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Influence of dietary fat saturation on lipid peroxidation of serum and low density lipoprotein in rats
Nutrition Research 22 (2002) 463– 472 www.elsevier.com/locate/nutres
Influence of dietary fat saturation on lipid peroxidation of serum and low density lipoprotein in rats Yi-Fa Lu*, Shuwen Lu Department of Nutrition and Food Sciences, Fu-Jen University, Hsinchuang, Taipei 24205, Taiwan Received 27 July 2001; accepted 31 August 2001
Abstract The purpose of this study was to investigate the influence of different P/S (polyunsaturated/ saturated) ratios on lipid peroxidation of plasma, liver and low-density lipoprotein (LDL) in rats, using dietary fats with different saturation levels. Male Sprague-Dawley rats weighing about 300 g were randomly divided into five groups and fed a semisynthetic diet containing 10% (w/w) fat for 6 weeks. The fat was composed of 100, 75, 50, 25, 0% corn oil and 0, 25, 50, 75, 100% lard (CO100, CO75, CO50, CO25 and LA groups, with P/S ratios of 3.81, 2.13, 1.21, 0.64, and 0.28, respectively) respectively. The body and liver weight of rats fed dietary fat with different P/S ratios were comparable among 5 groups. The CO100 group had higher plasma TBARS (thiobarbituric acidreactive substances) than the CO25 and LA groups. The TBARS of LDL in LA and CO25 groups were lower than those of the other (CO100, CO75 and CO50) groups. In addition, the rats fed P/S ratio ⬉1.21 (i.e., CO50, CO25, and LA groups) had lower LDL TBARS than rats fed P/S ratio ⬎1.21 (i.e., CO100 and CO75 groups) when LDL were incubated with Cu2⫹ at 37 °C for 2 or 5 h. On the other hand, rats fed diets with P/S ratio ⬉1.21 experienced significantly increase lag time of conjugated diene formation in LDL than those fed P/S ⬎1.21. The fatty acid content of 18:2 (n-6) in LDL was reduced by decreasing the ratio of P/S in dietary fat. The ratio of linoleic acid content to oleic acid content was inversely correlated with the lag time of LDL oxidation. However, different P/S ratio in the diet did not affect 18:1 (n ⫺ 9), vitamin E levels and the TBARS values in liver of rats. The results suggested that P/S ratios in the diet influenced the LDL oxidation, and the lipid peroxidation might have a threshold of P/S ratios between 1.21 and 0.64. © 2002 Elsevier Science Inc. All rights reserved. Keywords: LDL; Dietary fat; Lipid peroxidation; TBARS; Conjugated diene; P/S ratio
* Corresponding author. Tel.: ⫹886-2-2903-1111 ext 3615; fax: ⫹886-2-2902-1215. E-mail address: [email protected] (Y.-F. Lu). 0271-5317/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 2 7 1 - 5 3 1 7 ( 0 1 ) 0 0 4 0 4 - 3
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1. Introduction LDL (low density lipoprotein) is the major carrier of cholesterol in plasma, transporting it to the liver and peripheral tissues. In general, plasma levels of total cholesterol correlate highly with cholesterol concentrations of LDL [1]. Several lines of evidence indicate that diets rich in saturated fatty acids (SFA) raise serum lipid concentration [2– 4], while polyunsaturated fatty acids (PUFA) lower it [5]. Moreover, SFA increases plasma cholesterol concentrations twice as much as PUFA decreases it [6]. Avoiding excess intake of SFA has been recommended because hypercholesterolemia appears critical to atherogenic process and subsequently leads to serious cardiovascular disease. On the other hand, PUFA have some benefit of lowering total and LDL-cholesterol, and finally lowering the risk of cardiovascular disease. However, it is well known that PUFA are more susceptible to lipid peroxidation than SFA and the rate of oxidation is linearly dependent upon the PUFA concentration [7–9]. Furthermore, LDL oxidizability is dependent on the amount of related substrates, such as its fatty acid compositions, cholesterol contents, and antioxidant contents as well [7,10 –12]. Lipid oxidation usually results in decreasing membrane fluidity, cell injury, DNA damage and may cause the formation of atherosclerotic plaques [13]. Of course, antioxidants play an important role in preventing lipid oxidation and slowing the progression of atherosclerotic lesions [14]. On the other hand, animal studies have also demonstrated that the types and levels of dietary fat affect the susceptibility of lipid peroxidation and oxidative damage to cells [15]. The oxidation hypothesis of atherosclerosis suggests that circulating LDL is oxidized in vivo which leads to its enhanced uptake by macrophages inside the arterial system, and is believed to subsequently result in foam cell formation, one of the first stages of atherogenesis [16 –18]. Therefore, retardation of oxidatively modified LDL would be helpful in lowering the incidence of cardiovascular disease [19]. This work has attracted increasing interest during the last decades. Nevertheless, it is conceivable that the different constituents of LDL in relation to oxidation still remain unclear. The purpose of this investigation was to study the effects of different P/S (polyunsaturated/saturated) ratios on lipid oxidation of plasma, liver and LDL in rats, using corn oil and lard to prepare the different saturations of dietary fat. The study also investigated whether there is a maximum point of lipid peroxidation as P/S ratios changes in the diet.
2. Materials and methods 2.1. Animals and diets Thirty male Sprague-Dawley rats (National Laboratory Animal Breeding and Research Center, National Science Council, Taipei, Taiwan) weighing about 300 g were housed individually in stainless steel wire cages in a room maintained at 22–24°C with a controlled 12-h light— dark cycle. After 3 days adaptation to a commercial diet (#5001, PMI Feeds Inc., USA), rats were randomly divided into 5 groups (CO100, CO75, CO50, CO25 and LA groups with the P/S ratios 3.81, 2.13, 1.21, 0.64, and 0.28, respectively) and were given ad
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Table 1 Composition of experimental diets Ingredients 2
Casein Corn starch3 Sucrose Corn oil4 Lard5 ␣-Cellulose2 Mineral mixture2 Vitamin mixture2 Choline bitartrate6 DL-methionine6 Fatty acid composition7 (%) 14:0 16:0 18:0 18:1 (n ⫺ 9) 18:2 (n ⫺ 6) 18:3 (n ⫺ 3) P/S ratio8
Groups1 CO100
CO75
CO50
CO25
LA
20 45 15 10 — 5 3.5 1 0.2 0.3
20 45 15 7.5 2.5 5 3.5 1 0.2 0.3
20 45 15 5 5 5 3.5 1 0.2 0.3
20 45 15 2.5 7.5 5 3.5 1 0.2 0.3
20 45 15 — 10 5 3.5 1 0.2 0.3
0.1 10.8 2.1 26.3 57.9 1.0 3.81
0.8 15.2 4.7 29.8 45.8 1.0 2.13
1.2 19.9 7.3 33.6 33.6 0.9 1.21
1.6 24.7 9.9 37.5 21.1 0.8 0.64
1.9 27.7 11.7 39.9 13.2 0.7 0.28
CO100: 100% corn oil, CO75: 75% corn oil ⫹ 25% lard, CO50; 50% corn oil ⫹ 50% lard, CO25: 25% corn oil ⫹ 75% lard, LA: 100% lard. 2 Casein, ␣-cellulose, mineral premix and vitamin premix (ICN Pharmaceuticals, Inc., Cleveland, OH). 3 Corn starch (Sailer Brand, Netherlands). 4 Corn oil (Feng-Her Co., Taipei). 5 Lard (Jeng-Yi Co., Kaoshsiung, Taiwan). 6 Choline bitartrate and DL-methionine (Sigma). 7 Fatty acids comprising less than 1%, from duplicate assays, were omitted. 8 P: polyunsaturated fatty acids, S: saturated fatty acids. 1
libitum experimental diet (Table 1) and water for 6 weeks. The diet composition was according to the formula recommended by the American Institute of Nutrition [20]. The 10% fat comprised different ratios of corn oil and lard. Food intake and body weight were recorded daily and weekly, respectively. All animal experimental procedures were approved by the Animal Committee of Fu Jen University. 2.2. Sample preparation At the end of the experiment, animals were fasted overnight and sacrificed under diethyl ether anesthesia by withdrawing blood using a heparin-containing vacuum tube from the abdominal aorta between 9:00 am. and 11:00 am. Livers were removed immediately. Plasma and liver handling and storage were performed as previously described [21]. Liver homogenate was prepared with 10 volumes of ice-cold 10 mM phosphate buffer saline (PBS, pH 7.4) containing 1.15% KCl using a Potter-Elvehjem type homogenizer. Portions of the homogenates were measured immediately for thiobarbituric acid-reactive substances (TBARS).
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2.3. LDL isolation and oxidation LDL (1.019 –1.063 g/mL) was isolated by a sequential ultracentrifugation as previously described [22] using a 70.1 Ti rotor in a Beckman model L8 –70M ultracentrifuge (Palo Alto, CA). In brief, plasma, with previously added 0.1% EDTA solution to prevent lipid oxidation, was adjusted for density with KBr to 1.019 and centrifuged at 40,000 rpm for 16 h at 4°C. After removing the top layer, the density of the remaining fraction was adjusted to 1.063 and centrifuged at 40,000 rpm for 18 h. The top of the LDL fraction was collected, then dialyzed in the dark for 24 h at 4°C against 2 liters 10 mM PBS (pH 7.4) to eliminate KBr. The dialyzed LDL was diluted before the start of the oxidation with EDTA-free PBS buffer (pH 7.4) to a final concentration of 0.2 mg protein/mL. For conjugated diene assay, oxidation was initiated by addition of a freshly prepared CuCl2 solution (final concentration 10 M) and monitored continuously at 234 nm absorbance at room temperature on a Jasco V-530 specrophotometer (Jasco International Co., Ltd., Tokyo, Japan) [23]. Absorbance was recorded every 5 min for at least 4 h. The lag time was defined as the interval between the intercept of the linear least square slope of the curve with the initial absorbance axis, expressed in minutes. The in vitro oxidation of LDL was incubated with CuCl2 at a final concentration of 10 M at 37°C in a shaking water-bath for 0, 2 and 5 hours. The dialyzed and diluted LDL were stored under nitrogen gas in the dark at 4°C for a maximum of 1 week. 2.4. Chemical analysis The protein content of LDL was determined by the method of Lowry et al. [24] using bovine serum albumin as the standard. The TBARS formed during the previous step were assayed by addition of 2 volumes of 0.755% TBA reagent (30% trichloroacetic acid 10 mL/4 N HCl 1.25mL/2% BHT 112 L) and heated for 20 min at 100°C [25]. After cooling and centrifugation, the clear supernatant was measured at 532 nm. The lipid peroxidation in terms of TBARS was calculated using an extinction coefficient of 1.56 ⫻ 105 M⫺1cm⫺1. Plasma cholesterol was determined by commercial enzymatic kits (Boehringer Mannheim GmbH, Mannheim, Germany). LDL lipids were extracted [26] and fatty acids were analyzed after saponification and methylation [27]. Fatty acid analysis was performed by GLC using a Shimadzu GC-14A gas chromatograph equipped with flame ionization detector and a glass column (3 m ⫻ 2.0 mm) packed with 10% SP-2330 on Chromosorb WAW, 100/120 mesh (Supelco, Barcelona). Nitrogen was used as a carrier gas. The temperature of the oven and injection port were maintained at 190°C and 220°C, respectively. Peaks were identified by comparison of retention times with known methyl ester standards (Sigma Chemical Co.) and results were reported as area percentages using an integrator (Shimadzu C-R6A). Vitamin E contents of liver and sample corn oil were determined by reverse-phase HPLC as reported by Hatam and Kayden [28]. One gram of liver was first extracted by the method of Folch et al. [26] for lipids, then saponified in saturated KOH in a water bath (70°C), extracted with hexane, dried under nitrogen gas and resuspended in methanol/ascorbic acid before injection onto the high-performance liquid chromatograph. For analysis of sample corn oil, 0.5 g oil was directly saponified and extracted as described above. A Shimazu LC-10AD HPLC (Tokyo, Japan) was used with a 4 ⫻ 125 mm LiChrospher RP-18 column containing 5 m
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Table 2 Thiobarbituric acid reactive substances (TBARS) of plasma and liver, and liver ␣-tocopherol levels in rats fed different saturation of fat1,2 CO100 Plasma TBARS (nmol/mL) Liver TBARS (nmol/g) Liver ␣-tocopherol (nmol/g)
CO75
12.4 ⫾ 2.6 28.4 ⫾ 5.4 32.6 ⫾ 7.4
a
CO50
10.8 ⫾ 2.9 31.6 ⫾ 5.0 36.3 ⫾ 4.4
a,b
CO25
9.2 ⫾ 1.6 39.6 ⫾ 4.2 40.6 ⫾ 4.9
a,b
LA
8.1 ⫾ 1.3 34.4 ⫾ 4.4 38.2 ⫾ 5.5
b
8.2 ⫾ 1.2b 34.8 ⫾ 4.4 34.8 ⫾ 6.2
Values are means ⫾ SD, n ⫽ 6. Abbreviation: see footnote in Table 1. a,b Values in the same row not sharing common letters are significantly different at P ⬍ 0.05. 1 2
particles (E Merck, Darmstadt, Germany). Methanol was used as an eluting solvent at a flow rate of 1.0 mL/min. The ␣-tocopherol was detected at UV 292 nm (SPD-10AV UV-VIS Detector, Shimadzu). A pure standard material of ␣-tocopherol (Sigma Chemical Co., St. Louis, MO, USA) was used to construct a standard curve, and the retention time of a typical ␣-tocopherol peak was 5.5 ⫾ 0.2 min. Chemlab software (Scientific Information Service Co., Taipei, Taiwan) and a personal computer were used to integrate and run the data. 2.5. Statistical analysis The results were expressed as means ⫾ SD for six rats in each group. Data were analyzed by a one-way analysis of variance followed by inspection of all differences between pairs of means by Duncan’s multiple-range test. P ⬍ 0.05 was regarded to be statistically significant.
3. Results and discussion Rats weighing an initial average of 288 to 320 g, consumed 21.9 to 29.5 g/day of diets during 6-week feeding, and the final body weight was 475 to 531 g. The average of final body weight, liver weight and feed efficiency of rats fed dietary fat with different P/S ratios were comparable among the five groups. Assessment of lipid peroxidation by determining the formation of malondialdehyde (MDA), a secondary product of PUFA oxidation, was measured as MDA-2-thiobarbituric acid (TBA) complex (TBARS). This method is commonly used in clinical practice to measure human oxidative stress status due to its simplicity and sensitivity [29,30], although it has long been criticized as lacking specificity. The plasma TBARS of rats fed lard (LA) or 75% lard and 25% corn oil (CO25)-containing diets were significantly lower than rats fed corn oil (CO100) (Table 2). It seemed that dietary fat with P/S ratio below 0.64 effectively reduced lipid peroxidation (reduction of 34% compared to that for P/S ⫽ 3.81) of plasma in the rats. This is in agreement with the present concept that diets containing more PUFA would be more easily susceptible to oxidation. On the other hand, rats fed dietary fat with P/S ratio between 0.28 and 3.87 exhibited no change in liver TBARS or ␣-tocopherol content among the five groups (Table 2). The results
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Table 3 Lag time of conjugated diene formation and thiobarbituric acid-reactive substances (TBARS) formed during oxidation of LDL in rats fed different saturations of fat1,2 CO100
CO75
CO50
CO25
LA
Lag time (min)
151 ⫾ 22b,c 174 ⫾ 22a,b 201 ⫾ 29a,b 217 ⫾ 25a 130 ⫾ 18c LDL TBARS3 (nmol/mg protein) 0 hr 9.5 ⫾ 3.4a 7.3 ⫾ 2.0a 6.2 ⫾ 2.2a 4.3 ⫾ 1.6b 3.2 ⫾ 1.5b 2 hr 22.8 ⫾ 10.3a,* 20.9 ⫾ 1.6a,* 12.3 ⫾ 2.9b,* 12.4 ⫾ 2.9b,* 6.1 ⫾ 2.8b 5 hr 25.6 ⫾ 8.0a,* 26.6 ⫾ 4.5a,* 18.5 ⫾ 2.2b,* 20.4 ⫾ 2.7b,* 16.7 ⫾ 3.6b,* Values are means ⫾ SD, n ⫽ 6. Abbreviation: see footnote in Table 1. 3 LDL (200 g/mL) obtained from each group incubated with 10 M CuCl2 at 37°C. *Significantly different from the same group at 0 hr by Student’s t-test (P ⬍ 0.05). a,b Values in the same row not sharing common letters are significantly different at P ⬍ 0.05. 1 2
are consistent with the finding by Buckingham [9] that the malondialdehyde level in liver was not significantly influenced by elevated P/S ratios from 0.38 to 2.30, either when vitamin E was deficient or fed at increasing levels up to 2.5 times the recognized requirement for the rat. Although we did not restrict the intake of dietary vitamin E, the vitamin mixture (ICN) in sucrose provides vitamin E content in diet equal to 50 IU/kg. Even if corn oil is generally a vitamin E-rich oil and contains ␣-tocopherol at the level of 76 g/kg even in the CO100 group, the levels only corresponded to one seventh of the recommended requirement which is 5,000 IU/kg for growing rats. Table 3 shows that native LDL of rats fed lard or 75% lard and 25% corn oil (CO25)containing diets had lower TBARS than rats fed diets containing more than 50% corn oil-containing diet (CO100, CO75 and CO50 groups); however, no significant difference was seen among latter groups. Additionally, the TBARS values of LDL were lower in rats fed a diet containing corn oil below 50% (i.e., CO50, CO25 and LA groups) than those over 50% (CO100 and CO75 groups) when the LDL was incubated with Cu2⫹ (10 M) at 37°C after either 2 or 5 h. On the other hand, when compared to native LDL (without incubation), the LDL of rats fed corn oil-containing diets (i.e., CO100, CO75, CO50 and CO25 groups) incubated with Cu2⫹ had higher TBARS after 2 h incubation, while LDL of LA groups did not have higher TBARS values until 5 h incubation. The present study clearly demonstrated that both in plasma and LDL, the TBARS values were significantly decreased in CO25 and lard groups, suggesting that for reduction of lipid peroxidation in plasma and LDL, the P/S ratio of dietary fat should be as low as 0.64 and not exceed 1.21 at the maximum. The lag time, in LDL, for conjugated diene formation showed that diets containing corn oil below 50% (P/S ratio less than 1.21, including CO50, CO25 and LA groups) could effectively retard LDL peroxidation in the initial step of lipid peroxidation in rats suggesting that a more saturated fat diet would lengthen the LDL oxidation in the early stage (Table 3). The LDL oxidation is strongly dependent upon its PUFA and ␣-tocopherol content [11]. Undoubtedly, cholesterol, owing to its unsaturated double bond, producing cholesterol oxidation products, could also initiate LDL peroxidation [10]. The cholesterol concentrations of plasma in individual groups were comparable (average from 1.77 ⫾ 0.21 to 2.01 ⫾ 0.25
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Table 4 Effect of different of saturations of fat on fatty acid compositions of low density lipoprotein in rats1,2 Fatty acids
CO100
CO75
CO50
CO25
LA
14:0 16:0 16:1 (n ⫺ 18:0 18:1 (n ⫺ 18:2 (n ⫺ 18:3 (n ⫺ 20:3 (n ⫺ 20:4 (n ⫺ 20:5 (n ⫺ 22:5 (n ⫺ 22:6 (n ⫺ 18:2/18:1 ⌺PUFA UI3
0.4 ⫾ 0.1b 17.4 ⫾ 2.6 2.8 ⫾ 0.7 8.9 ⫾ 1.5b 20.8 ⫾ 3.0 22.0 ⫾ 2.6a 0.7 ⫾ 0.3 1.0 ⫾ 0.4 18.3 ⫾ 3.3 0.8 ⫾ 0.7a 1.2 ⫾ 0.3a 0.7 ⫾ 0.2 1.06 ⫾ 0.07a 44.9 ⫾ 4.3a 136 ⫾ 10a
0.3 ⫾ 0.1b 18.4 ⫾ 2.4 2.8 ⫾ 0.7 8.2 ⫾ 1.1b 21.8 ⫾ 2.1 19.6 ⫾ 1.1b 0.6 ⫾ 0.3 1.1 ⫾ 0.4 18.0 ⫾ 2.0 1.7 ⫾ 1.4a 1.2 ⫾ 0.3a 0.9 ⫾ 0.4 0.91 ⫾ 0.08b 42.6 ⫾ 3.9a 135 ⫾ 11a
(%) 3.2 ⫾ 1.1a 19.9 ⫾ 1.8 3.3 ⫾ 0.5 7.7 ⫾ 0.7b 20.6 ⫾ 1.4 14.9 ⫾ 1.4c 0.5 ⫾ 0.2 1.1 ⫾ 0.4 19.3 ⫾ 2.5 0.3 ⫾ 0.4b 0.7 ⫾ 0.1b 0.9 ⫾ 0.1 0.71 ⫾ 0.03c 37.5 ⫾ 3.2a,b 121 ⫾ 8a,b
2.7 ⫾ 0.7a 19.8 ⫾ 1.7 3.0 ⫾ 0.5 11.1 ⫾ 0.9a 21.9 ⫾ 2.1 10.3 ⫾ 0.9d 0.6 ⫾ 0.2 1.1 ⫾ 0.2 19.4 ⫾ 3.1 0.1 ⫾ 0.2b 1.4 ⫾ 1.2a 1.1 ⫾ 0.3 0.45 ⫾ 0.03d 34.5 ⫾ 3.5b 116 ⫾ 8b
5.3 ⫾ 3.7a 19.2 ⫾ 2.2 2.3 ⫾ 0.3 9.0 ⫾ 1.5a,b 23.6 ⫾ 5.1 10.7 ⫾ 1.5d 0.7 ⫾ 0.1 1.1 ⫾ 0.6 20.2 ⫾ 4.0 0.3 ⫾ 0.2b 0.9 ⫾ 0.2a,b 1.1 ⫾ 0.3 0.45 ⫾ 0.06d 35.1 ⫾ 4.1b 118 ⫾ 9b
7) 9) 6) 3) 6) 6) 3) 6) 3)
Values are means ⫾ SD, n ⫽ 6. Abbreviation: see footnote in Table 1. 3 UI: Unsaturation index ⫽ sum of the percentages of individual unsaturated fatty acids ⫻ number of double bond (s). a,b,c,d Values in the same row not sharing common letters are significantly different at P ⬍ 0.05. 1
2
mmol/L). The oxysterol contents in plasma, if any, might be not significantly different among the five groups. On the other hand, ␣-Tocopherol, a natural fat-soluble vitamin, is the most abundant antioxidant in LDL [31]. It has been demonstrated that the length of the lag time in LDL oxidation seems highly correlated with the ␣-tocopherol content of LDL [11] and heavy oxidation of LDL occurs only when the endogenous vitamin E contained in LDL is exhausted [32]. Other studies showed that the lag time of LDL is not linearly correlated with the ␣-tocopherol [33,34], especially using Cu2⫹ as a proxidant [15] or without antioxidant supplement [32]. Although the vitamin E content of diets in the present study was not severely restricted, and because LDL samples were too deficient to determine the ␣-tocopherol levels, the lag time of LDL autoxidation might mainly increase with increase in saturated fat as discussed in a later paragraph (Table 4). Moreover, it is worth noting that the longer the lag time of conjugated diene formation, the lower TABRS of LDL, as shown in Table 3. A negatively significant correlation was obtained between the lag time of LDL and LDL TBARS at zero time (Fig. 1). The significant difference for dietary fat containing less than 50% corn oil suggests that the lipid saturation of LDL played an important role in LDL autoxidation in this study. The saturation of FA composition of LDL in rats reflected on the increased saturation of dietary fat, i.e., the PUFA content (especially 18:2, n ⫺ 6) in LDL gradually decreased with the decrease of corn oil in diet (Table 4). Although oleic acid (18:1, n ⫺ 9) in LDL among the 5 groups was not significantly altered with the different saturation of dietary fat, the 18:2 (n ⫺ 6)/18:1 (n ⫺ 9) ratio of LDL decreased with the P/S ratio decrease in experimental diets. A significant correlation was found between the lag time of diene accumulation and
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Fig. 1. Relationship between the lag time of conjugated diene formation and LDL TBARS at zero time.
18:2/18:1 ratio (r ⫽ ⫺0.614, P ⬍ 0.01), resembling the results of a study of healthy humans for early LDL oxidizability [34], or vitamin E deficient subjects [12]. Esterbauer et al. [9] also pointed out that the ratio of PUFA to SFA might contribute to one of several factors influencing the LDL oxidative susceptibility. A strongly negative correlation (r ⫽ ⫺0.638, P ⬍ 0.01) was also observed between the lag time and unsaturation index of the fatty acids in LDL. The arachidonic acid (20:4, n ⫺ 6), derived from linoleic acid via desaturation and elongation, is found exclusively in animal products and can be oxidized to eicosanoids. Lenz et al. [35] have pointed out that oxidized LDL showed decreased content of linoleate and arachidonate with increased concentrations of TBARS. Although the fatty acids were not determined at the end of the incubation, the present study implied that dietary fat with different saturation did not affect the arachidonic acid content in native LDL among the 5 groups. It has been well established that free radical oxidation of LDL is accompanied by lipid peroxidation. Because LDL contains more complex lipids like the cholesterol esters, phospholipids and triacylglycerols, it might produce fatty acid hydroperoxides from the PUFA [35] or cholesterol oxidation products [10]. The cholesterol concentration of plasma in the rats was not significantly different among the 5 groups as previously discussed. Therefore, the major products at the earliest stages of LDL autoxidation, in vitro, might result from the PUFA hydroperoxides, and conjugated diene formation (Table 3). In this situation, the linoleic acid content in LDL might be one of the main factors influencing LDL peroxidation. The results from this study do not indicate a definit relationship to the development of atherosclerosis. The radical species initiating in vivo oxidation have not yet been identified. Moreover, the SD rats, having a very small amount of LDL, are not such good animal models for studying the process of atherosclerosis. Rats are, however, still extensively used in lipid metabolism studies, as their lipoprotein patterns are similar to a certain extent to those of human beings. The present study seems to consistently show that the autoxidation rate of
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LDL in vitro correlated with the extent of PUFA contents in LDL, suggesting that rats fed diet containing more saturated fat would be more effective in preventing plasma and LDL oxidation than rats fed diet containing more polyunsaturated fat. The ratio of 18:2 (n ⫺ 6)/18:1 (n ⫺ 9) in LDL appears to correlate with the rate of lipid peroxidation, and lipid peroxidation would have a threshold when P/S range in diet was from 1.21 to 0.64. In contrast, the role of arachidonic acid during the LDL oxidation process seemed negligible, although it is the main constituent of the lipid bilayer in cell membranes, and usually considered an important factor.
Acknowledgments The study was supported by a grant, NSC 88 –2314-B-030 – 003, from the National Science Council of Taiwan, ROC.
References [1] Grundy SM, Denke MA. Dietary influences on serum lipids and lipoproteins. J Lipid Res 1990;31:1149 –72. [2] Mattson FH, Grundy SM. Comparison of effects of dietary saturated, monounsaturated and polyunsaturated fatty acid on plasma lipids and lipoproteins in man. J Lipid Res 1985;26:194 –202. [3] Bonanoma A, Grundy SM. Effect of dietary stearic acid on plasma cholesterol and lipoprotein levels. N Engl J Med 1987;318:1244 – 8. [4] Grundy SM, Denke MA. Dietary influences on serum lipids and lipoproteins. J Lipid Res 1990;31:195–244. [5] Nestel PJ. Polyunsaturated fatty acids (n-3, n-6). Am J Clin Nutr 1987;45:1161–7. [6] Keys A, Anderson JT, Grande F. Serum cholesterol response to changes in the diet. IV. Particular saturated fatty acids in the diet. Metabolism 1965;14:776 – 87. [7] Thomas MJ, Thornburg T, Manning J, Hooper K, Rudel LL. Fatty acid composition of low-density lipoprotein influences its susceptibility to autoxidation. Biochemistry 1994;33:1828 –34. [8] Reaven P, Parthasarathy S, Grasse BJ, Miller E, Steinberg D, Witztum JL. Effects of oleate-rich and linoleate-rich diets on the susceptibility of low density lipoprotein to oxidative modification in mildly hypercholesterolemic subjects. J Clin Invest 1993;91:668 –76. [9] Buckingham KW. Effect of dietary polyunsaturated/saturated fatty acid ratio and dietary vitamin E on lipid peroxidation in the rat. J Nutr 1985;115:1425–35. [10] Esterbauer H, Gebicki J, Herbert P, Jurgens G. The role of lipid peroxidation and antioxidants in oxidative modification of LDL. Free Rad Biol Med 1992;13:341–90. [11] Esterbauer H, Striegl G, Pujl H, Rotheneder M. Continuous monitoring of in vivo oxidation of human low-density lipoprotein. Free Radic Res Comm 1989;6:67–75. [12] Kleinveld HA, Naber AHJ, Stalenhoef AFH, Demacker PNM. Oxidation resistance, oxidation rate, and extent of oxidation of human low-density lipoprotein depend on the ratio of oleic acid content to linoleic acid content: studies in vitamin E deficient subjects. Free Radic Biol Med 1993;15:273– 80. [13] Esterbauer H, Gebicki J, Puhl H, Jurgens G. The role of lipid peroxidation and antioxidants in oxidative modification of LDL. Free Radic Biol Med 1992;13:341–90. [14] Kita T, Nagano Y, Yokode M, Ishii K, Kume N, Ooshima A, Yoshida H, Kawai C. Probucol prevents the progression of atherosclerosis in Watanabe heritable hyperlipidemic rabbits, an animal model for familial hypercholesterolemia. Proc Natl Acad Sci USA 1987;84:5928 –31. [15] Thomas MJ, Rudel LL. Dietary fatty acids low density lipoprotein composition and oxidation and primate atherosclerosis. J Nutr 1996;126:1058S– 62S.
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[16] Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witztum JL. Beyond cholesterol. Modifications of low-density lipoprotein that increases its atherogenicity. N Engl J Med 1989;320:915–24. [17] Witztum JL, Steinberg D. Role of oxidized low-density lipoprotein in atherogenesis. J Clin Invest 1991; 88:1785–92. [18] Liu K, Cuddy TE, Pierce GN. Oxidative status of lipoproteins in coronary disease patients. Am Heart J 1992;123:285–90. [19] Gey KF, Puska P, Jordan P, Moser UK. Inverse correlation between plasma vitamin E and mortality from ischemic disease in cross-cultural epidemiology. Am J Clin Nutr 1991;53:326S–34S. [20] American Institute of Nutrition. Report of the AIN Ad Hoc Committee on standards for nutritional studies. J Nutr 1977;107:1340 – 8. [21] Lu YF, Lo YC. Effect of deep frying oil given with and without dietary cholesterol on lipid metabolism in rats. Nutr Res 1995;15:1783–92. [22] Imaizumi K, Lu YF, Sugano M. Characterization of serum apolipoprotein patterns in rats during suckling and post- weaning periods. Biochim Biophys Acta 1986;917:269 –78. [23] Esterbauer H, Striegl G, Puhl H, Rotheneder M. Continuous monitoring of in vitro oxidation of human low-density lipoprotein. Free Rad Res Comm 1989;6:67–75. [24] Lowry OH, Rosebrough MJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265–75. [25] Frostegard J, Nilsson J, Haegerstrand A, Wigzell H, Gidlund M. Oxidized low-density lipoprotein induces differentiation and adhesion of human monocytes and the monocytic cell line U937. Proc Natl Acad Sci USA 1990;87:904 – 8. [26] 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. [27] Matcalfe LD, Schmitz AA, Pelka JR. Rapid preparation of fatty acids methyl ester for gas chromatographic analysis. Anal Chem 1966;38:514 –23. [28] Hatam LJ, Kayden HJ. A high performance liquid chromatographic method for the determination of tocopherol in plasma, and cellular elements of the blood. J Lipid Res 1979;20:639 – 45. [29] Janero DR. Malondialdehyde and thiobarbituric acid-reactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury. Free Radic Biol Med 1990;9:515– 40. [30] Halliwell B, Chirico S. Lipid peroxidation: its mechanism, measurement, and significance. Am J Clin Nutr 1993;57:715S–25S. [31] Burton GW, Traber MG. Vitamin E: Antioxidant activity, biokinetics, and bioavailability. Ann Rev Nutr 1990;10:357– 82. [32] Jessup W, Rankin SM, De Whalley CV, Hoult JR, Scott J, Leake DS. Alpha-tocopherol consumption during low-density lipoprotein oxidation. Biochem J 1990;265:399 – 405. [33] Esterbauer H, Dieber-Rotheneder M, Striegl G, Waeg G. Role of vitamin E in preventing the oxidation of low-density lipoprotein. Am J Clin Nutr 1991;53:314S–21S. [34] Kontush A, Hubner C, Finckh B, Kohlschutter A, Beisiegel U. Low density lipoprotein oxidizability by copper correlates to its initial ubiquinol-10 and polyunsaturated fatty acid content. FEBS Lett 1994;341: 69 –73. [35] Lenz ML, Hughes H, Mitchell JR, Via DP, Guyton JR Taylor AA, Gotto AM, Smith CV. Lipid hydroperoxy and hydroxy derivatives in copper-catalyzed oxidation of low density lipoprotein. J Lipid Res 1990;31: 1043–50.
Influence of dietary fat saturation on lipid peroxidation of serum and low density lipoprotein in rats Yi-Fa Lu*, Shuwen Lu Department of Nutrition and Food Sciences, Fu-Jen University, Hsinchuang, Taipei 24205, Taiwan Received 27 July 2001; accepted 31 August 2001
Abstract The purpose of this study was to investigate the influence of different P/S (polyunsaturated/ saturated) ratios on lipid peroxidation of plasma, liver and low-density lipoprotein (LDL) in rats, using dietary fats with different saturation levels. Male Sprague-Dawley rats weighing about 300 g were randomly divided into five groups and fed a semisynthetic diet containing 10% (w/w) fat for 6 weeks. The fat was composed of 100, 75, 50, 25, 0% corn oil and 0, 25, 50, 75, 100% lard (CO100, CO75, CO50, CO25 and LA groups, with P/S ratios of 3.81, 2.13, 1.21, 0.64, and 0.28, respectively) respectively. The body and liver weight of rats fed dietary fat with different P/S ratios were comparable among 5 groups. The CO100 group had higher plasma TBARS (thiobarbituric acidreactive substances) than the CO25 and LA groups. The TBARS of LDL in LA and CO25 groups were lower than those of the other (CO100, CO75 and CO50) groups. In addition, the rats fed P/S ratio ⬉1.21 (i.e., CO50, CO25, and LA groups) had lower LDL TBARS than rats fed P/S ratio ⬎1.21 (i.e., CO100 and CO75 groups) when LDL were incubated with Cu2⫹ at 37 °C for 2 or 5 h. On the other hand, rats fed diets with P/S ratio ⬉1.21 experienced significantly increase lag time of conjugated diene formation in LDL than those fed P/S ⬎1.21. The fatty acid content of 18:2 (n-6) in LDL was reduced by decreasing the ratio of P/S in dietary fat. The ratio of linoleic acid content to oleic acid content was inversely correlated with the lag time of LDL oxidation. However, different P/S ratio in the diet did not affect 18:1 (n ⫺ 9), vitamin E levels and the TBARS values in liver of rats. The results suggested that P/S ratios in the diet influenced the LDL oxidation, and the lipid peroxidation might have a threshold of P/S ratios between 1.21 and 0.64. © 2002 Elsevier Science Inc. All rights reserved. Keywords: LDL; Dietary fat; Lipid peroxidation; TBARS; Conjugated diene; P/S ratio
* Corresponding author. Tel.: ⫹886-2-2903-1111 ext 3615; fax: ⫹886-2-2902-1215. E-mail address: [email protected] (Y.-F. Lu). 0271-5317/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 2 7 1 - 5 3 1 7 ( 0 1 ) 0 0 4 0 4 - 3
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1. Introduction LDL (low density lipoprotein) is the major carrier of cholesterol in plasma, transporting it to the liver and peripheral tissues. In general, plasma levels of total cholesterol correlate highly with cholesterol concentrations of LDL [1]. Several lines of evidence indicate that diets rich in saturated fatty acids (SFA) raise serum lipid concentration [2– 4], while polyunsaturated fatty acids (PUFA) lower it [5]. Moreover, SFA increases plasma cholesterol concentrations twice as much as PUFA decreases it [6]. Avoiding excess intake of SFA has been recommended because hypercholesterolemia appears critical to atherogenic process and subsequently leads to serious cardiovascular disease. On the other hand, PUFA have some benefit of lowering total and LDL-cholesterol, and finally lowering the risk of cardiovascular disease. However, it is well known that PUFA are more susceptible to lipid peroxidation than SFA and the rate of oxidation is linearly dependent upon the PUFA concentration [7–9]. Furthermore, LDL oxidizability is dependent on the amount of related substrates, such as its fatty acid compositions, cholesterol contents, and antioxidant contents as well [7,10 –12]. Lipid oxidation usually results in decreasing membrane fluidity, cell injury, DNA damage and may cause the formation of atherosclerotic plaques [13]. Of course, antioxidants play an important role in preventing lipid oxidation and slowing the progression of atherosclerotic lesions [14]. On the other hand, animal studies have also demonstrated that the types and levels of dietary fat affect the susceptibility of lipid peroxidation and oxidative damage to cells [15]. The oxidation hypothesis of atherosclerosis suggests that circulating LDL is oxidized in vivo which leads to its enhanced uptake by macrophages inside the arterial system, and is believed to subsequently result in foam cell formation, one of the first stages of atherogenesis [16 –18]. Therefore, retardation of oxidatively modified LDL would be helpful in lowering the incidence of cardiovascular disease [19]. This work has attracted increasing interest during the last decades. Nevertheless, it is conceivable that the different constituents of LDL in relation to oxidation still remain unclear. The purpose of this investigation was to study the effects of different P/S (polyunsaturated/saturated) ratios on lipid oxidation of plasma, liver and LDL in rats, using corn oil and lard to prepare the different saturations of dietary fat. The study also investigated whether there is a maximum point of lipid peroxidation as P/S ratios changes in the diet.
2. Materials and methods 2.1. Animals and diets Thirty male Sprague-Dawley rats (National Laboratory Animal Breeding and Research Center, National Science Council, Taipei, Taiwan) weighing about 300 g were housed individually in stainless steel wire cages in a room maintained at 22–24°C with a controlled 12-h light— dark cycle. After 3 days adaptation to a commercial diet (#5001, PMI Feeds Inc., USA), rats were randomly divided into 5 groups (CO100, CO75, CO50, CO25 and LA groups with the P/S ratios 3.81, 2.13, 1.21, 0.64, and 0.28, respectively) and were given ad
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Table 1 Composition of experimental diets Ingredients 2
Casein Corn starch3 Sucrose Corn oil4 Lard5 ␣-Cellulose2 Mineral mixture2 Vitamin mixture2 Choline bitartrate6 DL-methionine6 Fatty acid composition7 (%) 14:0 16:0 18:0 18:1 (n ⫺ 9) 18:2 (n ⫺ 6) 18:3 (n ⫺ 3) P/S ratio8
Groups1 CO100
CO75
CO50
CO25
LA
20 45 15 10 — 5 3.5 1 0.2 0.3
20 45 15 7.5 2.5 5 3.5 1 0.2 0.3
20 45 15 5 5 5 3.5 1 0.2 0.3
20 45 15 2.5 7.5 5 3.5 1 0.2 0.3
20 45 15 — 10 5 3.5 1 0.2 0.3
0.1 10.8 2.1 26.3 57.9 1.0 3.81
0.8 15.2 4.7 29.8 45.8 1.0 2.13
1.2 19.9 7.3 33.6 33.6 0.9 1.21
1.6 24.7 9.9 37.5 21.1 0.8 0.64
1.9 27.7 11.7 39.9 13.2 0.7 0.28
CO100: 100% corn oil, CO75: 75% corn oil ⫹ 25% lard, CO50; 50% corn oil ⫹ 50% lard, CO25: 25% corn oil ⫹ 75% lard, LA: 100% lard. 2 Casein, ␣-cellulose, mineral premix and vitamin premix (ICN Pharmaceuticals, Inc., Cleveland, OH). 3 Corn starch (Sailer Brand, Netherlands). 4 Corn oil (Feng-Her Co., Taipei). 5 Lard (Jeng-Yi Co., Kaoshsiung, Taiwan). 6 Choline bitartrate and DL-methionine (Sigma). 7 Fatty acids comprising less than 1%, from duplicate assays, were omitted. 8 P: polyunsaturated fatty acids, S: saturated fatty acids. 1
libitum experimental diet (Table 1) and water for 6 weeks. The diet composition was according to the formula recommended by the American Institute of Nutrition [20]. The 10% fat comprised different ratios of corn oil and lard. Food intake and body weight were recorded daily and weekly, respectively. All animal experimental procedures were approved by the Animal Committee of Fu Jen University. 2.2. Sample preparation At the end of the experiment, animals were fasted overnight and sacrificed under diethyl ether anesthesia by withdrawing blood using a heparin-containing vacuum tube from the abdominal aorta between 9:00 am. and 11:00 am. Livers were removed immediately. Plasma and liver handling and storage were performed as previously described [21]. Liver homogenate was prepared with 10 volumes of ice-cold 10 mM phosphate buffer saline (PBS, pH 7.4) containing 1.15% KCl using a Potter-Elvehjem type homogenizer. Portions of the homogenates were measured immediately for thiobarbituric acid-reactive substances (TBARS).
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2.3. LDL isolation and oxidation LDL (1.019 –1.063 g/mL) was isolated by a sequential ultracentrifugation as previously described [22] using a 70.1 Ti rotor in a Beckman model L8 –70M ultracentrifuge (Palo Alto, CA). In brief, plasma, with previously added 0.1% EDTA solution to prevent lipid oxidation, was adjusted for density with KBr to 1.019 and centrifuged at 40,000 rpm for 16 h at 4°C. After removing the top layer, the density of the remaining fraction was adjusted to 1.063 and centrifuged at 40,000 rpm for 18 h. The top of the LDL fraction was collected, then dialyzed in the dark for 24 h at 4°C against 2 liters 10 mM PBS (pH 7.4) to eliminate KBr. The dialyzed LDL was diluted before the start of the oxidation with EDTA-free PBS buffer (pH 7.4) to a final concentration of 0.2 mg protein/mL. For conjugated diene assay, oxidation was initiated by addition of a freshly prepared CuCl2 solution (final concentration 10 M) and monitored continuously at 234 nm absorbance at room temperature on a Jasco V-530 specrophotometer (Jasco International Co., Ltd., Tokyo, Japan) [23]. Absorbance was recorded every 5 min for at least 4 h. The lag time was defined as the interval between the intercept of the linear least square slope of the curve with the initial absorbance axis, expressed in minutes. The in vitro oxidation of LDL was incubated with CuCl2 at a final concentration of 10 M at 37°C in a shaking water-bath for 0, 2 and 5 hours. The dialyzed and diluted LDL were stored under nitrogen gas in the dark at 4°C for a maximum of 1 week. 2.4. Chemical analysis The protein content of LDL was determined by the method of Lowry et al. [24] using bovine serum albumin as the standard. The TBARS formed during the previous step were assayed by addition of 2 volumes of 0.755% TBA reagent (30% trichloroacetic acid 10 mL/4 N HCl 1.25mL/2% BHT 112 L) and heated for 20 min at 100°C [25]. After cooling and centrifugation, the clear supernatant was measured at 532 nm. The lipid peroxidation in terms of TBARS was calculated using an extinction coefficient of 1.56 ⫻ 105 M⫺1cm⫺1. Plasma cholesterol was determined by commercial enzymatic kits (Boehringer Mannheim GmbH, Mannheim, Germany). LDL lipids were extracted [26] and fatty acids were analyzed after saponification and methylation [27]. Fatty acid analysis was performed by GLC using a Shimadzu GC-14A gas chromatograph equipped with flame ionization detector and a glass column (3 m ⫻ 2.0 mm) packed with 10% SP-2330 on Chromosorb WAW, 100/120 mesh (Supelco, Barcelona). Nitrogen was used as a carrier gas. The temperature of the oven and injection port were maintained at 190°C and 220°C, respectively. Peaks were identified by comparison of retention times with known methyl ester standards (Sigma Chemical Co.) and results were reported as area percentages using an integrator (Shimadzu C-R6A). Vitamin E contents of liver and sample corn oil were determined by reverse-phase HPLC as reported by Hatam and Kayden [28]. One gram of liver was first extracted by the method of Folch et al. [26] for lipids, then saponified in saturated KOH in a water bath (70°C), extracted with hexane, dried under nitrogen gas and resuspended in methanol/ascorbic acid before injection onto the high-performance liquid chromatograph. For analysis of sample corn oil, 0.5 g oil was directly saponified and extracted as described above. A Shimazu LC-10AD HPLC (Tokyo, Japan) was used with a 4 ⫻ 125 mm LiChrospher RP-18 column containing 5 m
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Table 2 Thiobarbituric acid reactive substances (TBARS) of plasma and liver, and liver ␣-tocopherol levels in rats fed different saturation of fat1,2 CO100 Plasma TBARS (nmol/mL) Liver TBARS (nmol/g) Liver ␣-tocopherol (nmol/g)
CO75
12.4 ⫾ 2.6 28.4 ⫾ 5.4 32.6 ⫾ 7.4
a
CO50
10.8 ⫾ 2.9 31.6 ⫾ 5.0 36.3 ⫾ 4.4
a,b
CO25
9.2 ⫾ 1.6 39.6 ⫾ 4.2 40.6 ⫾ 4.9
a,b
LA
8.1 ⫾ 1.3 34.4 ⫾ 4.4 38.2 ⫾ 5.5
b
8.2 ⫾ 1.2b 34.8 ⫾ 4.4 34.8 ⫾ 6.2
Values are means ⫾ SD, n ⫽ 6. Abbreviation: see footnote in Table 1. a,b Values in the same row not sharing common letters are significantly different at P ⬍ 0.05. 1 2
particles (E Merck, Darmstadt, Germany). Methanol was used as an eluting solvent at a flow rate of 1.0 mL/min. The ␣-tocopherol was detected at UV 292 nm (SPD-10AV UV-VIS Detector, Shimadzu). A pure standard material of ␣-tocopherol (Sigma Chemical Co., St. Louis, MO, USA) was used to construct a standard curve, and the retention time of a typical ␣-tocopherol peak was 5.5 ⫾ 0.2 min. Chemlab software (Scientific Information Service Co., Taipei, Taiwan) and a personal computer were used to integrate and run the data. 2.5. Statistical analysis The results were expressed as means ⫾ SD for six rats in each group. Data were analyzed by a one-way analysis of variance followed by inspection of all differences between pairs of means by Duncan’s multiple-range test. P ⬍ 0.05 was regarded to be statistically significant.
3. Results and discussion Rats weighing an initial average of 288 to 320 g, consumed 21.9 to 29.5 g/day of diets during 6-week feeding, and the final body weight was 475 to 531 g. The average of final body weight, liver weight and feed efficiency of rats fed dietary fat with different P/S ratios were comparable among the five groups. Assessment of lipid peroxidation by determining the formation of malondialdehyde (MDA), a secondary product of PUFA oxidation, was measured as MDA-2-thiobarbituric acid (TBA) complex (TBARS). This method is commonly used in clinical practice to measure human oxidative stress status due to its simplicity and sensitivity [29,30], although it has long been criticized as lacking specificity. The plasma TBARS of rats fed lard (LA) or 75% lard and 25% corn oil (CO25)-containing diets were significantly lower than rats fed corn oil (CO100) (Table 2). It seemed that dietary fat with P/S ratio below 0.64 effectively reduced lipid peroxidation (reduction of 34% compared to that for P/S ⫽ 3.81) of plasma in the rats. This is in agreement with the present concept that diets containing more PUFA would be more easily susceptible to oxidation. On the other hand, rats fed dietary fat with P/S ratio between 0.28 and 3.87 exhibited no change in liver TBARS or ␣-tocopherol content among the five groups (Table 2). The results
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Table 3 Lag time of conjugated diene formation and thiobarbituric acid-reactive substances (TBARS) formed during oxidation of LDL in rats fed different saturations of fat1,2 CO100
CO75
CO50
CO25
LA
Lag time (min)
151 ⫾ 22b,c 174 ⫾ 22a,b 201 ⫾ 29a,b 217 ⫾ 25a 130 ⫾ 18c LDL TBARS3 (nmol/mg protein) 0 hr 9.5 ⫾ 3.4a 7.3 ⫾ 2.0a 6.2 ⫾ 2.2a 4.3 ⫾ 1.6b 3.2 ⫾ 1.5b 2 hr 22.8 ⫾ 10.3a,* 20.9 ⫾ 1.6a,* 12.3 ⫾ 2.9b,* 12.4 ⫾ 2.9b,* 6.1 ⫾ 2.8b 5 hr 25.6 ⫾ 8.0a,* 26.6 ⫾ 4.5a,* 18.5 ⫾ 2.2b,* 20.4 ⫾ 2.7b,* 16.7 ⫾ 3.6b,* Values are means ⫾ SD, n ⫽ 6. Abbreviation: see footnote in Table 1. 3 LDL (200 g/mL) obtained from each group incubated with 10 M CuCl2 at 37°C. *Significantly different from the same group at 0 hr by Student’s t-test (P ⬍ 0.05). a,b Values in the same row not sharing common letters are significantly different at P ⬍ 0.05. 1 2
are consistent with the finding by Buckingham [9] that the malondialdehyde level in liver was not significantly influenced by elevated P/S ratios from 0.38 to 2.30, either when vitamin E was deficient or fed at increasing levels up to 2.5 times the recognized requirement for the rat. Although we did not restrict the intake of dietary vitamin E, the vitamin mixture (ICN) in sucrose provides vitamin E content in diet equal to 50 IU/kg. Even if corn oil is generally a vitamin E-rich oil and contains ␣-tocopherol at the level of 76 g/kg even in the CO100 group, the levels only corresponded to one seventh of the recommended requirement which is 5,000 IU/kg for growing rats. Table 3 shows that native LDL of rats fed lard or 75% lard and 25% corn oil (CO25)containing diets had lower TBARS than rats fed diets containing more than 50% corn oil-containing diet (CO100, CO75 and CO50 groups); however, no significant difference was seen among latter groups. Additionally, the TBARS values of LDL were lower in rats fed a diet containing corn oil below 50% (i.e., CO50, CO25 and LA groups) than those over 50% (CO100 and CO75 groups) when the LDL was incubated with Cu2⫹ (10 M) at 37°C after either 2 or 5 h. On the other hand, when compared to native LDL (without incubation), the LDL of rats fed corn oil-containing diets (i.e., CO100, CO75, CO50 and CO25 groups) incubated with Cu2⫹ had higher TBARS after 2 h incubation, while LDL of LA groups did not have higher TBARS values until 5 h incubation. The present study clearly demonstrated that both in plasma and LDL, the TBARS values were significantly decreased in CO25 and lard groups, suggesting that for reduction of lipid peroxidation in plasma and LDL, the P/S ratio of dietary fat should be as low as 0.64 and not exceed 1.21 at the maximum. The lag time, in LDL, for conjugated diene formation showed that diets containing corn oil below 50% (P/S ratio less than 1.21, including CO50, CO25 and LA groups) could effectively retard LDL peroxidation in the initial step of lipid peroxidation in rats suggesting that a more saturated fat diet would lengthen the LDL oxidation in the early stage (Table 3). The LDL oxidation is strongly dependent upon its PUFA and ␣-tocopherol content [11]. Undoubtedly, cholesterol, owing to its unsaturated double bond, producing cholesterol oxidation products, could also initiate LDL peroxidation [10]. The cholesterol concentrations of plasma in individual groups were comparable (average from 1.77 ⫾ 0.21 to 2.01 ⫾ 0.25
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Table 4 Effect of different of saturations of fat on fatty acid compositions of low density lipoprotein in rats1,2 Fatty acids
CO100
CO75
CO50
CO25
LA
14:0 16:0 16:1 (n ⫺ 18:0 18:1 (n ⫺ 18:2 (n ⫺ 18:3 (n ⫺ 20:3 (n ⫺ 20:4 (n ⫺ 20:5 (n ⫺ 22:5 (n ⫺ 22:6 (n ⫺ 18:2/18:1 ⌺PUFA UI3
0.4 ⫾ 0.1b 17.4 ⫾ 2.6 2.8 ⫾ 0.7 8.9 ⫾ 1.5b 20.8 ⫾ 3.0 22.0 ⫾ 2.6a 0.7 ⫾ 0.3 1.0 ⫾ 0.4 18.3 ⫾ 3.3 0.8 ⫾ 0.7a 1.2 ⫾ 0.3a 0.7 ⫾ 0.2 1.06 ⫾ 0.07a 44.9 ⫾ 4.3a 136 ⫾ 10a
0.3 ⫾ 0.1b 18.4 ⫾ 2.4 2.8 ⫾ 0.7 8.2 ⫾ 1.1b 21.8 ⫾ 2.1 19.6 ⫾ 1.1b 0.6 ⫾ 0.3 1.1 ⫾ 0.4 18.0 ⫾ 2.0 1.7 ⫾ 1.4a 1.2 ⫾ 0.3a 0.9 ⫾ 0.4 0.91 ⫾ 0.08b 42.6 ⫾ 3.9a 135 ⫾ 11a
(%) 3.2 ⫾ 1.1a 19.9 ⫾ 1.8 3.3 ⫾ 0.5 7.7 ⫾ 0.7b 20.6 ⫾ 1.4 14.9 ⫾ 1.4c 0.5 ⫾ 0.2 1.1 ⫾ 0.4 19.3 ⫾ 2.5 0.3 ⫾ 0.4b 0.7 ⫾ 0.1b 0.9 ⫾ 0.1 0.71 ⫾ 0.03c 37.5 ⫾ 3.2a,b 121 ⫾ 8a,b
2.7 ⫾ 0.7a 19.8 ⫾ 1.7 3.0 ⫾ 0.5 11.1 ⫾ 0.9a 21.9 ⫾ 2.1 10.3 ⫾ 0.9d 0.6 ⫾ 0.2 1.1 ⫾ 0.2 19.4 ⫾ 3.1 0.1 ⫾ 0.2b 1.4 ⫾ 1.2a 1.1 ⫾ 0.3 0.45 ⫾ 0.03d 34.5 ⫾ 3.5b 116 ⫾ 8b
5.3 ⫾ 3.7a 19.2 ⫾ 2.2 2.3 ⫾ 0.3 9.0 ⫾ 1.5a,b 23.6 ⫾ 5.1 10.7 ⫾ 1.5d 0.7 ⫾ 0.1 1.1 ⫾ 0.6 20.2 ⫾ 4.0 0.3 ⫾ 0.2b 0.9 ⫾ 0.2a,b 1.1 ⫾ 0.3 0.45 ⫾ 0.06d 35.1 ⫾ 4.1b 118 ⫾ 9b
7) 9) 6) 3) 6) 6) 3) 6) 3)
Values are means ⫾ SD, n ⫽ 6. Abbreviation: see footnote in Table 1. 3 UI: Unsaturation index ⫽ sum of the percentages of individual unsaturated fatty acids ⫻ number of double bond (s). a,b,c,d Values in the same row not sharing common letters are significantly different at P ⬍ 0.05. 1
2
mmol/L). The oxysterol contents in plasma, if any, might be not significantly different among the five groups. On the other hand, ␣-Tocopherol, a natural fat-soluble vitamin, is the most abundant antioxidant in LDL [31]. It has been demonstrated that the length of the lag time in LDL oxidation seems highly correlated with the ␣-tocopherol content of LDL [11] and heavy oxidation of LDL occurs only when the endogenous vitamin E contained in LDL is exhausted [32]. Other studies showed that the lag time of LDL is not linearly correlated with the ␣-tocopherol [33,34], especially using Cu2⫹ as a proxidant [15] or without antioxidant supplement [32]. Although the vitamin E content of diets in the present study was not severely restricted, and because LDL samples were too deficient to determine the ␣-tocopherol levels, the lag time of LDL autoxidation might mainly increase with increase in saturated fat as discussed in a later paragraph (Table 4). Moreover, it is worth noting that the longer the lag time of conjugated diene formation, the lower TABRS of LDL, as shown in Table 3. A negatively significant correlation was obtained between the lag time of LDL and LDL TBARS at zero time (Fig. 1). The significant difference for dietary fat containing less than 50% corn oil suggests that the lipid saturation of LDL played an important role in LDL autoxidation in this study. The saturation of FA composition of LDL in rats reflected on the increased saturation of dietary fat, i.e., the PUFA content (especially 18:2, n ⫺ 6) in LDL gradually decreased with the decrease of corn oil in diet (Table 4). Although oleic acid (18:1, n ⫺ 9) in LDL among the 5 groups was not significantly altered with the different saturation of dietary fat, the 18:2 (n ⫺ 6)/18:1 (n ⫺ 9) ratio of LDL decreased with the P/S ratio decrease in experimental diets. A significant correlation was found between the lag time of diene accumulation and
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Fig. 1. Relationship between the lag time of conjugated diene formation and LDL TBARS at zero time.
18:2/18:1 ratio (r ⫽ ⫺0.614, P ⬍ 0.01), resembling the results of a study of healthy humans for early LDL oxidizability [34], or vitamin E deficient subjects [12]. Esterbauer et al. [9] also pointed out that the ratio of PUFA to SFA might contribute to one of several factors influencing the LDL oxidative susceptibility. A strongly negative correlation (r ⫽ ⫺0.638, P ⬍ 0.01) was also observed between the lag time and unsaturation index of the fatty acids in LDL. The arachidonic acid (20:4, n ⫺ 6), derived from linoleic acid via desaturation and elongation, is found exclusively in animal products and can be oxidized to eicosanoids. Lenz et al. [35] have pointed out that oxidized LDL showed decreased content of linoleate and arachidonate with increased concentrations of TBARS. Although the fatty acids were not determined at the end of the incubation, the present study implied that dietary fat with different saturation did not affect the arachidonic acid content in native LDL among the 5 groups. It has been well established that free radical oxidation of LDL is accompanied by lipid peroxidation. Because LDL contains more complex lipids like the cholesterol esters, phospholipids and triacylglycerols, it might produce fatty acid hydroperoxides from the PUFA [35] or cholesterol oxidation products [10]. The cholesterol concentration of plasma in the rats was not significantly different among the 5 groups as previously discussed. Therefore, the major products at the earliest stages of LDL autoxidation, in vitro, might result from the PUFA hydroperoxides, and conjugated diene formation (Table 3). In this situation, the linoleic acid content in LDL might be one of the main factors influencing LDL peroxidation. The results from this study do not indicate a definit relationship to the development of atherosclerosis. The radical species initiating in vivo oxidation have not yet been identified. Moreover, the SD rats, having a very small amount of LDL, are not such good animal models for studying the process of atherosclerosis. Rats are, however, still extensively used in lipid metabolism studies, as their lipoprotein patterns are similar to a certain extent to those of human beings. The present study seems to consistently show that the autoxidation rate of
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LDL in vitro correlated with the extent of PUFA contents in LDL, suggesting that rats fed diet containing more saturated fat would be more effective in preventing plasma and LDL oxidation than rats fed diet containing more polyunsaturated fat. The ratio of 18:2 (n ⫺ 6)/18:1 (n ⫺ 9) in LDL appears to correlate with the rate of lipid peroxidation, and lipid peroxidation would have a threshold when P/S range in diet was from 1.21 to 0.64. In contrast, the role of arachidonic acid during the LDL oxidation process seemed negligible, although it is the main constituent of the lipid bilayer in cell membranes, and usually considered an important factor.
Acknowledgments The study was supported by a grant, NSC 88 –2314-B-030 – 003, from the National Science Council of Taiwan, ROC.
References [1] Grundy SM, Denke MA. Dietary influences on serum lipids and lipoproteins. J Lipid Res 1990;31:1149 –72. [2] Mattson FH, Grundy SM. Comparison of effects of dietary saturated, monounsaturated and polyunsaturated fatty acid on plasma lipids and lipoproteins in man. J Lipid Res 1985;26:194 –202. [3] Bonanoma A, Grundy SM. Effect of dietary stearic acid on plasma cholesterol and lipoprotein levels. N Engl J Med 1987;318:1244 – 8. [4] Grundy SM, Denke MA. Dietary influences on serum lipids and lipoproteins. J Lipid Res 1990;31:195–244. [5] Nestel PJ. Polyunsaturated fatty acids (n-3, n-6). Am J Clin Nutr 1987;45:1161–7. [6] Keys A, Anderson JT, Grande F. Serum cholesterol response to changes in the diet. IV. Particular saturated fatty acids in the diet. Metabolism 1965;14:776 – 87. [7] Thomas MJ, Thornburg T, Manning J, Hooper K, Rudel LL. Fatty acid composition of low-density lipoprotein influences its susceptibility to autoxidation. Biochemistry 1994;33:1828 –34. [8] Reaven P, Parthasarathy S, Grasse BJ, Miller E, Steinberg D, Witztum JL. Effects of oleate-rich and linoleate-rich diets on the susceptibility of low density lipoprotein to oxidative modification in mildly hypercholesterolemic subjects. J Clin Invest 1993;91:668 –76. [9] Buckingham KW. Effect of dietary polyunsaturated/saturated fatty acid ratio and dietary vitamin E on lipid peroxidation in the rat. J Nutr 1985;115:1425–35. [10] Esterbauer H, Gebicki J, Herbert P, Jurgens G. The role of lipid peroxidation and antioxidants in oxidative modification of LDL. Free Rad Biol Med 1992;13:341–90. [11] Esterbauer H, Striegl G, Pujl H, Rotheneder M. Continuous monitoring of in vivo oxidation of human low-density lipoprotein. Free Radic Res Comm 1989;6:67–75. [12] Kleinveld HA, Naber AHJ, Stalenhoef AFH, Demacker PNM. Oxidation resistance, oxidation rate, and extent of oxidation of human low-density lipoprotein depend on the ratio of oleic acid content to linoleic acid content: studies in vitamin E deficient subjects. Free Radic Biol Med 1993;15:273– 80. [13] Esterbauer H, Gebicki J, Puhl H, Jurgens G. The role of lipid peroxidation and antioxidants in oxidative modification of LDL. Free Radic Biol Med 1992;13:341–90. [14] Kita T, Nagano Y, Yokode M, Ishii K, Kume N, Ooshima A, Yoshida H, Kawai C. Probucol prevents the progression of atherosclerosis in Watanabe heritable hyperlipidemic rabbits, an animal model for familial hypercholesterolemia. Proc Natl Acad Sci USA 1987;84:5928 –31. [15] Thomas MJ, Rudel LL. Dietary fatty acids low density lipoprotein composition and oxidation and primate atherosclerosis. J Nutr 1996;126:1058S– 62S.
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Y.-F. Lu, S. Lu / Nutrition Research 22 (2002) 463– 472
[16] Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witztum JL. Beyond cholesterol. Modifications of low-density lipoprotein that increases its atherogenicity. N Engl J Med 1989;320:915–24. [17] Witztum JL, Steinberg D. Role of oxidized low-density lipoprotein in atherogenesis. J Clin Invest 1991; 88:1785–92. [18] Liu K, Cuddy TE, Pierce GN. Oxidative status of lipoproteins in coronary disease patients. Am Heart J 1992;123:285–90. [19] Gey KF, Puska P, Jordan P, Moser UK. Inverse correlation between plasma vitamin E and mortality from ischemic disease in cross-cultural epidemiology. Am J Clin Nutr 1991;53:326S–34S. [20] American Institute of Nutrition. Report of the AIN Ad Hoc Committee on standards for nutritional studies. J Nutr 1977;107:1340 – 8. [21] Lu YF, Lo YC. Effect of deep frying oil given with and without dietary cholesterol on lipid metabolism in rats. Nutr Res 1995;15:1783–92. [22] Imaizumi K, Lu YF, Sugano M. Characterization of serum apolipoprotein patterns in rats during suckling and post- weaning periods. Biochim Biophys Acta 1986;917:269 –78. [23] Esterbauer H, Striegl G, Puhl H, Rotheneder M. Continuous monitoring of in vitro oxidation of human low-density lipoprotein. Free Rad Res Comm 1989;6:67–75. [24] Lowry OH, Rosebrough MJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265–75. [25] Frostegard J, Nilsson J, Haegerstrand A, Wigzell H, Gidlund M. Oxidized low-density lipoprotein induces differentiation and adhesion of human monocytes and the monocytic cell line U937. Proc Natl Acad Sci USA 1990;87:904 – 8. [26] 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. [27] Matcalfe LD, Schmitz AA, Pelka JR. Rapid preparation of fatty acids methyl ester for gas chromatographic analysis. Anal Chem 1966;38:514 –23. [28] Hatam LJ, Kayden HJ. A high performance liquid chromatographic method for the determination of tocopherol in plasma, and cellular elements of the blood. J Lipid Res 1979;20:639 – 45. [29] Janero DR. Malondialdehyde and thiobarbituric acid-reactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury. Free Radic Biol Med 1990;9:515– 40. [30] Halliwell B, Chirico S. Lipid peroxidation: its mechanism, measurement, and significance. Am J Clin Nutr 1993;57:715S–25S. [31] Burton GW, Traber MG. Vitamin E: Antioxidant activity, biokinetics, and bioavailability. Ann Rev Nutr 1990;10:357– 82. [32] Jessup W, Rankin SM, De Whalley CV, Hoult JR, Scott J, Leake DS. Alpha-tocopherol consumption during low-density lipoprotein oxidation. Biochem J 1990;265:399 – 405. [33] Esterbauer H, Dieber-Rotheneder M, Striegl G, Waeg G. Role of vitamin E in preventing the oxidation of low-density lipoprotein. Am J Clin Nutr 1991;53:314S–21S. [34] Kontush A, Hubner C, Finckh B, Kohlschutter A, Beisiegel U. Low density lipoprotein oxidizability by copper correlates to its initial ubiquinol-10 and polyunsaturated fatty acid content. FEBS Lett 1994;341: 69 –73. [35] Lenz ML, Hughes H, Mitchell JR, Via DP, Guyton JR Taylor AA, Gotto AM, Smith CV. Lipid hydroperoxy and hydroxy derivatives in copper-catalyzed oxidation of low density lipoprotein. J Lipid Res 1990;31: 1043–50.