Comparison between High-PUFA and Low-PUFA Fats on Lipid Peroxidation and LDL Oxidation

Christine M. Seppanen, Hyejung Cho, A. Saari Csallany

Department of Food Science and Nutrition, University of Minnesota, Minneapolis and St. Paul, USA.

DOI: 10.4236/fns.2013.45074                

Abstract

This study was conducted to determine the effects of a low polyunsaturated fatty acid (PUFA) 21 diet versus a high-PUFA diet on lipid peroxidation and on low density and very low density lipoprotein (LDL + VLDL) oxidation in vivo. Rats were fed 10% beef tallow (BT) or 10% soybean oil (SO) diet for 21 weeks. Lipid peroxidation was measured by assessing urinary 24 excretions of secondary lipid peroxidation products, by HPLC and by measuring thiobarbituric acid reactive substances (TBARS) in liver tissue. Plasma LDL + VLDL in vivo oxidation was measured by conjugated diene concentration and TBARS; ex vivo resistance to copper-induced oxidation was also assessed. Total urinary aldehydes, twelve individual urinary aldehydes, and TBARS in the liver were significantly lower in the BT group compared to the SO group. Plasma LDL + VLDL was significantly more resistant to copper-induced ex vivo oxidation to the BT group compared to the SO group. However, in vivo plasma LDL + VLDL oxidation levels measured as conjugated dienes and by TBARS were not significantly different. In general, the low-PUFA BT diet appears to have a protective effect on in vivo lipid peroxidation compared to the high-PUFA diet, but not on in vivo plasma LDL + VLDL oxidation.

Keywords

Lipid Peroxidation; Oxidized LDL; Beef Tallow; Rats; Soybean Oil; TBARS

1. Introduction

Lipid peroxidation, the oxidative deterioration of polyunsaturated fatty acids (PUFA), proceeds through an autocatalytic free radical induced chain reaction [1-3], and this is associated with the loss of membrane PUFA and the formation of hydroperoxides, free radical intermediates, and numerous secondary lipid peroxidation products such as aldehydes and related carbonyl compounds [4]. Lipid peroxidation can impair cell functions by altering macromolecules including proteins and nucleic acids [5]. Increased in lipid peroxidation in vivo is associated with several human diseases (heart disease, diabetes, cancer, cataract, and others) and is of considerable interest from a public health point of view [6]. The release of toxic aldehydic degradation products, byproducts of PUFA metabolism, could result in significant damage to cells present in the arterial walls and may be related to atherosclerotic lesions. Oxidative modification of low-density lipoprotein (LDL), which results in the formation of oxidized-LDL particles, has been implicated in the pathogenesis of atherosclerosis and heart disease [7-11]. During the process of LDL oxidation, PUFA undergo peroxidative decomposition, resulting in the formation of secondary lipid oxidation products such as aldehydes and ketones [12]. Aldehydes may conjugate to apo B and phospholipids of LDL and these changes result in a shift in the metabolic fate of LDL, causing it to be recognized by scavenger receptors on macrophages rather than by LDL-specific receptors [13-15]. Since scavenger receptors are not down regulated by an increase in the intracellular cholesterol content, cholesterol accumulation and foam cell formation can result [10,11]. Factors that have been reported to affect the susceptibility of LDL to oxidation include antioxidant concentration [16-21] and fatty acid composition [22-30].

Dietary factors which have been shown to influence the susceptibility of LDL particles to oxidation relate to the level of consumption of various types of fatty acids. In general, it appears that diets rich in monounsaturated fatty acids are associated with the inhibition of the oxidation of LDL particles, while diets rich in PUFA, specifically w-6 fatty acids, appear to promote LDL oxidation [31]. To date, the relative effects of low-PUFA containing dietary fats, such as beef tallow, which contains about 20% stearic acid and 42% monounsaturated fatty acid isomers (mostly oleic acid), on LDL oxidation have not been thoroughly investigated. Stearic acid has not been shown to increase plasma cholesterol as other saturated fatty acids do [32]. Thus, beef tallow is an ideal low-PUFA dietary fat to compare to a high-PUFA fat such as soybean oil because it is expected to decrease the susceptibility of LDL to oxidation and it should have no effect on plasma cholesterol.

Previous investigations in our laboratory have shown the beneficial effects of dietary beef fat [33], as compared to a high-PUFA soybean oil diet with respect to general in vivo lipid peroxidation. Dietary consumption of beef fat resulted in reduced susceptibility of serum to oxidation and also reduced whole body oxidation as measured by urinary excretion of secondary lipid oxidation products, without increased serum cholesterol or triglyceride levels in rats.

The focus of the current investigation was on LDL oxidation. The purpose of this research was to determine if consumption of a low-PUFA beef tallow diet would provide a protective effect against LDL oxidation and in vivo lipid peroxidation as compared to a high-PUFA soybean oil containing diet.

2. Materials and Methods

2.1. Animals and Diets

Weanling Sprague-Dawley female rats (Harlan Sprague Dawley, Indianapolis, IN) were divided into two groups of ten animals each and received either of two dietary treatments that differed only in the fat source (Table 1) for 21 weeks. The beef tallow diet (BT) contained low polyunsaturated fatty acids (PUFA) (~4%) and the soybean oil diet (SO) contained high PUFA (~58% [all 2- or 3-double bonded]) [33]. Total dietary fat was 10% by weight of diet corresponding to consumption of 20% of calories from fat, which represented a moderate to lowfat diet. The diets were equalized for vitamin E content to produce results based on fatty acid difference and not

Table 1. Composition of experimental diets fed to rats for 21 weeks.

antioxidant consumption.

During the 21-week feeding period, food and water were provided ad libitum and body weight and 24-hr food intake were measured weekly. At the end of the feeding period, the animals were placed in metabolic cages, fasted for 48 hrs with access to water ad libitum during which time the urine was collected in the second 24-hr period. After fasting, the rats were anesthetized with isoflurane and blood was collected by cardiac puncture. Blood was collected into tubes containing anticoagulant (EDTA Disodium salt, Fisher Biotech, Fair Lawn, NJ) (~1 mg/mL blood), and then centrifuged (6 min at 2200× g, room temperature) to separate the plasma. The plasma was collected and 10 µL per mL plasma of 600 g/L sucrose was added to prevent LDL aggregation [34]. Liver tissue was also collected. Plasma, liver tissue, and urine were all stored at −70˚C until use. The University of Minnesota Institutional Animal Care and Use Committee (IACUC) approved all protocols used in this study.

2.2. Urinary Aldehydes

Urine analysis was conducted using the method developed in this laboratory for the excretion of lipophilic aldehydes which are secondary lipid peroxidation products [35-37]. Three mL of urine sample was combined with an equal volume of freshly prepared dinitrophenylhydrazine (DNPH) reagent [37]. The urine and DNPH mixture was incubated overnight at room temperature in the dark to form hydrazone derivatives of aldehydes (DNP-hydrazones) and related carbonyl compounds. The DNPhydrazones were extracted with CH₂C_l2. The polar and nonpolar DNP-hydrazones, and osozones (sugar DNPH derivatives) were separated by thin-layer chromatography on silica gel plates developed with CH₂C_l2 and the corresponding aldehyde bands were eluted with methanol. The polar and nonpolar DNP-hydrazone fractions were each separately chromatographed on a reverse phase C₁₈ HPLC column (Ultrasphere ODS C₁₈, 25 cm × 4.6 mm i.d., 5 µm) with gradient elution. The polar compounds (PC) were eluted with an initial isocratic mobile phase of 50:50 methanol:water (v/v) for 10 min followed by a linear gradient to 100% methanol. The nonpolar compounds (NPC) were eluted with initial isocratic mobile phase of 75:25 methanol:water (v/v) for 10 min followed by a linear gradient to 100% 112 methanol and the UV detection wavelength was 378 nm. All solvents used were HPLC grade. The method sensitivity was less than 1 ng for the individual derivatives per 10 μl injection to the HPLC column.

2.3. Urinary Creatinine

Urinary creatinine was analyzed by the method of Heinegard and Tiderstrom [38]. Creatinine was reacted with picric acid under alkaline conditions and the absorbance was determined at 500 nm. The absorbance was translated to concentration based on the absorbance of a known concentration of creatinine.

2.4. Thiobarbituric Acid Reactive Substances (TBARS)

The method of Uchiyama and Mikasa [39] was used to determine the thiobarbituric acid reactive (TBARS) products of in vivo lipid peroxidation in the liver. Results are expressed as malondialdehyde (MDA) equivalents.

2.5. Lipoprotein Assays

2.5.1. Lipoprotein Isolation

Plasma low-density lipoproteins (LDL) plus the very low density lipoproteins (VLDL) were isolated by a short-run density-gradient ultracentrifugation method [40]. Plasma (2 mL) was placed in a polycarbonate centrifuge tube (Beckman Instruments, Inc., Palo Alto, CA) and 4 mL saline (d = 1.1416 g/mL) containing 0.1 g/mL EDTA was added to adjust the density of the solution to 1.053 g/mL. The tubes were centrifuged 8 hrs at 150,000× g at 15˚C. After centrifugation, the LDL + VLDL containing fraction at the top of the tube was collected. EDTA was removed from the samples by rapid filtration through disposable Sephadex G-25 desalting columns (PD-10, Amersham Biosciences, Piscataway, NJ). Samples were stored at 4˚C until further analysis. All analyses were completed in <1 week.

2.5.2. Protein Concentration of Lipoproteins

The protein content of the LDL + VLDL fraction was determined by the Bradford method [34]. Aliquots (100 µL) of EDTA-free lipoprotein were incubated with 2 mL of Bradford reagent containing Coomassie brilliant blue (100 mg/L) for 10 min. The absorbance was read at 595 nm. Protein concentration was based on bovine serum albumin (35 mg/dL) standard.

2.5.3. In Vivo Oxidation of Lipoproteins

The method of Ahutopa et al. [41] was used to determine the extent of in vivo LDL + VLDL oxidation by measuring the amount of conjugated dienes in the isolated LDL + VLDL fraction. Lipids were extracted from the LDL + VLDL samples with chloroform:methanol (2:1 v/v). Aliquots of LDL + VLDL solution (250 µL) were combined with 4 mL water, then washed three times with 2 mL chloroform:methanol (2:1 v/v). The solvent phases were collected and further washed two more times with 8 mL water. The solvent was evaporated under N₂ gas and the lipids were redissolved in 1.5 mL cyclohexane. The absorbance of each sample was determined at 234 nm and absorbance units were converted to molar units using the molar extinction coefficient of 2.95 × 104 M⁻¹·cm⁻¹. Results are reported as nmol conjugated dienes in the LDL + VLDL fraction per mg LDL + VLDL protein.

2.5.4. Copper-Induced ex Vivo Oxidation of Lipoproteins

A modified method of Kleinveld et al. [40] was used to complete the copper-induced ex vivo oxidation of LDL + VLDL and the response to this oxidation was measured. Aliquots of the plasma LDL + VLDL fractions (200 µL) were placed in a cuvette with 0.6 mL phosphate-buffered saline (0.01 M, pH 7.4). Oxidation was initiated by the addition of CuSO₄·5H₂O solution (final concentration of 20 µmol/L). The formation of conjugated dienes was monitored by measuring the absorbance at 234 nm at 37˚C every 2 min for 6 hrs. The lag time was defined as the time before the onset of rapid oxidation and conjugated diene formation. This value is reported in minutes. The maximal rate of oxidation was calculated from the slope of the absorbance curve during the time of rapid formation of conjugated dienes. The maximum concentration of conjugated dienes was also determined and is reported as nmol dienes per mg LDL + VLDL protein.

2.5.5. Cholesterol and Triglyceride Concentration in Lipoproteins

The amount of cholesterol and triglycerides in the LDL + VLDL fraction were determined using commercially available test kits (cholesterol, Roche Diagnostics, Indianapolis, IN; triglycerides, Wako Chemicals USA, Inc. Richmond, VA). The results are expressed as mg LDL + VLDL cholesterol or triglyceride per mg LDL + VLDL protein.

2.6. Statistical Analyses

All statistical analyses were performed using GraphPad Prism version 4.00 for Windows (GraphPad Software, San Diego, CA). The Student’s t-test was used to determine statistically significant differences between the dietary treatment groups for all analyses. Differences were considered significant when p < 0.05.

3. Results

In this experiment, 20 rats were fed either a 10% beef tallow (BT) or 10% soybean oil (SO) containing diet for 21 weeks, while all other dietary ingredients were identical. Table 1 shows the composition of the diets. The fatty acid composition of the dietary fats was previously reported [35]. Figure 1 represents the average body weight of the animals, as monitored weekly during the feeding period. Average daily food intake was also monitored weekly. There was no significant difference between the two diet groups in either body weight (p = 0.0907) or food intake (p = 0.1128).

Figure 2 presents the summary of the urinary excretion of non-polar carbonyl compounds (NPC) by rats fed BT or SO diets. The bars represent duplicate measurements of the individually separated and quantified carbonyl compounds. The following aldehydes and ketones were identified in the urine and are in good agreement in our previously published results [37,38]; butanal, 2-butanone, pentanal, 2-pentanone, 2-hexenal, hexanal, 2,4- heptadienal, 2-heptenal, octanal, 2,4-decadieanal, and decanal. Two of the measured compounds are not yet identified. The results are expressed as µg hexanal equivalents per mg urinary creatinine and are reported in the order of elution from the HPLC column. In general, increased excretion of aldehydes was observed in most cases due to increased in vivo oxidation in response to the SO diet as compared to the BT diet and statistical significances were observed for three compounds: decanal and the unidentified compounds E and L.

Conflicts of Interest

The authors declare no conflicts of interest.

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