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The Journal of Nutrition Vol. 128 No. 1 January 1998,
pp. 116-122
School of Nutrition and Health Science, Taipei Medical College and * Graduate Institute of Home Economics, Chinese Cultural University, Taipei, Taiwan, R.O.C.
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ABSTRACT |
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Abstract
Introduction
Methods
Results
Discussion
References
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To investigate the effect of dietary oxidized frying oil (OFO) on tissue retention of vitamin C, and to explore the effect of vitamin C supplementation on tissue vitamin E concentrations and lipid peroxidation, male weanling guinea pigs were divided into four groups. Guinea pigs were fed 15% OFO diets supplemented with vitamin C at 300, 600 or 1500 mg/kg diet. Control animals were fed a diet containing 15% fresh untreated soybean oil with 300 mg/kg of vitamin C. After 60 d of feeding, body weight gain, food intake, feed efficiency and plasma triglyceride concentration were significantly lower in guinea pigs fed OFO diets than in controls (P < 0.05). However, plasma cholesterol concentration was highest in guinea pigs fed the OFO diet supplemented with 300 mg/kg vitamin C. Increasing vitamin C in OFO diets significantly reduced plasma cholesterol concentration. Plasma and tissue vitamins C and E concentrations were significantly lower in the OFO-fed guinea pigs receiving 300 mg/kg vitamin C than in controls. Greater levels of supplemental vitamin C increased tissue vitamins C and E. Guinea pigs fed OFO diets had significantly higher tissue levels of thiobarbituric acid reactive substances (TBARS) (P < 0.05) than controls. Our results demonstrated that OFO feeding, which impaired 
-tocopherol retention and increased TBARS, could be alleviated somewhat by vitamin C supplementation.
A high intake of fat is prevalent in most developed countries. It is associated with a high incidence of some chronic diseases, especially cardiovascular disease and certain types of cancer (Committee on Diet and Health 1989). Fried foods are an important fat source although their relative contribution is difficult to evaluate. Thermally oxidized fat is generally considered to contain potentially toxic lipid peroxidation products (Kubow 1992 Vitamin C exerts a powerful antioxidant effect on biological water-soluble compartments and represents an outstanding antioxidant in plasma; it reacts directly with superoxide anion (O2 We reported previously that rats fed a diet containing 15% oxidized frying oil (OFO)4 had significantly lower This study was designed to assess the influence of OFO on vitamin C status in guinea pigs which, like humans, lack an ascorbate synthetic pathway. The study also examined whether supplementary vitamin C can alleviate the impaired nutritional status in guinea pigs fed OFO.
Preparation of oxidized frying oil (OFO).
Soybean oil (President, Tainan, Taiwan) purchased from a local supermarket was subjected to the following frying process: 9 kg of soybean oil was poured into a cast iron wok (60 cm i.d., 18 cm central depth) and heated on a gas stove which was adjusted to maintain the oil temperature at 205 ± 5°C. Potatoes were peeled and cut into sticks (10 × 1.5 × 1.5 cm), then fried in the oil. Every 30 min, a batch of potato sticks (~100 g) was fried for 2.5-3 min. The potato frying process was performed for 6 h/d and was repeated for four successive days. The resultant frying oil (OFO) was stored at
Analysis of the test oils.
The OFO was analyzed for acid value, peroxide value and UV absorbance at 233 nm as reported in our previous study (Liu and Huang 1995a Formulation of the test diets.
Four diets were formulated according to recommendations for guinea pigs (NRC 1978), detailed in Table 1. The OFO diets contained 15 g/100 g of oxidized frying oil with 300, 600 and 1500 mg vitamin C/kg to yield the D300, D600 and D1500 diets, respectively. The control diet contained 15 g/100 g fresh soybean oil with 300 mg vitamin C/kg (diet F300). Diets were prepared by mixing the powdered ingredients and were stored at Care of animals.
Twenty-four male weanling guinea pigs (N:HART) were purchased from the Laboratory Animal Center, College of Medicine, National Taiwan University. They were housed individually in stainless steel wire cages in a room maintained at 23 ± 2°C with a controlled 12-h light:dark cycle. The guinea pigs were fed a nonpurified diet (Teklad 0736 guinea pig diet, Teklad, Madison, WI) for 1 wk, and the control diet (F300) for 1~2 wk until they grew steadily. They were then randomly assigned to four dietary groups described in Table 1. All guinea pigs had free access to food and tap water. Body weight and feed intake were recorded weekly. NIH guidelines for care were followed (NRC 1985).
Tissue sampling and preparation.
After 60 d of feeding, guinea pigs were anesthetized with sodium pentobarbital. Blood was collected from the abdominal vena cava into a heparin-containing tube and analyzed for hematologic variables. Plasma was separated by centrifugation at 1000 × g for 10 min, and stored at Analysis.
Fresh blood samples were analyzed for hematocrit by centrifugation at 12,000 g for 5 min at 25°C in a capillary tube and for hemoglobin by the cyanomethemoglobin method (Oser 1965
Statistical analysis.
Significant difference between groups was analyzed by ANOVA and Duncan`s multiple range test with the use of the General Linear Model of the SAS package (SAS Institute, Cary, NC). Linear regression analysis was employed to assess the effect of dietary vitamin C on blood variables and on the tissue concentrations of vitamin C, vitamin E and TBARS among the three OFO-fed groups. Differences of P < 0.05 were considered significant.
Analysis of the test oils.
The quality of soybean oil declined considerably throughout the frying process. The acid value and UV absorbance at 233 nm showed a steady increase with prolonged frying. Concentrations of total polar compounds and non-urea adductable fractions were found to be 42.3 and 16.7%, respectively, after the 24-h process; these values were 7.6- and 10-fold higher, respectively, than in fresh soybean oil.
Animal growth.
After 60 d of feeding, body weight gain and food intake of guinea pigs fed the OFO diet were significantly lower than those of the controls (Table2). The feed efficiencies of guinea pigs fed the OFO diets (D300 and D600) were significantly lower than those of guinea pigs fed the control diet. However, supplementation with 1500 mg vitamin C/kg (D1500) significantly (P < 0.05) improved feed efficiency to a value that was not different than that of the control group. Diarrhea was observed in all guinea pigs fed OFO diets but not in controls.
Hematologic and lipid values in blood.
The OFO-fed guinea pigs (D300) had lower hematocrits and hemoglobin concentrations than controls (Table 3). However, guinea pigs fed D600 and D1500 did not differ from controls. Plasma GOT and GPT activities were significantly higher in OFO-fed groups than controls, and vitamin C supplementation lowered both values in a dose-dependent manner (r < Tissue and plasma vitamin C.
Guinea pigs fed the D300 diet had lower vitamin C concentration than controls in all tissues (Table 4). On the other hand, guinea pigs fed OFO supplemented with higher levels of vitamin C (D600, D1500) generally had higher vitamin C concentrations in plasma and tissues other than brain than the control and D300 groups.
Tissue vitamin E.
Guinea pigs fed D300 diets had lower vitamin E ( Tissue TBARS (thiobarbituric acid reactive substances).
Generally, dietary OFO caused a significantly elevation of the TBARS concentrations in the tissues of guinea pigs (Table 6). Guinea pigs fed the D300 diet had the highest tissue TBARS concentrations. When dietary vitamin C level increased (D600 and D1500), lower TBARS concentrations were found in tissues compared with the D300 group except in heart and testis of the D600 group.
Correlations by linear regression analysis.
Table 7 shows correlation coefficients from linear regression analysis between dietary vitamin C, and tissue vitamin C, vitamin E and TBARS among the three OFO-fed groups. There was a significant positive correlation between dietary vitamin C and tissue vitamin C in all tissues, and between dietary vitamin C and tissue vitamin E concentration except in heart, spleen and testis. In contrast, a significant inverse relationship was found between dietary vitamin C and tissue TBARS except in brain, heart and testis.
The frying process oxidized the soybean oil and generated 42.3% total polar compounds and 16.7% non-urea adductable fractions in the oil. Values from our OFO sample were in the range of those obtained from street vendors in the Taipei area (Hau et al. 1987). TBARS was 10-fold greater after a 24-h frying process, which generated numerous secondary products of lipid peroxidation.
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
).
), hydroxyl radical (OH·) and various lipid hydroperoxides (Frei et al. 1989, Jacob 1995, Yu 1994). Vitamin E is the major lipid-soluble chain-breaking antioxidant that protects biomembranes against oxidative injury. The interaction between vitamin C and vitamin E is interesting. Some sparing actions of vitamin C on vitamin E or synergism between these two vitamins has been shown in model systems (Niki et al. 1982 and 1985); however, direct and convincing evidence of these mechanisms in a biological system has yet to be demonstrated.
-tocopherol concentrations in plasma and most tissues than rats fed a diet containing a similar level of fresh soybean oil (Liu and Huang 1995a). These OFO-fed rats also had accelerated body -tocopherol catabolism/turnover in a radioisotope study (Liu and Huang 1996). We also found increased urinary vitamin C in the OFO-fed rats (Liu 1993).
MATERIALS AND METHODS
Abstract
Introduction
Methods
Results
Discussion
References
20°C until the test diets were prepared.
View this table:
Table 1.
Composition of test diets
View this table:
Table 2.
Body weight gain, food intake and feed efficiency of guinea pigs fed fresh soybean oil or oxidized frying oil diets supplemented with 300, 600 or 1500 mg vitamin C/kg for 60 d1,2
). Total polar compounds (Waltking and Wessel 1981) and the non-urea adductable fractions (Firestone et al. 1961) were also determined.
20°C. The oil portion was blended in each week before feeding.
30°C until assayed. Liver, kidney, heart, spleen, lung, brain, adrenal gland, testis, epididymal fat pad and vastus lateralis muscle tissues were excised, weighed, quick-frozen in liquid nitrogen and stored at 30°C.
). Plasma glutamate-oxaloacetate transaminase (GOT, EC 2.6.1.1), and glutamate-pyruvate transaminase (GPT, EC 2.6.1.2) activities were determined by SCE (Scandinavian Committee on Enzymes)-UV methods using commercial kits (Randox Lab, Crumlin, Antrium, UK). Plasma cholesterol and triglyceride concentrations were also determined by enzymatic-colorimetric methods using commercial kits (Randox Lab).
). Either 0.5 mL plasma or 1 mL tissue homogenate (25%, homogenized in 0.01 mol/L phosphate buffer, pH 7.4) was mixed with an equal amount of 0.62 mol/L trichloroacetic acid (TCA) and then centrifuged for 10 min at 3500 × g. The supernatant (0.25 mL), 0.25 mL deionized water and 0.1 mL DTC solution (0.4 g thiourea, 0.05 g CuSO4·5H2O, and 3 g 2,4- dinitrophenyl hydrazine in 4.5 mol/L H2SO4) were mixed and incubated in a 37°C water bath for 3 h. After incubation, 0.75 mL ice-cold 8.17mol/L H2SO4 was added and allowed to stand at room temperature for 30 min. The absorbance was then measured at 520 nm.
). Briefly, 1 mL tissue homogenate was deproteinized with 1 mL absolute ethanol (containing 79 mmol/L pyrogallol), saponified with saturated KOH and extracted with n-hexane (containing 1.25 g/L BHT). The vitamin E in the n-hexane layer was evaporated under nitrogen and the residue solubilized in 100 µL methanol for HPLC analysis. For plasma vitamin E analysis, 0.5 mL plasma was mixed with 2 mL absolute ethanol, 0.1 mL of 12 mol/L HCl and 6 mL n-hexane. The reverse-phase HPLC analyses were performed with a 4 × 125 mm Lichrosphere 100RP-18 column (Merck, Darmstadt, Frankfurt, Germany) containing 5-µm particles protected by a guard column with a UV detector at 292 nm. Chemlab (Shiunn-Hwa, Taipei, Taiwan) software was used on an IBM compatible personal computer to integrate and process the data.
View this table:
Table 3.
Blood analysis of guinea pigs fed fresh soybean oil or oxidized frying oil diets supplemented with 300, 600 or 1500 mg vitamin C/kg for 60 d1,2
View this table:
Table 4.
Tissue vitamin C concentrations of guinea pigs fed fresh soybean oil or oxidized frying oil diets supplemented with 300, 600 or 1500 mg vitamin C/kg for 60 d1,2
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
View this table:
Table 5.
Tissue -tocopherol concentration of guinea pigs fed fresh soybean oil or oxidized frying oil diets supplemented with 300, 600 or 1500 mg vitamin C/kg for 60 d1,2
0.70, P < 0.05). Plasma triglyceride concentration was significantly lower in guinea pigs fed OFO diets than in controls. Plasma cholesterol concentration was higher in the D300 group than in all other groups. Increasing vitamin C in OFO diets (D600 and D1500) significantly reduced plasma cholesterol. Evaluation of correlation coefficients demonstrated that dietary vitamin C had a significant effect on all blood and plasma values except for plasma triglyceride in OFO-fed guinea pigs.
-tocopherol) concentration in all tissues than controls (Table 5). However, when the dietary vitamin C level was raised to 600 and 1500 mg/kg, guinea pigs generally had higher -tocopherol concentrations in plasma and tissues than the D300 group. Guinea pigs supplemented with 1500 mg vitamin C/kg had essentially the same plasma vitamin E concentration as the control group. There were no significant differences in -tocopherol concentration in tissues between the D1500 and F300 groups except in brain and liver.
View this table:
Table 6.
Thiobarbituric acid-reactive substances in tissues from guinea pigs fed fresh soybean oil or oxidized frying oil diet with 300, 600 or 1500 mg vitamin C/kg1,2
View this table:
Table 7.
Correlations between dietary vitamin C and tissue vitamin C, tissue vitamin E, and tissue thiobarbituric acid reactive substances (TBARS) levels in guinea pigs fed with oxidized frying oil (OFO) diets
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
, Liu and Huang 1995a). However, in this study, body weight gain, food intake and feed efficiency were all significantly lower in OFO-fed guinea pigs, especially in the D300 and D600 groups. Higher dietary vitamin C restored the feed efficiency to the level of that of the control group. Similarly, higher dietary vitamin C significantly improved several blood variables such as hemoglobin, hematocrit, and plasma GOT and GPT activities in OFO-fed guinea pigs. Kato et al. (1977) showed that growth depression of guinea pigs caused by polychlorinated biphenyls (PCB) was appreciably ameliorated by dietary vitamin C. Our results demonstrated that vitamin C is not only an essential nutrient for the growth of guinea pigs, but it also strongly protected against pathologic conditions associated with oxidative stress. Our previous studies showed that plasma cholesterol and triglyceride levels were significantly lower in OFO-fed rats than in controls as a result of a reduction in the absorption of fat (Liu and Huang 1995a and 1995b). In this study, we found a similar reduction of plasma triglyceride concentration in OFO-fed groups compared with controls. In contrast, the plasma cholesterol concentration of the OFO-fed group (D300) was higher than that of the control group. Vitamin C is necessary for the activation of 7-hydroxylase, and vitamin C deficiency attenuates hepatic cholesterol-7-hydroxylase, a rate limiting enzyme responsible for the transformation of cholesterol to bile acid (Holloway and Rivers 1981). A negative correlation between plasma cholesterol concentration and dietary vitamin C has been reported (Jayachandran et al. 1996). The decrease in plasma cholesterol due to vitamin C supplementation observed in this study may be attributed to the activation of 7-hydroxylase by vitamin C.
, Kato and Yoshida 1979). Vitamin C is a hydrophilic antioxidant that reacts directly with O2, OH· and various lipid hydroperoxides. This and previous studies have shown that ingestion of thermally oxidized oil results in higher tissue lipid peroxidation as judged by higher tissue TBARS levels (Huang et al. 1988, Liu and Huang 1995a). Like xenobiotics and ethanol administration, OFO feeding can induce hepatic microsomal cytochrome P-450 (Huang et al. 1988 and 1989). It is believed that free radicals can be generated in the microsomal cytochrome P-450-catalyzed reactions. Therefore supplementation of vitamin C could increase the radical scavenging potential resulting from OFO-feeding, thereby protecting cells against radical-induced lipid peroxidation.
and 1985). Niki (1989) summarized some of the research that showed that vitamin C can reduce the tocopheroxyl radical to regenerate vitamin E by using a liposomal membrane system or homogeneous solutions. Chan et al. (1991) showed that vitamin C appeared to chemically regenerate the oxidized tocopherol in human platelet homogenates.
). A similar sparing effect of vitamin C on vitamin E was reported to occur in a mutant strain of Wistar rats that lacks vitamin C synthetic capacity (Igarashi et al. 1991). However, several studies reported that no interaction between the two vitamins. Burton and co-workers (1990) reported that vitamin C intake had no significant effect on vitamin E levels in blood or tissues of guinea pigs irrespective of their vitamin E status. In rats, excess vitamin C intake lowered tissue antioxidant potential during marginal vitamin E intake, thus suggesting an increased vitamin E requirement when vitamin C intake was high (Chen 1981). No "sparing effect" of vitamin C on vitamin E was reported in weanling pigs fed low vitamin E-selenium diets (Yen et al. 1985).
and 1996). The data of this study showed a significant positive correlation between dietary vitamin C levels and tissue vitamin E concentrations among the three OFO-fed groups and that dietary supplementary vitamin C raised the depleted vitamin E levels of OFO-fed guinea pigs. These data imply that vitamin C has an effective antioxidant ability as well as a sparing action on vitamin E during oxidative stress in a biological system.
and 1993). Lower vitamin C status may lead to elevated levels of tissue TBARS (Chakraborty et al. 1994, Mukhopadhyay et al. 1995). The present data also showed that supplementation of high dietary vitamin C in the OFO-fed diet significantly reduced tissue levels of TBARS in guinea pigs. This evidence clearly shows that vitamin C can suppress lipid peroxidation, thus protecting the cells from oxidative stress.
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FOOTNOTES |
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Manuscript received 5 February 1997. Initial reviews completed 19 March 1997. Revision accepted 9 September 1997.
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LITERATURE CITED |
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Abstract
Introduction
Methods
Results
Discussion
References
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-tocopherol in the male guinea pig at three dietary levels of vitamin C and two levels of vitamin E. Evidence that vitamin C dose not "spare" vitamin E in vivo.
Lipids
1990;
25:199-210
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-tocopherol retention in male rats is compromised by feeding diets containing oxidized frying oils.
J. Nutr.
1995a;
125:3071-3080
- tocopherol depletion and radioisotope tracer excretion in vitamin E-deficient rats.
J. Nutr.
1996;
126:2227-2235
-tocopherol.
Ann. N.Y. Acad. Sci.
1989;
370:186-199
-chromanoxy radical by glutathione and vitamin C. Chem. Lett. 789-792. This article has been cited by other articles:
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 A. Sulzle, F. Hirche, and K. Eder Thermally Oxidized Dietary Fat Upregulates the Expression of Target Genes of PPAR{alpha} in Rat Liver J. Nutr., June 1, 2004; 134(6): 1375 - 1383. [Abstract] [Full Text] [PDF]
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 J. A. Beckman, A. B. Goldfine, M. B. Gordon, L. A. Garrett, J. F. Keaney Jr., and M. A. Creager Oral antioxidant therapy improves endothelial function in Type 1 but not Type 2 diabetes mellitus Am J Physiol Heart Circ Physiol, December 1, 2003; 285(6): H2392 - H2398. [Abstract] [Full Text] [PDF]
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N. Khan-Merchant, M. Penumetcha, O. Meilhac, and S. Parthasarathy Oxidized Fatty Acids Promote Atherosclerosis Only in the Presence of Dietary Cholesterol in Low-Density Lipoprotein Receptor Knockout Mice J. Nutr., November 1, 2002; 132(11): 3256 - 3262. [Abstract] [Full Text] [PDF]
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M. L. Fernandez Guinea Pigs as Models for Cholesterol and Lipoprotein Metabolism J. Nutr., January 1, 2001; 131(1): 10 - 20. [Abstract] [Full Text]
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K. Eder and G. I. Stangl Plasma Thyroxine and Cholesterol Concentrations of Miniature Pigs Are Influenced by Thermally Oxidized Dietary Lipids1 J. Nutr., January 1, 2000; 130(1): 116 - 121. [Abstract] [Full Text]
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