ALAN C. TSAI, GEESJE M. TRIE 2 ANDC. R.-s. LIN Human Nutrition Program, School of Public Health, The University of Michigan, Ann Arbor, Michigan 48109 ABSTRACT The effect of cholesterol feeding on liver and aortic nonenzymatic lipid peroxidation and glutathione peroxidase activities, and on liver microsomal NADPH-dependent lipid peroxidation, codeine hydroxylation and cytochrome P-450 levels was examined in rats and guinea pigs. One percent cholesterol was added to a casein-sucrose-soybean oil basal diet for rats or a stock diet with 2% soybean oil for guinea pigs. The effect of vitamin E and cholestyramine was also examined in some experiments. Cholesterol feeding increased the rate of lipid peroxidation in liver and aortic homogenate both in rats and guinea pigs when fed non-vitamin E supplemented basal diets. Vitamin E supplementation prevented the in crease in the aorta, but not as completely in the liver in rats, while the reverse was true in guinea pigs. The effect of cholestyramine was dependent on the level of vitamin E in the diet. Cholesterol feeding decreased gluta thione peroxidase activities in rats and guinea pigs. In guinea pigs, choles terol feeding also markedly decreased liver microsomal NADPH-dependent lipid peroxidation, codein hydroxylation and cytochrome P-450 levels espe cially when fed non-vitamin E supplemented basal diets. In rats, cholesterol feeding reduced liver microsomal NADPH-dependent lipid peroxidation and in some cases, increased microsomal codeine hydroxylation activities, but had no effect on microsomal cytochrome P-450 levels. Vitamin E sup plementation increased liver and serum cholesterol levels in guinea pigs, but had no such effect in rats. Results of this study indicate that cholesterol feeding can result in various metabolic alterations in rats and guinea pigs. The implication of these alterations in atherogenesis requires further in vestigations. J. Nutr. 107: 310-319, 1977. INDEXING KEY WORDS cholesterol feeding •tissue lipid peroxi dation •glutathione peroxidase •microsomal NADPH-dependent lipid peroxidation •microsomal codeine hydroxylation •microsomal cyto chrome P-450 •vitamin E •aorta In a previous communication from this laboratory (1), cholesterol feeding was shown to result in an increase in the rate of lipid peroxidation and a decrease in glutathione peroxidase activity in rat liver, Since lipid peroxidation has been shown to be a deteriorative process to tissues (2, 3) and glutathione peroxidase a tissue lipid peroxide-decomposing system (4-6), it was important to determine whether such
changes may also occur in the aorta, and thus, play some role in the etiology of atherosclerosis. In the present study, the effect of cholesterol feeding was compared in rats and guinea pigs. The latter is known Receivedfor publicationMay 24, 1976. .¿Ä^S H^^ÕA gen, The Netherlands. 310
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Effect of Cholesterol Feeding on Tissue Lipid Peroxidation, Glutathione Peroxidase Activity and Liver Microsomal Functions in Rats and Guinea Pigs1
METABOLIC ALTERATIONS IN CHOLESTEROL FED ANIMALS
METHODS
Rat experiments. Male Sprague-Dawley rats 3 weighing approximately 130 g initi ally were used in all four rat experiments in this study. They were housed in stain less steel cages with raised wire floors under an environment of controlled light ing and temperature. Food and water were provided ad libitum. Food consumption and body weight were recorded periodi cally. Composition of the basal diets is shown in table 1. The design, treatment and feeding duration of each experiment are indicated in the tables of results. Guinea pig experiments. Male guinea pigs 4 weighing 200 to 250 g were randomly allotted to treatment groups and housed in galvanized cages with raised wire floors under an environment of controlled light ing and temperature. Food and water were provided ad libitum. Body weight gain was recorded periodically. Food consumption was not recorded because of excessive food wastage, especially in the cholesterol-fed groups. The basal diet was a pelleted guinea pig stock diet5 plus 2% soybean
TABLE 1 Composition of basal diets for rat experiments Ingredient
Basal diet A
Casein Soybean oil1 Sucrose Mineral mix* Vitamin mix1 Choline Cl« dl-Methionine4 Fiber5 if-ascorbic acid*
g/100g 20 10 60.2 4 0.4 0.2 0.2 5 0.02
Basal diets used in experiments 1-4 Experiment
Diet
Composition
1 2
A B
3 4
C D
As above A + vitamin K (100 IU/kg diet) A + cholesterol (1%) Same as A, but with differ ent oil1
1For experiments 1, 2, and 3, Jaxmar pure vege table oil, Anderson Clayton Foods, Dallas, Texas 75200. For experiment 4, Wesson salad oil. 2 Wes son's modified Osborne-Mendel salt mixture, Gen eral Biochemicals, Chagrin Falls, Ohio. Contained: (g/kg salt mixture) NaCl, 105; KC1, 120; KH,PO4, 310; Ca (PO4)2, 149; CaCOi, 210; MgSO4, 90; FePO4-4H2O, 14.7; MnSO4, 0.02; KzAl(SO4)4-24 H2O, 0.09; CuSO4-5H,O, 0.39; NaF, 0.57; Kl, 0.05 (from Science, 75, 339. 1932). ' See reference (1) for composition. *From Sigma Chemical Com pany, St. Louis, Missouri. *Alphacel, from ICN Pharmaceuticals, Inc., Cleveland, Ohio 44128.
oil. The design, treatment and feeding du ration of each experiment are indicated in the tables of results. In all rat and guinea pig experiments, cholesterol was dissolved in hot soybean oil before being thoroughly mixed into the diet. Soybean oil for the control diets was heated the same way. All animals were killed by decapitation. Samples of serum, liver and aorta were taken immediately for laboratory determinations. Laboratory determinations. Approxi mately 2 g of fresh liver was homogenized in 13 ml of potassium phosphate buffer (75 niM, pH 7.0). An aliquot (0.5 ml) of 3 Rnts were purchased from Spartan Research Aniinnls. Haslett. Michigan 4S840. ' Guiñen pigs were purchased from Charles River Brpiling Laboratorios, Inc., Wilmington, Massachu setts 01887. 6 Wayne guinea pig diet. Allied Mills, Inc., Chicago, Illinois GOGOG.
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to be a species relatively more susceptible to atherosclerosis than the rat when fed an atherogenetic diet. Since alteration in cho lesterol metabolism such as during bile duct ligation, has been shown to affect microsomal functions in experimental ani mals (7-9), and since the microsome is the site for cholesterol catabolism (10, 11), microsomal parameters including NADPHdependent lipid peroxidation, codeine hydroxylation, cytochrome P-450 level, and microsomal protein levels were investigated in cholesterol-fed animals. The activity of glucose-6-phosphate dehydrogenase which has been observed to be depressed in cho lesterol fed rats (1) was also examined in this study. Further, since vitamin E has been shown to play an important role in controlling the rate of tissue lipid peroxida tion (12-16) and microsomal NADPHdependent lipid peroxidation (17, 18), as well as drug metabolizing activities (12, 19-21), the effect of vitamin E supplemen tation was also investigated. Cholestyramine feeding, which interrupts the enterohepatic circulation of cholesterol, was em ployed to study the effect of cholesterol depletion on these parameters.
311
312
TSAI, THIE AND LIN TABLE 2 Results of cholesterol feeding and vitamin E supplementation on lipid peroxidation, enzyme activities and other parameters in rats fed diets for 30 days (experiment Õ)1
E134 Vitamin
4'357 ± 4358 ± ±1519.5± ±1520.1 22.9± 23.6± ± 0.1267 0.2376 ±102.4± 717 ± 0.642 0.687± 3.124± ±2425
4344 ± ±2619.3 22.9± 0.0266 ± 52.3± 0.642± 620 ±
A134 Initial body wt., gFinal
gFoodbody wt.,
g/dayLiver intake, wt.Serum wt., g/100 g body mlLiver cholesterol, mg/100 mg/gLiver cholesterol, mg/gNon-enzymatic long-chain F.A., lipidperoxidation, tissue MA/gtissueLiver mi
±12443 4360 ± ±62418 ±55336 ±511.8± changeAorta ±562.7 hr2hrNet :0 13 ± 0.57.4± 2.30.3± ± 4.65.6± 4.97.0± changeGlutathione 0.38.2± peroxidaseLiver4Aorta4Serum4Liver 0.31.1± 0.80.8± 0.42.8± 0.13.0± 0.42.3 0.21.6± DH'Liver G-6-P 16.1± 0.64.0± 6-PG DH'Basal 0.6+ ± 0.6+ hr2hrNet : 0
Choles terol E133 vitamin 4347 ± ±1120.0 23.5 ± 0.274 ±
±820 796 ± ±3020
584 ± 2152 ± ±4063 ±40131 ±382.1± ±412.6 1.32.2± 0.41.4 ± 0.40.1± ±0.8-1.2 0.77.0 ± 0.38.9± 0.51.1± 0.33.1± 0.22.2± 0.46.1 ± 1.3+
ECh, ENSCh, ECh,Ch X EChN3NSChCb E, Ch X
0.71.05± ± 0.33.0 0.21.1 ± 0.53.4 ± ±0.8AOV1NSNSNSChCbChChNSCh,
1Cholesterol-supplemented diets contained 1% cholesterol, vitamin E-supplemented diets contained 100 IU vitamin E/kg diet as dl-a-tocopheryl succinate. * Analysis of variance. Ch = significant cholesterol effect. E = significant vitamin E effect. NS = not significant. 3 Mean ±so for six rats. *Amóles NADPH oxidized/g tissue/minute or /ml serum/minute. ' AmólesNADPH formed/g tissue/minute.
the homogenate was removed for the deter mination of non-enzymatic lipid peroxida tion (1). The remaining homogenate was centrifuged at 10,000 X g for 15 minutes and the precipitate was discarded. The supernatant was re-centrifuged at 100,000 X g for 90 minutes. The supernatant was removed for the assay of glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 6phosphogluconate dehydrogenase ( EC 1.1.1.44) (22) and glutathione peroxidase (EC 1.11.1.9) (23) at 23°while the microsomal pellet was used for the determina tion of microsomal activities. The pellet was washed once with 1.157o KC1 solution and then suspended in 2 ml of 1.15% KC1 solution and 2 ml of potassium phosphate buffer (75 mM, pH 7.4) and kept frozen until time of analysis (within 3 days). Microsomal NADPH-dependent lipid peroxidation was determined according to the method of Orrenius (24). The amount of malonaldehyde (MA) formed was mea sured by the thiobarbituric acid (TEA) procedure (13). Microsomal codeine hydroxylation activity was determined by the
procedure of Carpenter and Howard (21). The reaction mixture, with codeine as sub strate, was incubated for 10 minutes. The amount of formaldehyde was determined according to the method of Nash (25). Microsomal cytochrome P-450 was deter mined by the procedure of Omura and Sato (26). In some experiments, the aorta was ho mogenized in 3 ml potassium phosphate buffer (75 mM, pH 7.0). An aliquot (0.5 ml ) of the homogenate was used for deter mining the rate of lipid peroxidation in the aorta. The remaining solution was centri fuged at 50,000 X g for 90 minutes at 4° and the supernatant was then removed for glutathione peroxidase assay (23). Microsomal protein and liver soluble protein were determined by the method of Lowry et al. (27). Serum vitamin E level was determined by the procedure of Hashim and Schuttringer (28). Liver vitamin E and microsomal vitamin E levels were determined by the method of Walcizaki and Imai (14). The rate of nonenzymatic lipid peroxidation was deter-
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Choles terol134
METABOLIC
ALTERATIONS
IN CHOLESTEROL
RESULTS RaÃ-experiments. Results of rat experi ments are shown in tables 2 to 5. Choles terol feeding did not significantly affect growth or food intake, but resulted in ele vated serum and liver cholesterol levels and liver long-chain fatty acid levels. Dietary vitamin E supplementation did not affect growth, food intake, serum and liver cho lesterol or liver long-chain fatty acid levels (tables 2 and 4). Inclusion of cholestyramine for a period of 7 days had no effect on growth or food intake, but significantly reduced serum and liver cholesterol levels and decreased liver long-chain fatty acid concentrations in rats fed a cholesterolsupplemented diet (table 4). Cholesterol feeding increased the rate (net changes) of non-enzymatic lipid peroxidation in the liver and aorta in ex periment 1 (table 2). Vitamin E effectively
313
reduced the rate of tissue lipid peroxida tion and prevented the elevation due to cholesterol feeding in the aorta (table 2). Cholestyramine feeding tended to decrease the rate of tissue lipid peroxidation when rats were fed a non-vitamin E-supplemented basal diet, but resulted in an in crease when the rats were fed a vitamin E-supplemented diet (table 4). Both cholesterol and vitamin E decreased the rate of liver microsomal NADPHdependent lipid peroxidation (table 5), but cholestyramine feeding led to an in crease (table 4). Cholesterol feeding did not affect the rate of codeine hydroxylation in experiment 2 (table 3), but in creased the rate in experiment 4 (table 5). Vitamin E supplementation increased the rate of microsomal codeine hydroxylation (tables 4 and 5). The effect of cholestyra mine feeding on microsomal hydroxylation was dependent on the level of dietary vitamin E. Cholestyramine feeding de creased microsomal codeine hydroxylation only when rats were fed a vitamin E sup plemented diet (table 4). Supplementation of cholesterol, vitamin E or cholestyramine did not significantly affect the level of cytochrome P-450 in liver microsomes nor the level of microsomal protein (tables 4
TABLE 3 Results of cholesterol feeding on liver microsomal activities, liver and serum glufathione peroxidase activities and other parameters in rats fed diets for 137 days (experiment £)' Basal B gFinal body wt., Initial gFood body wt., g/dayLiver intake, wt.Serum wt., g/100 g body mlLiver cholesterol, mg/100 mg/gLiver cholesterol, mg/gLiver long-chain F.A., mg/gLiver soluble protein, mg/gLiver reduced GSH, :NADPH-dependent microsomal parameters peroxidation,OD-532/mg lipid protein/hrCodeine
HCHOformed hydroxylation, nmoles /minCytochrome /mg protein proteinMicrosomal P-450, nmoles/mg liverGlutathione protein, mg/g peroxidase5LiverSerum124
4»603± ±6820.6 23.4 ± 0.2133 ± ±212.8 0.332 ± 498 ± ±1027 2.51.4 ± 0.381.67± ±
0.60.93 0.118 ± 1.48.9± 1.23.4± ± 0.3125
+ Cholesterol 3587 ± ±3519.9 24.6 ± 0.2«165 ± ±18«38 6.8«94 ± ±18«74 ±10*24 20.58± ± 0.3*1.32± 0.50.89 0.1618± 3.36.2± 0.8«2.5 ± ± 0.2*
1 The basal diet in this experiment contained 100 IU vitamin E/kg diet as dl-alpha-tocopheryl succinate. Cholesterol-supplemented diet contained 1% cholesterol. 2 Mean±SD for eight rats. 8AmólesNADPH oxidized/g tissue/minute or /ml serum/minute. *Significantly different from basal group (P < 0.05).
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mined by TEA method as described in a previous publication (1). Serum choles terol, liver cholesterol, liver long-chain fatty acid levels were determined as de scribed previously (29). Statistics. Results of each experiment were statistically analyzed according to the design of the experiment (30).
FED ANIMALS
314
TSAI, THIE AND LIN TABLE 4
gFinal Initial body wt., gFood body wt., g/dayI.iver intake, wt.Serum wt., g/100 g body mlLiver cholesterol, mg/100 mg/gLiver cholesterol, mg/gLiver long-chain F.A., mg/gNon-enzymatic soluble protein, peroxidation.Mg lipid tissueLiver: MA/g hr2hrNet 0 changeAorta: Ohr2hr 'Net changeLiver parametersNADPH-dependent microsomal peroxidation.OD-532/mg lipid protein/hrCodeine nmolesHCHO/formed/mg hydroxylation, protein/minCytochrome proteinMicrosomal P-450, nmoles /mg protein, mg/g liver135
+ Cholestyramine vitamin E
BasalC
+ Cholestyramine
V375 ± ±1719.6 25.3 ± 0.1151 ± ±1717.1 4.863 ± ±23102 ±1330
2370 ± ±1818.9 24.6 ± 0.2100 ± ±242.9 0.342 ± ±7106 ±1022
3378 ± 1219.2± 15.3 ± 0.2154 ± 3319.0± 4.968 ± 17113 ± 2919 ±
3380 ± ±2118.4 24.6 ± 0.4102 ± 63.6 ± 1.236 ± ±5102 815 ±
±10593 ±81563 ±772.6 0.711 ± ±108.4 9.82.54 ±
±5474 ±39452 ±360.6 1.17.3± 2.56.7± 1.83.51 ±
6210 ± ±108191 ±1072.2 l.S5.3± 1.53.1± 1.71.2±
±2350 ±30335 ±321.0 0.75.8± 1.74.8± 1.72.36 ±
0.41.54± ±
0.41.88± ±
0.50.84± 0.115.3 ± 1.2133
0.60.88± 0.215.6 ± 1.1131
+ Vitamin E
AOV
CtE,E X CtCtEE, E X
CtE,E X
0.61.48± ±
0.82.3± 0.60.96 ± 0.115.0± ± 2.4134
CtE
CtNSNS X 0.60.74± 0.216.7 ± 3.8NSN8N8CtCtCtCtN8EE,
1 The basal diet contained 1% cholesterol. Vitamin E-supplemented diets contained 100 IU vitamin tocopheryl Buccinate. C h olea ty ramine (3%) was fed to rats during the last 7 days of the feeding period only. E = significant vitamin E effect. Ct = significant cholestyramine effect. * Meaniso for eight rats.
E/kg diet as dl-alphaa Analysis of variance.
TABLE 5 Results of cholesterol feeding and ritamin E supplementation on microsomal activities and other parameters in rats fed diets for 21 days (experiment 4)1 E124 Vitamin
E125 vitamin
3367 ± ±2618.6 25.5± 0.6149± ±150.7 0.119± 7.273± ±3964 ±232.0
3350 ± ±1018.7 14.8± 0.2110± ±141.9± 0.32.5 0.472± ±2626 30.4±
2381 ± ±1220.5 25.9± 0.3147± ±131.7± 0.421 6.694± 766 ± ±150.7
0.41.7±
0.62.1 ±
0.41.9± ±
0.42.6 ±
ECh.E, Ch X
0.30.8±
0.30.7± ±
0.70.8±
0.80.7 ±
ENSNSECh
D122 gFinal body wt., Initial gFood body wt., g/dayLiver intake, wt.Serum wt., K/100 g body mlSerumcholesterol, mg/100 mlLiver vitamin E, mg/100 mg/gLiver cholesterol, pg/gLiver vitamin E, rng/gLiver long-chain F.A.. parametersNADPH-dependent microsomal peroxidation,OD-532/mg lipid protein/hrCodeine HCHOformed/mg hydroxylation, nmoles protein/minCytochrome nmoles/mgproteinMicrosomal P-450, liverMicrosomal protein, mg/g liverMicrosomal vitamin E, Mg/g cholesterol, mg/g liverBasal
1>355 ± ±1919.1 14.7 ± 0.2100 ± ±131.1 0.42.5± 0.641 ± ±1028 ±83
0.118 ± 0.120 27.0± 17.5 ± 2.40.54 ± 4.81.0± ± ± 0.1-(-Cholesterol1230.4+
EChCh,
EChCh,
4.720 0.118± 118 ± 219 ± 4.70.6 ± 3.91.1± ± ± 0.1-(-Cholesterol 0.2ACV»N8NSNSChChCh,
1Basal diet was basal diet A with Wesson oil instead of Jaxmar pure vegetable oil. Cholesterol-supplemented diet contained 1% cholesterol. Vitamin E treated groups received intra peritoneal injections of 10 IU vitamin E in oil as dl-alpha-tocopherol twice weekly. «Analysisof variance. Ch = significant cholesterol effect; E = significant Vitamin E effect. NS = not significant. ' Mean±l so for seven rata.
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ResidÃ-sof cholestyramine feeding and vitamin E supplementation on tissue lipid peroxidation, microsomal activities and other parameters in rats fed cholesterol-enriched diets for 33 days (experiment 3)1
METABOLIC ALTERATIONS IN CHOLESTEROL FED ANIMALS
315
TABLE 6 Results of cholesterol-feeding on liver lipid peroxidation, enzyme activities and other parameters in guinea pigs fed diets for 72 days (experiment 6)1
changeLiver NADP-dependentlipid microsomal peroxidation,OD-532/mg protein/hrMicrosomal liverLiver protein, mg/g peroxidase3Serum glutathione glutatbione peroxidase*185
+ Cholesterol
±11*639 ±494.1 0.92.2 ± 0.130 ± 1.2107± ±1466 4.80.19± ± 0.044.9 0.317.9 ±
17546 ± 56*8.3 ± 3.1«38 ± 6.3«137 ± 24«99 ± 18145 ± 35«0.16± ± 0.095.4 0.548.7 ±
0.835.9 ± 1.918.0 ± 1.440.46 ± 0.148.2 ± 1.517.3 ± 3.00.29 ± ±0.08186
23.5«204.7 ± ±114«156 94«O.OC± ± 0.09«4.8 1.3«10.2 ± 2.3«0.28 ± ± 0.08
1The basal diet was a guinea pig stock diet plus 2% soybean oil. The cholesterol-supplemented diet was the basal diet plus 1% cholesterol. 2Mean±sr>for six guinea pigs. 3Amóles NADPtt oxidized/g tissue/ minute or/ml serum/minute. *Significantly different than the basal group, (P < O.Oö).
and 5). The concentration of microsomal vitamin E was increased by vitamin E sup plementation, but was unaffected by cho lesterol feeding (table 5). Cholesterol reeding decreased the activ ity of glutathione peroxidase in the liver (tables 2 and 3). It also decreased the activity of this enzyme in the serum in a long-term experiment (137 days), but had no effect in a short-term experiment (30 days). Cholesterol feeding also decreased the activity of liver glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. Dietary vitamin E supple mentation had no effect on these enzymes. Cholesterol feeding reduced the level of vitamin E in the serum, but increased the level in the liver. Vitamin E supplementa tion increased vitamin E levels both in the serum and in the liver. Guinea pig experiments. Results of guinea pig experiments are shown in tables 6 and 7. Cholesterol feeding significantly reduced body weight gain and increased liver weight weight, liver cholesterol, liver longchain fatty acid, liver vitamin E and serum cholesterol levels. It appeared to decrease liver soluble protein levels, but had no sig nificant effect on serum vitamin E levels.
Dietary supplementation of cholesterol markedly elevated the rate of tissue lipid peroxidation in the liver and aorta when the guinea pigs were fed the basal diet (table 7). Vitamin E administration com pletely prevented this cholesterol-feeding effect in the liver, but the prevention was not complete in the aorta. Dietary supplementation of cholesterol also drastically reduced the rate of liver microsomal NADPH-dependent lipid per oxidation when the guinea pigs were fed the basal diet. When the guinea pigs were fed the same diet supplemented with vita min E, the rate of microsomal NADPHdependent lipid peroxidation in the liver was very low and the activity was slightly increased by cholesterol feeding. Liver microsomal codeine hydroxylation was sig nificantly reduced by cholesterol feeding, but was not influenced by vitamin E ad ministration. Liver microsomal cytochrome P-450 and microsomal protein levels were significantly reduced by cholesterol feed ing. Vitamin E supplementation also re duced liver microsomal protein levels, but had no effect on cytochrome P-450 levels. Cholesterol feeding decreased the activity of glutathione peroxidase in the liver, but
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gFinal Initial body wt., gLiverbody wt., wt.Liver wt., g/100 g body mg/gLiver cholesterol, mg/gLiver long-chain F.A., mg/gSerum soluble protein, mlSerum cholesterol, mg/100 mlSerum vitamin E, mg/100 mlNon-enzymatic protein, mg/100 peroxidation,jig liver lipid liverOhr2hrNet MA/g
Basal
316
TSAI, TRIE AND LIN
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changes may also occur in the aorta, and thus, play some role in the etiology of atherosclerosis. In the present study, the effect of cholesterol feeding was compared in rats and guinea pigs. The latter is known Receivedfor publicationMay 24, 1976. .¿Ä^S H^^ÕA gen, The Netherlands. 310
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Effect of Cholesterol Feeding on Tissue Lipid Peroxidation, Glutathione Peroxidase Activity and Liver Microsomal Functions in Rats and Guinea Pigs1
METABOLIC ALTERATIONS IN CHOLESTEROL FED ANIMALS
METHODS
Rat experiments. Male Sprague-Dawley rats 3 weighing approximately 130 g initi ally were used in all four rat experiments in this study. They were housed in stain less steel cages with raised wire floors under an environment of controlled light ing and temperature. Food and water were provided ad libitum. Food consumption and body weight were recorded periodi cally. Composition of the basal diets is shown in table 1. The design, treatment and feeding duration of each experiment are indicated in the tables of results. Guinea pig experiments. Male guinea pigs 4 weighing 200 to 250 g were randomly allotted to treatment groups and housed in galvanized cages with raised wire floors under an environment of controlled light ing and temperature. Food and water were provided ad libitum. Body weight gain was recorded periodically. Food consumption was not recorded because of excessive food wastage, especially in the cholesterol-fed groups. The basal diet was a pelleted guinea pig stock diet5 plus 2% soybean
TABLE 1 Composition of basal diets for rat experiments Ingredient
Basal diet A
Casein Soybean oil1 Sucrose Mineral mix* Vitamin mix1 Choline Cl« dl-Methionine4 Fiber5 if-ascorbic acid*
g/100g 20 10 60.2 4 0.4 0.2 0.2 5 0.02
Basal diets used in experiments 1-4 Experiment
Diet
Composition
1 2
A B
3 4
C D
As above A + vitamin K (100 IU/kg diet) A + cholesterol (1%) Same as A, but with differ ent oil1
1For experiments 1, 2, and 3, Jaxmar pure vege table oil, Anderson Clayton Foods, Dallas, Texas 75200. For experiment 4, Wesson salad oil. 2 Wes son's modified Osborne-Mendel salt mixture, Gen eral Biochemicals, Chagrin Falls, Ohio. Contained: (g/kg salt mixture) NaCl, 105; KC1, 120; KH,PO4, 310; Ca (PO4)2, 149; CaCOi, 210; MgSO4, 90; FePO4-4H2O, 14.7; MnSO4, 0.02; KzAl(SO4)4-24 H2O, 0.09; CuSO4-5H,O, 0.39; NaF, 0.57; Kl, 0.05 (from Science, 75, 339. 1932). ' See reference (1) for composition. *From Sigma Chemical Com pany, St. Louis, Missouri. *Alphacel, from ICN Pharmaceuticals, Inc., Cleveland, Ohio 44128.
oil. The design, treatment and feeding du ration of each experiment are indicated in the tables of results. In all rat and guinea pig experiments, cholesterol was dissolved in hot soybean oil before being thoroughly mixed into the diet. Soybean oil for the control diets was heated the same way. All animals were killed by decapitation. Samples of serum, liver and aorta were taken immediately for laboratory determinations. Laboratory determinations. Approxi mately 2 g of fresh liver was homogenized in 13 ml of potassium phosphate buffer (75 niM, pH 7.0). An aliquot (0.5 ml) of 3 Rnts were purchased from Spartan Research Aniinnls. Haslett. Michigan 4S840. ' Guiñen pigs were purchased from Charles River Brpiling Laboratorios, Inc., Wilmington, Massachu setts 01887. 6 Wayne guinea pig diet. Allied Mills, Inc., Chicago, Illinois GOGOG.
Downloaded from https://academic.oup.com/jn/article-abstract/107/2/310/4769025 by University of California, San Francisco user on 01 January 2019
to be a species relatively more susceptible to atherosclerosis than the rat when fed an atherogenetic diet. Since alteration in cho lesterol metabolism such as during bile duct ligation, has been shown to affect microsomal functions in experimental ani mals (7-9), and since the microsome is the site for cholesterol catabolism (10, 11), microsomal parameters including NADPHdependent lipid peroxidation, codeine hydroxylation, cytochrome P-450 level, and microsomal protein levels were investigated in cholesterol-fed animals. The activity of glucose-6-phosphate dehydrogenase which has been observed to be depressed in cho lesterol fed rats (1) was also examined in this study. Further, since vitamin E has been shown to play an important role in controlling the rate of tissue lipid peroxida tion (12-16) and microsomal NADPHdependent lipid peroxidation (17, 18), as well as drug metabolizing activities (12, 19-21), the effect of vitamin E supplemen tation was also investigated. Cholestyramine feeding, which interrupts the enterohepatic circulation of cholesterol, was em ployed to study the effect of cholesterol depletion on these parameters.
311
312
TSAI, THIE AND LIN TABLE 2 Results of cholesterol feeding and vitamin E supplementation on lipid peroxidation, enzyme activities and other parameters in rats fed diets for 30 days (experiment Õ)1
E134 Vitamin
4'357 ± 4358 ± ±1519.5± ±1520.1 22.9± 23.6± ± 0.1267 0.2376 ±102.4± 717 ± 0.642 0.687± 3.124± ±2425
4344 ± ±2619.3 22.9± 0.0266 ± 52.3± 0.642± 620 ±
A134 Initial body wt., gFinal
gFoodbody wt.,
g/dayLiver intake, wt.Serum wt., g/100 g body mlLiver cholesterol, mg/100 mg/gLiver cholesterol, mg/gNon-enzymatic long-chain F.A., lipidperoxidation, tissue MA/gtissueLiver mi
±12443 4360 ± ±62418 ±55336 ±511.8± changeAorta ±562.7 hr2hrNet :0 13 ± 0.57.4± 2.30.3± ± 4.65.6± 4.97.0± changeGlutathione 0.38.2± peroxidaseLiver4Aorta4Serum4Liver 0.31.1± 0.80.8± 0.42.8± 0.13.0± 0.42.3 0.21.6± DH'Liver G-6-P 16.1± 0.64.0± 6-PG DH'Basal 0.6+ ± 0.6+ hr2hrNet : 0
Choles terol E133 vitamin 4347 ± ±1120.0 23.5 ± 0.274 ±
±820 796 ± ±3020
584 ± 2152 ± ±4063 ±40131 ±382.1± ±412.6 1.32.2± 0.41.4 ± 0.40.1± ±0.8-1.2 0.77.0 ± 0.38.9± 0.51.1± 0.33.1± 0.22.2± 0.46.1 ± 1.3+
ECh, ENSCh, ECh,Ch X EChN3NSChCb E, Ch X
0.71.05± ± 0.33.0 0.21.1 ± 0.53.4 ± ±0.8AOV1NSNSNSChCbChChNSCh,
1Cholesterol-supplemented diets contained 1% cholesterol, vitamin E-supplemented diets contained 100 IU vitamin E/kg diet as dl-a-tocopheryl succinate. * Analysis of variance. Ch = significant cholesterol effect. E = significant vitamin E effect. NS = not significant. 3 Mean ±so for six rats. *Amóles NADPH oxidized/g tissue/minute or /ml serum/minute. ' AmólesNADPH formed/g tissue/minute.
the homogenate was removed for the deter mination of non-enzymatic lipid peroxida tion (1). The remaining homogenate was centrifuged at 10,000 X g for 15 minutes and the precipitate was discarded. The supernatant was re-centrifuged at 100,000 X g for 90 minutes. The supernatant was removed for the assay of glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 6phosphogluconate dehydrogenase ( EC 1.1.1.44) (22) and glutathione peroxidase (EC 1.11.1.9) (23) at 23°while the microsomal pellet was used for the determina tion of microsomal activities. The pellet was washed once with 1.157o KC1 solution and then suspended in 2 ml of 1.15% KC1 solution and 2 ml of potassium phosphate buffer (75 mM, pH 7.4) and kept frozen until time of analysis (within 3 days). Microsomal NADPH-dependent lipid peroxidation was determined according to the method of Orrenius (24). The amount of malonaldehyde (MA) formed was mea sured by the thiobarbituric acid (TEA) procedure (13). Microsomal codeine hydroxylation activity was determined by the
procedure of Carpenter and Howard (21). The reaction mixture, with codeine as sub strate, was incubated for 10 minutes. The amount of formaldehyde was determined according to the method of Nash (25). Microsomal cytochrome P-450 was deter mined by the procedure of Omura and Sato (26). In some experiments, the aorta was ho mogenized in 3 ml potassium phosphate buffer (75 mM, pH 7.0). An aliquot (0.5 ml ) of the homogenate was used for deter mining the rate of lipid peroxidation in the aorta. The remaining solution was centri fuged at 50,000 X g for 90 minutes at 4° and the supernatant was then removed for glutathione peroxidase assay (23). Microsomal protein and liver soluble protein were determined by the method of Lowry et al. (27). Serum vitamin E level was determined by the procedure of Hashim and Schuttringer (28). Liver vitamin E and microsomal vitamin E levels were determined by the method of Walcizaki and Imai (14). The rate of nonenzymatic lipid peroxidation was deter-
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Choles terol134
METABOLIC
ALTERATIONS
IN CHOLESTEROL
RESULTS RaÃ-experiments. Results of rat experi ments are shown in tables 2 to 5. Choles terol feeding did not significantly affect growth or food intake, but resulted in ele vated serum and liver cholesterol levels and liver long-chain fatty acid levels. Dietary vitamin E supplementation did not affect growth, food intake, serum and liver cho lesterol or liver long-chain fatty acid levels (tables 2 and 4). Inclusion of cholestyramine for a period of 7 days had no effect on growth or food intake, but significantly reduced serum and liver cholesterol levels and decreased liver long-chain fatty acid concentrations in rats fed a cholesterolsupplemented diet (table 4). Cholesterol feeding increased the rate (net changes) of non-enzymatic lipid peroxidation in the liver and aorta in ex periment 1 (table 2). Vitamin E effectively
313
reduced the rate of tissue lipid peroxida tion and prevented the elevation due to cholesterol feeding in the aorta (table 2). Cholestyramine feeding tended to decrease the rate of tissue lipid peroxidation when rats were fed a non-vitamin E-supplemented basal diet, but resulted in an in crease when the rats were fed a vitamin E-supplemented diet (table 4). Both cholesterol and vitamin E decreased the rate of liver microsomal NADPHdependent lipid peroxidation (table 5), but cholestyramine feeding led to an in crease (table 4). Cholesterol feeding did not affect the rate of codeine hydroxylation in experiment 2 (table 3), but in creased the rate in experiment 4 (table 5). Vitamin E supplementation increased the rate of microsomal codeine hydroxylation (tables 4 and 5). The effect of cholestyra mine feeding on microsomal hydroxylation was dependent on the level of dietary vitamin E. Cholestyramine feeding de creased microsomal codeine hydroxylation only when rats were fed a vitamin E sup plemented diet (table 4). Supplementation of cholesterol, vitamin E or cholestyramine did not significantly affect the level of cytochrome P-450 in liver microsomes nor the level of microsomal protein (tables 4
TABLE 3 Results of cholesterol feeding on liver microsomal activities, liver and serum glufathione peroxidase activities and other parameters in rats fed diets for 137 days (experiment £)' Basal B gFinal body wt., Initial gFood body wt., g/dayLiver intake, wt.Serum wt., g/100 g body mlLiver cholesterol, mg/100 mg/gLiver cholesterol, mg/gLiver long-chain F.A., mg/gLiver soluble protein, mg/gLiver reduced GSH, :NADPH-dependent microsomal parameters peroxidation,OD-532/mg lipid protein/hrCodeine
HCHOformed hydroxylation, nmoles /minCytochrome /mg protein proteinMicrosomal P-450, nmoles/mg liverGlutathione protein, mg/g peroxidase5LiverSerum124
4»603± ±6820.6 23.4 ± 0.2133 ± ±212.8 0.332 ± 498 ± ±1027 2.51.4 ± 0.381.67± ±
0.60.93 0.118 ± 1.48.9± 1.23.4± ± 0.3125
+ Cholesterol 3587 ± ±3519.9 24.6 ± 0.2«165 ± ±18«38 6.8«94 ± ±18«74 ±10*24 20.58± ± 0.3*1.32± 0.50.89 0.1618± 3.36.2± 0.8«2.5 ± ± 0.2*
1 The basal diet in this experiment contained 100 IU vitamin E/kg diet as dl-alpha-tocopheryl succinate. Cholesterol-supplemented diet contained 1% cholesterol. 2 Mean±SD for eight rats. 8AmólesNADPH oxidized/g tissue/minute or /ml serum/minute. *Significantly different from basal group (P < 0.05).
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mined by TEA method as described in a previous publication (1). Serum choles terol, liver cholesterol, liver long-chain fatty acid levels were determined as de scribed previously (29). Statistics. Results of each experiment were statistically analyzed according to the design of the experiment (30).
FED ANIMALS
314
TSAI, THIE AND LIN TABLE 4
gFinal Initial body wt., gFood body wt., g/dayI.iver intake, wt.Serum wt., g/100 g body mlLiver cholesterol, mg/100 mg/gLiver cholesterol, mg/gLiver long-chain F.A., mg/gNon-enzymatic soluble protein, peroxidation.Mg lipid tissueLiver: MA/g hr2hrNet 0 changeAorta: Ohr2hr 'Net changeLiver parametersNADPH-dependent microsomal peroxidation.OD-532/mg lipid protein/hrCodeine nmolesHCHO/formed/mg hydroxylation, protein/minCytochrome proteinMicrosomal P-450, nmoles /mg protein, mg/g liver135
+ Cholestyramine vitamin E
BasalC
+ Cholestyramine
V375 ± ±1719.6 25.3 ± 0.1151 ± ±1717.1 4.863 ± ±23102 ±1330
2370 ± ±1818.9 24.6 ± 0.2100 ± ±242.9 0.342 ± ±7106 ±1022
3378 ± 1219.2± 15.3 ± 0.2154 ± 3319.0± 4.968 ± 17113 ± 2919 ±
3380 ± ±2118.4 24.6 ± 0.4102 ± 63.6 ± 1.236 ± ±5102 815 ±
±10593 ±81563 ±772.6 0.711 ± ±108.4 9.82.54 ±
±5474 ±39452 ±360.6 1.17.3± 2.56.7± 1.83.51 ±
6210 ± ±108191 ±1072.2 l.S5.3± 1.53.1± 1.71.2±
±2350 ±30335 ±321.0 0.75.8± 1.74.8± 1.72.36 ±
0.41.54± ±
0.41.88± ±
0.50.84± 0.115.3 ± 1.2133
0.60.88± 0.215.6 ± 1.1131
+ Vitamin E
AOV
CtE,E X CtCtEE, E X
CtE,E X
0.61.48± ±
0.82.3± 0.60.96 ± 0.115.0± ± 2.4134
CtE
CtNSNS X 0.60.74± 0.216.7 ± 3.8NSN8N8CtCtCtCtN8EE,
1 The basal diet contained 1% cholesterol. Vitamin E-supplemented diets contained 100 IU vitamin tocopheryl Buccinate. C h olea ty ramine (3%) was fed to rats during the last 7 days of the feeding period only. E = significant vitamin E effect. Ct = significant cholestyramine effect. * Meaniso for eight rats.
E/kg diet as dl-alphaa Analysis of variance.
TABLE 5 Results of cholesterol feeding and ritamin E supplementation on microsomal activities and other parameters in rats fed diets for 21 days (experiment 4)1 E124 Vitamin
E125 vitamin
3367 ± ±2618.6 25.5± 0.6149± ±150.7 0.119± 7.273± ±3964 ±232.0
3350 ± ±1018.7 14.8± 0.2110± ±141.9± 0.32.5 0.472± ±2626 30.4±
2381 ± ±1220.5 25.9± 0.3147± ±131.7± 0.421 6.694± 766 ± ±150.7
0.41.7±
0.62.1 ±
0.41.9± ±
0.42.6 ±
ECh.E, Ch X
0.30.8±
0.30.7± ±
0.70.8±
0.80.7 ±
ENSNSECh
D122 gFinal body wt., Initial gFood body wt., g/dayLiver intake, wt.Serum wt., K/100 g body mlSerumcholesterol, mg/100 mlLiver vitamin E, mg/100 mg/gLiver cholesterol, pg/gLiver vitamin E, rng/gLiver long-chain F.A.. parametersNADPH-dependent microsomal peroxidation,OD-532/mg lipid protein/hrCodeine HCHOformed/mg hydroxylation, nmoles protein/minCytochrome nmoles/mgproteinMicrosomal P-450, liverMicrosomal protein, mg/g liverMicrosomal vitamin E, Mg/g cholesterol, mg/g liverBasal
1>355 ± ±1919.1 14.7 ± 0.2100 ± ±131.1 0.42.5± 0.641 ± ±1028 ±83
0.118 ± 0.120 27.0± 17.5 ± 2.40.54 ± 4.81.0± ± ± 0.1-(-Cholesterol1230.4+
EChCh,
EChCh,
4.720 0.118± 118 ± 219 ± 4.70.6 ± 3.91.1± ± ± 0.1-(-Cholesterol 0.2ACV»N8NSNSChChCh,
1Basal diet was basal diet A with Wesson oil instead of Jaxmar pure vegetable oil. Cholesterol-supplemented diet contained 1% cholesterol. Vitamin E treated groups received intra peritoneal injections of 10 IU vitamin E in oil as dl-alpha-tocopherol twice weekly. «Analysisof variance. Ch = significant cholesterol effect; E = significant Vitamin E effect. NS = not significant. ' Mean±l so for seven rata.
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ResidÃ-sof cholestyramine feeding and vitamin E supplementation on tissue lipid peroxidation, microsomal activities and other parameters in rats fed cholesterol-enriched diets for 33 days (experiment 3)1
METABOLIC ALTERATIONS IN CHOLESTEROL FED ANIMALS
315
TABLE 6 Results of cholesterol-feeding on liver lipid peroxidation, enzyme activities and other parameters in guinea pigs fed diets for 72 days (experiment 6)1
changeLiver NADP-dependentlipid microsomal peroxidation,OD-532/mg protein/hrMicrosomal liverLiver protein, mg/g peroxidase3Serum glutathione glutatbione peroxidase*185
+ Cholesterol
±11*639 ±494.1 0.92.2 ± 0.130 ± 1.2107± ±1466 4.80.19± ± 0.044.9 0.317.9 ±
17546 ± 56*8.3 ± 3.1«38 ± 6.3«137 ± 24«99 ± 18145 ± 35«0.16± ± 0.095.4 0.548.7 ±
0.835.9 ± 1.918.0 ± 1.440.46 ± 0.148.2 ± 1.517.3 ± 3.00.29 ± ±0.08186
23.5«204.7 ± ±114«156 94«O.OC± ± 0.09«4.8 1.3«10.2 ± 2.3«0.28 ± ± 0.08
1The basal diet was a guinea pig stock diet plus 2% soybean oil. The cholesterol-supplemented diet was the basal diet plus 1% cholesterol. 2Mean±sr>for six guinea pigs. 3Amóles NADPtt oxidized/g tissue/ minute or/ml serum/minute. *Significantly different than the basal group, (P < O.Oö).
and 5). The concentration of microsomal vitamin E was increased by vitamin E sup plementation, but was unaffected by cho lesterol feeding (table 5). Cholesterol reeding decreased the activ ity of glutathione peroxidase in the liver (tables 2 and 3). It also decreased the activity of this enzyme in the serum in a long-term experiment (137 days), but had no effect in a short-term experiment (30 days). Cholesterol feeding also decreased the activity of liver glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. Dietary vitamin E supple mentation had no effect on these enzymes. Cholesterol feeding reduced the level of vitamin E in the serum, but increased the level in the liver. Vitamin E supplementa tion increased vitamin E levels both in the serum and in the liver. Guinea pig experiments. Results of guinea pig experiments are shown in tables 6 and 7. Cholesterol feeding significantly reduced body weight gain and increased liver weight weight, liver cholesterol, liver longchain fatty acid, liver vitamin E and serum cholesterol levels. It appeared to decrease liver soluble protein levels, but had no sig nificant effect on serum vitamin E levels.
Dietary supplementation of cholesterol markedly elevated the rate of tissue lipid peroxidation in the liver and aorta when the guinea pigs were fed the basal diet (table 7). Vitamin E administration com pletely prevented this cholesterol-feeding effect in the liver, but the prevention was not complete in the aorta. Dietary supplementation of cholesterol also drastically reduced the rate of liver microsomal NADPH-dependent lipid per oxidation when the guinea pigs were fed the basal diet. When the guinea pigs were fed the same diet supplemented with vita min E, the rate of microsomal NADPHdependent lipid peroxidation in the liver was very low and the activity was slightly increased by cholesterol feeding. Liver microsomal codeine hydroxylation was sig nificantly reduced by cholesterol feeding, but was not influenced by vitamin E ad ministration. Liver microsomal cytochrome P-450 and microsomal protein levels were significantly reduced by cholesterol feed ing. Vitamin E supplementation also re duced liver microsomal protein levels, but had no effect on cytochrome P-450 levels. Cholesterol feeding decreased the activity of glutathione peroxidase in the liver, but
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gFinal Initial body wt., gLiverbody wt., wt.Liver wt., g/100 g body mg/gLiver cholesterol, mg/gLiver long-chain F.A., mg/gSerum soluble protein, mlSerum cholesterol, mg/100 mlSerum vitamin E, mg/100 mlNon-enzymatic protein, mg/100 peroxidation,jig liver lipid liverOhr2hrNet MA/g
Basal
316
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