and liver were extracted (6) and analyzed for total cholesterol (7) and triglyceride (8). Aliquots of liver extracts from each rat in each group were pooled, transesterified, and the fatty acid methyl esters analyzed by gas chromatography. Mucosa from a weighed strip of small intestine from each rat were scraped free, extracted (6) and analyzed for individual triglyceride content (8) and fatty acid spectrum. RESULTS The necropsy results are presented in Table 3. Weight gain and liver weight were similar for all five groups. Serum cholesterol and triglycerides were also similar. Liver cholesterol levels were slightly, but not significantly, elevated in rats fed PNOR. Liver triglycerides of rats fed IW or JJ were significantly (p < 0.05) lower than those of rats fed CO or PNO. Liver triglycerides of rats fed PNOR were lower than those of rats fed CO or PNO by 26 and 31%, respectively.
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TABLE 3 N e c r o p s y Data (:t: SEM) of Rats Fed Various Oils (6/group)
Oil* CO
PNO
Weight gain, g 89_+ 3 Liver weight, g 7.9-+ 0.2 Relative liver weight 2.92+ 0.05
79• 7.4•
4 0.3
2.81 •
0.11
PNOR 86+ 4 7.9_+ 0.3
IW 88 + 4 7.6 _+ 0.4
2.94_+ 0.12 2.82 _+ 0.09
Serum (mg/dl) Cholesterol Triglycerides
63_+ 3 175• 5
61+ 7 18I+ 7
Liver (g/100 g) Cholesterol Triglycerides
0.48_+ 0.04 2.64_+ 0.34ac
0.49+ 0.05 0.55_+ 0.06 2.86_+ 0.58bd 1.96 _+ 0.56
67_+ 5 175• 4
60 + 5 176 • 5
]J 79 -+ 9 7.1 • 0.3 2.75 _+ 0.19
70 -+ 4 180 _+ 8
0.44 _+ 0.04 0.47 _+ 0.06 1.20 _+ 0.26ab 0.75 _+ 0.20cd
Values bearing s a m e letter are significantly (p < 0.05) different. *See footnote, Table 2.
Liver fatty acid spectra are given in Table 4. Levels of palm• a n d stearic acids w e r e similar in all five groups. Oleic acid levels w e r e lowest in livers of rats fed CO or IW. Linoleic acid was highest in the livers of rats fed corn oil.
TABLE 4 Liver Fatty Acids in Rats Fed Various Oils* Oil Fatty acid (%) 14:0 16:0 18:0 18:1 18:2 18:3 20:0 22:0 Other
CO
PNO
PNOR
IW
JJ
9.8 23.8 17.6 12.2 25.9 0.5
trace 27.3 21.6 29.3 15.6 trace
trace 23.3 20.2 26.9 17.3 trace
trace 27.8 25.8 17.4 19.3 0.5
trace 24.4 25.5 25.3 12.9 0.4
0.4 9.9
6.3
12.3
trace 9.3
trace 11.5
*See footnote, Table 2.
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The total fecal fat levels (determined gravimetrically) are presented in Table 5. There were no significant differences in fat excretion after 10 days on the various regimens. At 20 days rats fed peanut oil excreted significantly more fat than those fed CO, but other differences were not significant. There were no significant differences in fat excretion between days 10 and 20 in any group. TABLE 5 Fecal Fat ( m g / g ) in Rats Fed Various Oils Sample Group*
10 Day
CO PNO PNOR IW JJ
48.3 44.7 48.2 53.9 46.5
+ 2.0 • 3.0 • 5.0 • 13.0 + 6.0
20 Day 42.5 54.0 42.6 47.2 46.7
• 2.3 :t: 3.0 • 4.7 + 3.4 _+ 4.3
*See footnote, Table 2. Tables 6 and 7 detail the fatty acid spectra of the fecal samples collected on days 10 and 20. At 10 days palmitic acid was significantly lower in group JJ compared to all other groups. Stearic acid was lower in groups PNO, PNOR and IW than in groups CO and JJ. Excretion of oleic acid was highest and of linoleic acid lowest in group CO. At 20 days palmitic acid excretion was highest in groups P N O and IW and stearic and oleic acids lowest. Linoleic acid excretion was highest in g r o u p CO and linolenic excretion lowest. In all groups palmitic and stearic acid excretion rose, and o!eic and linoleic excretion fell between days 10 and 20. TABLE 6 Fecal Fatty Acids (% • SEM) in Rats Fed Various Oils (10 Day) Group* Fatty acid 14:0 16:0 18:0 18:1 18:2 18:3 20:0 22:0 22:1 24:0
0.2 26.4 21.3 35.2 12.1 2.2
CO • • + • + •
0.1 0.9 1.5 2.5 1.4 0.5
1.0 + 0.2 1.8 + 0.4
*See footnote, Table 2.
PNO trace 25.2 + 1.5 14.7 + 1.0 27.5 • 2.5 3.7 • 0.3 6.3 • 0.3
PNOR trace 21.2 + 0.5 17.0 + 1.7 33.2 + 3.2 2.2 • 0.6 7.1 + 1.3
IW trace 28.2 + 1.1 16.3 + 0.7 29.6 + 1.8 5.7 • 0.9 6.1 • 0.6
JJ trace 12.8 + 4.1 24.1 + 4.3 33.3 • 9.0 1.3 • 0.4 9.1 • 1.4
14.0 • 1.5 0.3 + 0.1 8.4 • 1.8
11.9 + 1.1 0.6 + 0.1 6.8 • 1.6
10.3 • 1.1 trace 3.7 • 0.6
12.1 • 2.7 trace 6.3 • 1.5
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D. KRITCHEVSKY et al.
TABLE 7 Fecal Fatty Acids (% • SEM) in Rats Fed Various Oils (20 Days) Group* Fatty Acid 14:0 16:0 18:0 18:1 18:2 18:3 20:0 22:0 22:1 24:0
(30 0.3 24.7 33.8 22.8 11.6 2.8
• + • • • •
PNO 0.2 5.2 7.1 4.7 1.4 0.4
-
1.3 • 0.4 trace 2.7 • 0.9
0.3 45.4 20.1 10.8 0.2 7.6
• + + • • +
0.2 8.2 4~ 3.7 0.1 0.8 0.7 • 0.4 9.7 • 2.4 5.2 + 1.4
PNOR 0.1 23.0 24.9 21.4 0.2 6.2 1.9 13.5 0.2 8.7
+ • • • • + • + + •
0.1 0.6 1.8 2.1 0.1 0.3 0.6 1.1 0.1 0.9
IW
JJ
0.1 + 0.1 34.6 + 0.9 22.5 • 1.3 14.1 • 1.5 trace 8.0 + 0.5 2.0 + 0.4 13.5 • 0.9
trace 22.4 + 1.1 30.1 + 1.4 19.0 • 2.6 0.1 + 0.1 8.7 • 0.5 1.8 + 0.4 11.9 • 0.9
5.2 •
0.4
6.2 •
0.6
*See footnote, Table 2.
The fecal steroid excretion data are s u m m a r i z e d in Table 8. Neutral steroid excretion was similar for all five dietary groups. Acidic steroid excretion was similar in the rats fed intact peanut oil of whatever origin (PNO, IW, JJ). Acidic steroid excretion in rats fed PNOR was reduced by about a third and reduced even further in rats fed CO. There appears to be no relation between steroid excretion and fatty acid composition of the fat. TABLE 8 Fecal Neutral and Acidic Steroids in Rats Fed Various Oils Fecal steroids (mg/g) Group*
Neutral
Acidic
(30 PNO PNOR IW
21 18 25 23 21
105 255 180 265 241
*See footnote, Table 2. The data on triglyceride levels of gut mucosa and their fatty acid spectra are given in Table 9. Rats fed CO or PNOR had the lowest levels of mucosal triglycerides. The highest levels of mucosal triglycerides were f o u n d in rats fed PNO or IW. Mucosal triglycerides in g r o u p PNO were significantly higher than those in rats fed PNOR, JJ or CO. Rats fed IW had significantly higher mucosal triglycerides than those fed PNOR or CO.
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TABLE 9 Gut Mucosal Triglycerides ( m g / g • SEM) and Fatty Acids (% • SEM) in Rats Fed Various Oils Group* CO
PNO
PNOR
IW
JJ
Triglycerides
10.8 + 1.7
19.5 • 1.6
10.5 • 1.8
17.6 • 1.8
14.6 + 1.1
Fatty Acids (%) 14:0 16:0 18:0 18:1 18:2 18:3 20:0 22:0 Other
44.6 17.2 6.8 10.9 16.8 1.0 0.4 0.3 4.1
45.4 + 2.7 16.4 + 2.8 6.1 + 0.4 17.0 + 1.2 10.4 • 5.9 0.3 • 0.1 0.1 • 0.1 trace 4.1 • 0.4
45.7 + 4.9 16.7 + 2.8 7.2 • 1.0 15.6 + 0.9 9.7 • 0.5 0.3 + 0.2 0.1 + 0.1 trace 5.3 • 0.4
39.4 16.8 8.2 15.9 13.7 0.4
1.7 1.6 0.6 2.0 1.1 0.2
35.2 + 4.4 10.1 + 3.3 11.9 + 1.1 24.0 + 5.3 9.6 + 1.7 trace
0,2 0.5
trace 9.4 • 0.9
+ + + • • + • + •
1.8 1.8 0.6 1.3 1.6 0.2 0.1 0.1 0.9
+ + • • • • 0.4 • 5.5 •
*See footnote, Table 2.
The two groups of rats with lowest levels of mucosal triglycerides excreted the least a m o u n t of acidic steroid, but fecal acidic steroid excretion in the four PNO-fed groups was fairly consistent (235 + 19 m g / g ) compared to that observed in the rats fed CO. DISCUSSION One of the aims of this s t u d y was to determine if the 18:1/18:2 ratio of various p e a n u t oils could affect lipid metabolism in rats. For comparison, we also fed rats CO and PNOR which has the same 18:1/18:2 ratio as PNOR but different triglyceride structure. The only significant effect observed on necropsy was the lower liver triglycerides in rats fed PNOR, IW or JJ. Gut mucosal triglycerides were lowest in rats fed CO or PNOR. In Table 10 w e have compared 18:1/18:2 ratios of liver, gut and feces with the ratio observed in the dietary fat. In general, liver 18:1/18:2 ratios resemble those of the fat. One might a d d u c e a similar conservation of fatty acid ratio in the gut mucosa albeit there are elevations of 44, 87, and 34% in groups CO, IW and JJ compared with the starting levels. In the feces there is a m a r k e d elevation of 18:1/18:2 levels suggesting conservation of linoleic acid or that it might be a substrate of choice for metabolism of the intestinal flora. The major differences between groups were observed in the liver and mucosal triglycerides. Retention of triglycerides in the mucosa m a y be related to the atherogenic effects of certain fats. This possibility remains to be tested.
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D. KRITCHEVSKY et al.
TABLE 10 Oleic/Linoleic Acid Ratios in Rats Fed Various Oils Group* Site
(30
PNO
PNOR
IW
JJ
Diet Liver Gut Feces - 10D Feces - 20D
0.45 0.47 0.65 2.9 2.0
1.55 1.88 1.63 7.4 5.4
1.55 1.55 1.61 15.1 107
1.02 0.90 1.16 5.2 1410
3.06 1.96 2.50 25.6 271
*See footnote, Table 2. ACKNOWLEDGEMENTS Supported, in part, by a grant (HL03299) and a Research Career Award (HL00734) from the National Institutes of Health, grant CRCR-1-1068 from USDASEA, and by funds from the Commonwealth of Pennsylvania. REFERENCES 1.
Gresham GA, Howard AN. The independent production of atherosclerosis and thrombosis in the rat. Brit J Exp Pathol 1960; 41:359-402.
2.
Kritchevsky D, Tepper SA, Vesselinovitch D, Wissler RW. Cholesterol vehicle in experimental atherosclerosis 11. Peanut oil. Atherosclerosis 1971; 14:53-64.
.
Vesselinovitch D, Getz GS, Hughes RH, Wissler RW. Atherosclerosis in the rhesus monkey fed three food fats. Atherosclerosis 1974; 20:303-21.
4.
Kritchevsky D, Tepper SA, Scott DA, Klurfeld DM, Vesselinovitch D, Wissler RW. Cholesterol vehicle in experimental atherosclerosis 18. Comparison of North American, African and South American peanut oils. Atherosclerosis 1981; 38:291-9.
.
Kritchevsky D, Tepper SA, Vesselinovitch D, Wissler RW. Cholesterol vehicle in experimental atherosclerosis 13. Randomized peanut oil. Atherosclerosis 1973; 17:22543.
.
Folch J, Lees M, Sloane-Stanley GH. A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 1957; 226:497-509.
.
Rudel LL, Morris MD. Determination of cholesterol using o-phthalaldehyde. J. Lipid Res 1973; 14:364-6.
8.
Levy AL, Keyloun C. Measurement of triglycerides using nonane extraction and colorimetry. Adv Automated Anal 1972; 1:487-502. Accepted for publication April 4, 1990.
INFLUENCE OF PEANUT OIL OF DIFFERENT ORIGINS ON LIPID METABOLISM IN RATS David Kritchevsky, PhD 1., Shirley A. Tepper 1, Elaina Ryder, MS 1, Larry M. Davidson 1, Josephine Miller, PhD 2, R. E. Worthington, PhD 2, and David M. Klurfeld, PhD 1 1Wistar Institute 'of Anatomy and Biology, Philadelphia, PA 19104 2Department of Food Science, University of Georgia, Agriculture Experiment Station, Experiment, GA 30212
ABSTRACT Mechanism(s) underlying the unexpected atherogenicity of peanut oil are obscure. Triglyceride structure a n d / o r composition may contribute to atherogenicity. The studies reported here were designed to explore further effects on lipid metabolism. We have compared in rats the effects on lipid metabolism of: corn oil (CO), commercial peanut oil (PNO), randomized PNO (PNOR), and oils prepared from two specific cultivars of peanut, India White (IW) and Jenkins Jumbo (JJ). The oils (15%) were fed for 3 weeks in a semipurified diet. There were no differences in weight gain, liver weight, or serum lipids. Liver cholesterol levels were not significantly different, but liver triglycerides were lower in rats fed IW or JJ. Feces were collected at 10 and 20 days. Fecal fat (%) was similar in all groups. All rats excreted increasing amounts of 16:0 and 18:0, but less 18:1 and 18:2, suggesting conservation of the more unsaturated fatty acids. Fecal excretion of neutral steroids was similar in the five dietary groups, but acidic steroid excretion was lower in rats fed CO and PNO than in the other groups. Triglyceride content of the intestinal mucosa differed significantly, being (mg/g): CO - 10.8 + 1.7; PNO - 19.15 + 1.6; PNOR - 10.5 + 1.8; IW - 17.6 + 1.8; and JJ 14.6 + 0.9. In every group, mucosal 18:1/18:2 was higher than that of CO fed. Retention of triglycerides in the mucosa may be related to atherogenic effects. KEY WORDS: Fatty acids, gut mucosal lipids, liver lipids, oleic/linoleic acid ratio, peanut oil, serum lipids.
* To whom correspondence and reprint requests should be addressed: David Kritchevsky, Wistar Institute of Anatomy and Biology, 3601 Spruce St., Philadelphia, PA, USA. 997
998
D. KRITCHEVSKY et al.
INTRODUCTION Peanut oil (PNO) has been shown to be unexpectedly atherogenic in rats (1), rabbits (2) and monkeys (3). Comparison of the atherogenic effects of peanut oils from North America, South America and Africa showed the South American oil (which had the lowest 18:1/18:2 ratio) to be the most atherogenic for rabbits fed cholesterol (4). In an effort to investigate further the possibility that the 18:1/18:2 ratio may affect lipid metabolism, we compared the effects on lipid metabolism in rats of corn oil (CO) (18:1/18:2 = 0.45), PNO (18:1/18:2 = 1.55), and oils prepared from two special cultivars of peanut. These oils are designated as India White (IW) (18:1/18:2 = 1.02) and Jenkins Jumbo (JJ) (18:1/18:2 = 3.06). Randomized peanut oil (PNOR), which has the same fatty acid composition as PNO but different triglyceride structure, has been shown not to be atherogenic for rabbits (5). This oil was also fed to a group of rats to determine if its effects differed from those of native peanut oil. Our findings are the basis of this report. METHODS AND MATERIALS Male Wistar rats were randomized into groups of six rats each. The average starting weight of the groups was 182 + 0.3 g. Rats were kept in individual cages in an air-conditioned (21~ room maintained on a 12-hour light-dark cycle. The rats were fed a semipurified diet containing 15% fat (Table 1). The diet provided 411 kcal/100 g. The fats used were CO, PNO, PNOR, IW and JJ. Food and water were provided ad libitum. The diets were mixed to our specifications and pelleted by Dyets, Inc. (Bethlehem, PA). The fatty acid spectra of the fats used are detailed in Table 2. TABLE 1 Composition of Diet Ingredient*
%
% of Calories
Fat Casein Sucrose Cellulose Mineral Mix (AIN-76) Vitamin Mix (AIN-76A)
15 20 49 10 5 1
32.8 19.5 47.7
*Diet provides 411 kcal/100 g. Feces from each rat were collected after 10 and 20 days on the diets. Each individual sample was lyophilized, powdered and extracted with acidified chloroform-methanol (2:1). The extracted fat was dried over anhydrous sodium sulfate, transesterified and the methyl esters subjected to gas chromatography. The fatty acid methyl esters were analyzed using a temperature programmed (175-2050C) Hewlett-Packard Model 5830A gas chromatograph with a six-foot column of 10% SP2300 on 80/100 Supelcoport (Supelco, Inc., Bellefont, PA). Fatty acids were identified and quantitated by comparison with authentic standards (Nu Chek Prep, Inc., Elysian, MN).
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TABLE 2 Fatty Acid Composition of Oils Fatty Acid 14:0 16:0 16:1 18:0 18:1 18:2 18:3 20:0 20:1 20:4 22:0 24:0 Other
CO 10.5
26.8 59.6 1.0 0.4 0.2 0.1 0.3 1.1
PNO
PNO-IW
PNO-JJ
0.1 10.8 0.2 2.3 46.8 30.2
11.4 0.1 4.3 37.6 37.0
7.8 0.1 4.2 60.9 19.9
2.0 1.7
2.4 1.0
2.0 1.0
3.3 1.9 0.7
4.2 1.5 0.5
2.6 1.1 0.4
*CO, corn oil; PNO, peanut oil; PNOR, randomized peanut oil; IW, India White; JJ, Jenkins Jumbo. Aliquots (200 mg) of pooled feces from each group (1200 mg total) were saponified for 3 hours at 70~ in 10 ml of 1N KOH in 50% ethyl alcohol. The neutral steroids were extracted with petroleum ether (4 x 10 ml), and the extracts were dried over anh.-Na2SO4 and taken to dryness. The weighed residue was designated as neutral steroid. The aqueous residue from the petroleum ether extraction was acidified to pH 3 with conc. HC1. The acidified solution was extracted with ethyl ether (4 x 10 ml), and the residue remaining after evaporation of the ether was designated acidic steroid. At 21 days the rats were fasted for 18 hours, then killed by barbiturate injection. Serum and liver were extracted (6) and analyzed for total cholesterol (7) and triglyceride (8). Aliquots of liver extracts from each rat in each group were pooled, transesterified, and the fatty acid methyl esters analyzed by gas chromatography. Mucosa from a weighed strip of small intestine from each rat were scraped free, extracted (6) and analyzed for individual triglyceride content (8) and fatty acid spectrum. RESULTS The necropsy results are presented in Table 3. Weight gain and liver weight were similar for all five groups. Serum cholesterol and triglycerides were also similar. Liver cholesterol levels were slightly, but not significantly, elevated in rats fed PNOR. Liver triglycerides of rats fed IW or JJ were significantly (p < 0.05) lower than those of rats fed CO or PNO. Liver triglycerides of rats fed PNOR were lower than those of rats fed CO or PNO by 26 and 31%, respectively.
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TABLE 3 N e c r o p s y Data (:t: SEM) of Rats Fed Various Oils (6/group)
Oil* CO
PNO
Weight gain, g 89_+ 3 Liver weight, g 7.9-+ 0.2 Relative liver weight 2.92+ 0.05
79• 7.4•
4 0.3
2.81 •
0.11
PNOR 86+ 4 7.9_+ 0.3
IW 88 + 4 7.6 _+ 0.4
2.94_+ 0.12 2.82 _+ 0.09
Serum (mg/dl) Cholesterol Triglycerides
63_+ 3 175• 5
61+ 7 18I+ 7
Liver (g/100 g) Cholesterol Triglycerides
0.48_+ 0.04 2.64_+ 0.34ac
0.49+ 0.05 0.55_+ 0.06 2.86_+ 0.58bd 1.96 _+ 0.56
67_+ 5 175• 4
60 + 5 176 • 5
]J 79 -+ 9 7.1 • 0.3 2.75 _+ 0.19
70 -+ 4 180 _+ 8
0.44 _+ 0.04 0.47 _+ 0.06 1.20 _+ 0.26ab 0.75 _+ 0.20cd
Values bearing s a m e letter are significantly (p < 0.05) different. *See footnote, Table 2.
Liver fatty acid spectra are given in Table 4. Levels of palm• a n d stearic acids w e r e similar in all five groups. Oleic acid levels w e r e lowest in livers of rats fed CO or IW. Linoleic acid was highest in the livers of rats fed corn oil.
TABLE 4 Liver Fatty Acids in Rats Fed Various Oils* Oil Fatty acid (%) 14:0 16:0 18:0 18:1 18:2 18:3 20:0 22:0 Other
CO
PNO
PNOR
IW
JJ
9.8 23.8 17.6 12.2 25.9 0.5
trace 27.3 21.6 29.3 15.6 trace
trace 23.3 20.2 26.9 17.3 trace
trace 27.8 25.8 17.4 19.3 0.5
trace 24.4 25.5 25.3 12.9 0.4
0.4 9.9
6.3
12.3
trace 9.3
trace 11.5
*See footnote, Table 2.
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The total fecal fat levels (determined gravimetrically) are presented in Table 5. There were no significant differences in fat excretion after 10 days on the various regimens. At 20 days rats fed peanut oil excreted significantly more fat than those fed CO, but other differences were not significant. There were no significant differences in fat excretion between days 10 and 20 in any group. TABLE 5 Fecal Fat ( m g / g ) in Rats Fed Various Oils Sample Group*
10 Day
CO PNO PNOR IW JJ
48.3 44.7 48.2 53.9 46.5
+ 2.0 • 3.0 • 5.0 • 13.0 + 6.0
20 Day 42.5 54.0 42.6 47.2 46.7
• 2.3 :t: 3.0 • 4.7 + 3.4 _+ 4.3
*See footnote, Table 2. Tables 6 and 7 detail the fatty acid spectra of the fecal samples collected on days 10 and 20. At 10 days palmitic acid was significantly lower in group JJ compared to all other groups. Stearic acid was lower in groups PNO, PNOR and IW than in groups CO and JJ. Excretion of oleic acid was highest and of linoleic acid lowest in group CO. At 20 days palmitic acid excretion was highest in groups P N O and IW and stearic and oleic acids lowest. Linoleic acid excretion was highest in g r o u p CO and linolenic excretion lowest. In all groups palmitic and stearic acid excretion rose, and o!eic and linoleic excretion fell between days 10 and 20. TABLE 6 Fecal Fatty Acids (% • SEM) in Rats Fed Various Oils (10 Day) Group* Fatty acid 14:0 16:0 18:0 18:1 18:2 18:3 20:0 22:0 22:1 24:0
0.2 26.4 21.3 35.2 12.1 2.2
CO • • + • + •
0.1 0.9 1.5 2.5 1.4 0.5
1.0 + 0.2 1.8 + 0.4
*See footnote, Table 2.
PNO trace 25.2 + 1.5 14.7 + 1.0 27.5 • 2.5 3.7 • 0.3 6.3 • 0.3
PNOR trace 21.2 + 0.5 17.0 + 1.7 33.2 + 3.2 2.2 • 0.6 7.1 + 1.3
IW trace 28.2 + 1.1 16.3 + 0.7 29.6 + 1.8 5.7 • 0.9 6.1 • 0.6
JJ trace 12.8 + 4.1 24.1 + 4.3 33.3 • 9.0 1.3 • 0.4 9.1 • 1.4
14.0 • 1.5 0.3 + 0.1 8.4 • 1.8
11.9 + 1.1 0.6 + 0.1 6.8 • 1.6
10.3 • 1.1 trace 3.7 • 0.6
12.1 • 2.7 trace 6.3 • 1.5
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D. KRITCHEVSKY et al.
TABLE 7 Fecal Fatty Acids (% • SEM) in Rats Fed Various Oils (20 Days) Group* Fatty Acid 14:0 16:0 18:0 18:1 18:2 18:3 20:0 22:0 22:1 24:0
(30 0.3 24.7 33.8 22.8 11.6 2.8
• + • • • •
PNO 0.2 5.2 7.1 4.7 1.4 0.4
-
1.3 • 0.4 trace 2.7 • 0.9
0.3 45.4 20.1 10.8 0.2 7.6
• + + • • +
0.2 8.2 4~ 3.7 0.1 0.8 0.7 • 0.4 9.7 • 2.4 5.2 + 1.4
PNOR 0.1 23.0 24.9 21.4 0.2 6.2 1.9 13.5 0.2 8.7
+ • • • • + • + + •
0.1 0.6 1.8 2.1 0.1 0.3 0.6 1.1 0.1 0.9
IW
JJ
0.1 + 0.1 34.6 + 0.9 22.5 • 1.3 14.1 • 1.5 trace 8.0 + 0.5 2.0 + 0.4 13.5 • 0.9
trace 22.4 + 1.1 30.1 + 1.4 19.0 • 2.6 0.1 + 0.1 8.7 • 0.5 1.8 + 0.4 11.9 • 0.9
5.2 •
0.4
6.2 •
0.6
*See footnote, Table 2.
The fecal steroid excretion data are s u m m a r i z e d in Table 8. Neutral steroid excretion was similar for all five dietary groups. Acidic steroid excretion was similar in the rats fed intact peanut oil of whatever origin (PNO, IW, JJ). Acidic steroid excretion in rats fed PNOR was reduced by about a third and reduced even further in rats fed CO. There appears to be no relation between steroid excretion and fatty acid composition of the fat. TABLE 8 Fecal Neutral and Acidic Steroids in Rats Fed Various Oils Fecal steroids (mg/g) Group*
Neutral
Acidic
(30 PNO PNOR IW
21 18 25 23 21
105 255 180 265 241
*See footnote, Table 2. The data on triglyceride levels of gut mucosa and their fatty acid spectra are given in Table 9. Rats fed CO or PNOR had the lowest levels of mucosal triglycerides. The highest levels of mucosal triglycerides were f o u n d in rats fed PNO or IW. Mucosal triglycerides in g r o u p PNO were significantly higher than those in rats fed PNOR, JJ or CO. Rats fed IW had significantly higher mucosal triglycerides than those fed PNOR or CO.
PEANUT OILS AND LIPIDS IN RATS
1003
TABLE 9 Gut Mucosal Triglycerides ( m g / g • SEM) and Fatty Acids (% • SEM) in Rats Fed Various Oils Group* CO
PNO
PNOR
IW
JJ
Triglycerides
10.8 + 1.7
19.5 • 1.6
10.5 • 1.8
17.6 • 1.8
14.6 + 1.1
Fatty Acids (%) 14:0 16:0 18:0 18:1 18:2 18:3 20:0 22:0 Other
44.6 17.2 6.8 10.9 16.8 1.0 0.4 0.3 4.1
45.4 + 2.7 16.4 + 2.8 6.1 + 0.4 17.0 + 1.2 10.4 • 5.9 0.3 • 0.1 0.1 • 0.1 trace 4.1 • 0.4
45.7 + 4.9 16.7 + 2.8 7.2 • 1.0 15.6 + 0.9 9.7 • 0.5 0.3 + 0.2 0.1 + 0.1 trace 5.3 • 0.4
39.4 16.8 8.2 15.9 13.7 0.4
1.7 1.6 0.6 2.0 1.1 0.2
35.2 + 4.4 10.1 + 3.3 11.9 + 1.1 24.0 + 5.3 9.6 + 1.7 trace
0,2 0.5
trace 9.4 • 0.9
+ + + • • + • + •
1.8 1.8 0.6 1.3 1.6 0.2 0.1 0.1 0.9
+ + • • • • 0.4 • 5.5 •
*See footnote, Table 2.
The two groups of rats with lowest levels of mucosal triglycerides excreted the least a m o u n t of acidic steroid, but fecal acidic steroid excretion in the four PNO-fed groups was fairly consistent (235 + 19 m g / g ) compared to that observed in the rats fed CO. DISCUSSION One of the aims of this s t u d y was to determine if the 18:1/18:2 ratio of various p e a n u t oils could affect lipid metabolism in rats. For comparison, we also fed rats CO and PNOR which has the same 18:1/18:2 ratio as PNOR but different triglyceride structure. The only significant effect observed on necropsy was the lower liver triglycerides in rats fed PNOR, IW or JJ. Gut mucosal triglycerides were lowest in rats fed CO or PNOR. In Table 10 w e have compared 18:1/18:2 ratios of liver, gut and feces with the ratio observed in the dietary fat. In general, liver 18:1/18:2 ratios resemble those of the fat. One might a d d u c e a similar conservation of fatty acid ratio in the gut mucosa albeit there are elevations of 44, 87, and 34% in groups CO, IW and JJ compared with the starting levels. In the feces there is a m a r k e d elevation of 18:1/18:2 levels suggesting conservation of linoleic acid or that it might be a substrate of choice for metabolism of the intestinal flora. The major differences between groups were observed in the liver and mucosal triglycerides. Retention of triglycerides in the mucosa m a y be related to the atherogenic effects of certain fats. This possibility remains to be tested.
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D. KRITCHEVSKY et al.
TABLE 10 Oleic/Linoleic Acid Ratios in Rats Fed Various Oils Group* Site
(30
PNO
PNOR
IW
JJ
Diet Liver Gut Feces - 10D Feces - 20D
0.45 0.47 0.65 2.9 2.0
1.55 1.88 1.63 7.4 5.4
1.55 1.55 1.61 15.1 107
1.02 0.90 1.16 5.2 1410
3.06 1.96 2.50 25.6 271
*See footnote, Table 2. ACKNOWLEDGEMENTS Supported, in part, by a grant (HL03299) and a Research Career Award (HL00734) from the National Institutes of Health, grant CRCR-1-1068 from USDASEA, and by funds from the Commonwealth of Pennsylvania. REFERENCES 1.
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Kritchevsky D, Tepper SA, Vesselinovitch D, Wissler RW. Cholesterol vehicle in experimental atherosclerosis 11. Peanut oil. Atherosclerosis 1971; 14:53-64.
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Kritchevsky D, Tepper SA, Vesselinovitch D, Wissler RW. Cholesterol vehicle in experimental atherosclerosis 13. Randomized peanut oil. Atherosclerosis 1973; 17:22543.
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