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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:3255-3262.)
© 1997 American Heart Association, Inc.

Articles

Tetradecylthioacetic Acid Inhibits the Oxidative Modification of Low Density Lipoprotein and 8-Hydroxydeoxyguanosine Formation In Vitro

Ziad A. Muna; Khaled Doudin; Jon Songstad; Rune J. Ulvik; ; Rolf K. Berge

From the Department of Clinical Biology, Division of Biochemistry, University of Bergen, Haukeland University Hospital, N-5021 Bergen, Norway (Z.A.M., R.J.U., R.K.B) and the Department of Chemistry, University of Bergen, N-5007 Bergen, Norway (K.D., J.S.).

Correspondence to Ziad A. Muna, Department of Clinical Biology, Division of Biochemistry, University of Bergen, Haukeland University Hospital, N-5021 Bergen, Norway. E-mail ziad.muna{at}ikb.uib.no

	Abstract

Top Abstract Introduction Methods Synthesis of... LDL Preparation and Storage... Copper Ion–Induced LDL... Preincubation Electrophoretic Mobility Measurement of Lipid Peroxides Comparing Methods for Detecting... Analysis of 8-OH-dG Scavenging of DPPH Radical Wavelength Spectra Measurement The Effect of TTA... Interactions Between LDL and... Results Discussion References

 
Abstract Oxidative modification of low-density lipoprotein (LDL) is thought to play a key role in the formation of foam cells and in initiation and progression of atherosclerotic plaque. The hypolipidemic 3-thia fatty acids contain a sulfur atom and might therefore possess reducing (antioxidant) properties. Consequently, the effects of 3-thia fatty acids on the susceptibility of LDL particles to undergo oxidative modification in vitro were studied. Tetradecylthioacetic acid (TTA), incorporated into the LDL particle and increased the lag time of copper ion induced LDL oxidation in a dose-dependent manner. 80 µmol/L TTA reduced the generation of lipid peroxides during copper ion induced LDL oxidation (for 2 hours) by 100%, 2,2'-azobis-(2,4-dimethylvaleronitrile) induced LDL oxidation by 64%, and 2,2'-azobis-(2-amidinopropane hydrochloride) induced LDL oxidation (for 6 hours) by 21%. The electrophoretic mobility of the oxidized LDL was reduced by TTA in both copper ion and azo-compounds initiated oxidation. This fatty acid analogue was effectively able to reduce in a dose dependent manner the formation of 8-hydroxydeoxyguanosine from 2-deoxyguanosine with ascorbic acid as the radical producer. TTA bound copper(II) ions and did not reduce copper(II) to copper(I). It failed to scavenge the 1.1-diphenyl-2-picrylhydrazyl radicals. The results suggest that the modification of LDL in the lipid and protein moieties can be significantly reduced by TTA. This acid may exert its antioxidant effect partially through metal ion binding and through free radical scavenging.

Key Words: tetradecylthioacetic acid • 3-thia fatty acids • oxidized LDL • atherosclerosis • lipid peroxidation • copper reduction.

	Introduction

Top Abstract Introduction Methods Synthesis of... LDL Preparation and Storage... Copper Ion–Induced LDL... Preincubation Electrophoretic Mobility Measurement of Lipid Peroxides Comparing Methods for Detecting... Analysis of 8-OH-dG Scavenging of DPPH Radical Wavelength Spectra Measurement The Effect of TTA... Interactions Between LDL and... Results Discussion References

 
Excess of triacylglycerol and cholesterol in the blood accelerates the development of atherosclerotic coronary heart disease.¹ Reduction of high blood concentrations of lipids by drugs or by diet has therefore for several years been accepted as a routine therapeutic measure for people at risk. In addition, a new therapeutic strategy based on the use of antioxidants seems to get increasing support.² Antioxidants may reduce the susceptibility of LDL to oxidative modification and thus inhibit the appearance of foam cells and the formation of fatty streaks, which are the first steps in the atherosclerotic process.³ A possible role for redox reactions in the pathophysiology of atherosclerosis in vivo has for instance been suggested by the inhibiting effect of antioxidant therapy on the progression of atherosclerosis in WHHL rabbits.^4,5 The significance of LDL-oxidation as an early event in the pathogenesis of atherosclerosis,⁶ has stimulated the search for new agents with antioxidant properties and the hope of developing new and better drugs for treating coronary heart disease and other diseases caused by vascular atherosclerosis.

Recently, the effect of sulfur substituted fatty acid analogues on lipid metabolism and blood lipids has been extensively studied.⁷ Long-chain thia fatty acids are activated to their CoA thioesters in the endoplasmic reticulum (ER).⁸ They cannot be ß-oxidized but are metabolized by extra mitochondrial ß-oxidation and sulfur oxidation in the ER followed by peroxisomal ß-oxidation to short sulfinyl dicarboxylic acids.⁹ In vivo, long-chain 3-thia fatty acids are incorporated into phospholipids,¹⁰ particularly in heart and hepatocytes. Among plasma lipoproteins, they were found within VLDL and LDL.¹¹ TTA has been reported as one of the most potent lipid lowering of the monocarboxylic 3-thia fatty acids, causing reduction in both cholesterol (data to be published) and triacylglycerol in rats.¹² TTA induces proliferation of peroxisomes and mitochondria^7,13–15 that may lead to increased oxidative stress in the body. For instance, when rats were treated with peroxisome proliferators (ciprofibrate and perfluorodecanoic acid) there was an increase in 8-hydroxy-deoxyguanosine (8-OH-dG), which is a marker of oxidative damage of DNA.^16–18 In order to reveal redox properties of TTA, we tested the compound in two quite different experimental models: the effect of TTA on oxidative modification of LDL caused by copper and nonmetal oxidants, and its effect on the ascorbate induced oxidation of the DNA-fragment 2-deoxyguanosine. In both systems TTA behaved as an antioxidant.

	Methods

Top Abstract Introduction Methods Synthesis of... LDL Preparation and Storage... Copper Ion–Induced LDL... Preincubation Electrophoretic Mobility Measurement of Lipid Peroxides Comparing Methods for Detecting... Analysis of 8-OH-dG Scavenging of DPPH Radical Wavelength Spectra Measurement The Effect of TTA... Interactions Between LDL and... Results Discussion References

 
Chemicals
2,2'-azobis-(2-amidinopropane hydrochloride) (AAPH) and 2,2'-azobis-(2,4-dimethylvaleronitrile) (AMVN) were purchased from Polysciences Inc. All other chemicals were from common commercial sources and were of reagent grade.

Fatty Acid Analogues
The different 3-thia fatty acids were synthesized as previously described.¹⁹ These were dissolved in ethanol or in potassium hydroxide and added in the indicated concentrations simultaneously with the oxidative agent, or as indicated.

	Synthesis of 1-[14C]-Tetradecylthioacetic Acid

Top Abstract Introduction Methods Synthesis of... LDL Preparation and Storage... Copper Ion–Induced LDL... Preincubation Electrophoretic Mobility Measurement of Lipid Peroxides Comparing Methods for Detecting... Analysis of 8-OH-dG Scavenging of DPPH Radical Wavelength Spectra Measurement The Effect of TTA... Interactions Between LDL and... Results Discussion References

 
1-Tetradecanethiol was distilled at 84°C to 86°C/0.01 mm Hg and [¹⁴C]-bromoacetic acid (1 mCi bromoacetic acid (55 mCi/mmol)) was distilled at 117°C to 118°C/15 mm Hg. All other chemicals were flushed with argon. 42 mg tetradecanethiol were placed in a round bottom flask. 2 mL methanol were added and the mixture was stirred under argon to prevent oxidation of the sulfides. After 15 minutes 1.70 mL of 0.75 mol/L KOH in methanol were carefully added. The reaction mixture was then stirred for 20 minutes. 19 mg [¹⁴C]-bromoacetic acid were dissolved in 10 mL methanol. The solution was allowed to evaporate to 5 mL under a flush of nitrogen after which 1.68 mL of 757 mmol/L KOH were added. The reaction mixture was bubbled through with argon and boiled for 24 hours. 48 mg of 37% HCl in 10 mL water were then added. This led to precipitation of the product. Before the mixture was set at 4°C to continue precipitation, pH was measured and seen to have a value of 4 to 5. 1-[¹⁴C]-tetradecylthioacetic acid was transferred to a G-4 filter, washed with distilled water, and recrystalized from dichloromethane in 10% pentane. It had a melting point of 63°C and the yield was 78%.

	LDL Preparation and Storage Time

Top Abstract Introduction Methods Synthesis of... LDL Preparation and Storage... Copper Ion–Induced LDL... Preincubation Electrophoretic Mobility Measurement of Lipid Peroxides Comparing Methods for Detecting... Analysis of 8-OH-dG Scavenging of DPPH Radical Wavelength Spectra Measurement The Effect of TTA... Interactions Between LDL and... Results Discussion References

 
LDL was prepared from fresh human plasma obtained from healthy volunteers by sequential ultracentrifugation, as described in detail elsewhere.^20,21 Briefly, LDL was taken as the 1.019 to 1.063 density fraction. The LDL fractions were dialyzed extensively against 150 mmol/L sodium chloride, 16 mmol/L sodium phosphate and 4 mmol/L potassium phosphate, pH 7.4, bubbled with nitrogen. Protein was measured by the Bio-Rad Protein assay (Bio-Rad Laboratory) using bovine serum albumin as a standard. The purity of the LDL preparation was evaluated with agarose gel electrophoresis, using the Beckman, Paragon system.

After being dialyzed it was necessary to test the stability of the LDL under storage conditions. Four different LDL preparations obtained from 4 different healthy donors were subjected to copper ion oxidation after different storage intervals (0 to 6 days, 0 to 4°C and 7 days, -80°C). The lag time, the half-time for the maximum diene production (T_1/2), the rate of propagation, and the maximum diene production were monitored. The results showed that the LDL stored at 0 to 4°C gave values that were within the experimental error of those obtained with freshly prepared LDL, while the oxidation parameters of LDL stored at -80°C were outside experimental error (data not shown). Therefore, we used LDL stored at 4°C and within 6 days after it was dialyzed.

	Copper Ion–Induced LDL Oxidation and Monitoring of Conjugated Dienes

Top Abstract Introduction Methods Synthesis of... LDL Preparation and Storage... Copper Ion–Induced LDL... Preincubation Electrophoretic Mobility Measurement of Lipid Peroxides Comparing Methods for Detecting... Analysis of 8-OH-dG Scavenging of DPPH Radical Wavelength Spectra Measurement The Effect of TTA... Interactions Between LDL and... Results Discussion References

 
The kinetics of LDL oxidation and the conjugated diene method have been described by others.^22,23 However, we optimized the experimental conditions in our own laboratory with the following main results. The increase in LDL protein concentration caused an increase in the lag time and T_1/2, and a delay and higher maximum of diene production. When the copper ion concentration was increased, the lag time decreased, however, not beyond a minimum level. The initial and the maximum diene concentrations remained virtually constant for a fixed LDL protein concentration (data not shown). 25 µg/mL LDL protein was enough to obtain sufficient analytical sensitivity with a change in the absorbance at 234 nm of about 1. 10 µmol/L Copper ion was the corresponding optimal concentration, and 37°C was the optimal reaction temperature. The oxidation was initiated as described earlier.²⁴ Basically, 25 µg LDL-protein were placed in 1 mL quartz cuvette to which calcium magnesium free phosphate buffered solution (PBS) at pH 7.4 was added. TTA and the other compounds tested, were added as specified. The oxidation was initiated by adding freshly prepared CuCl₂X2H₂O solution to a final concentration of 10 µmol/L. Oxidation was performed at 37°C in a single-beam UV-spectrophotometer (Shimadzu MPS-2000), with a capacity of measuring 6 samples simultaneously. Absorbance was recorded every 2 minutes up to 3 hours. The initial absorbance was set to an arbitrary value and then the increase was recorded over the time period.

	Preincubation

Top Abstract Introduction Methods Synthesis of... LDL Preparation and Storage... Copper Ion–Induced LDL... Preincubation Electrophoretic Mobility Measurement of Lipid Peroxides Comparing Methods for Detecting... Analysis of 8-OH-dG Scavenging of DPPH Radical Wavelength Spectra Measurement The Effect of TTA... Interactions Between LDL and... Results Discussion References

 
25 µg/mL LDL protein and 20 µmol/L TTA (final concentrations) were preincubated at 37°C for 1 hour before oxidation was initiated by the addition of 10 µmol/L copper ions. The reaction was monitored by measuring the change in absorbance at 234 nm.

	Electrophoretic Mobility

Top Abstract Introduction Methods Synthesis of... LDL Preparation and Storage... Copper Ion–Induced LDL... Preincubation Electrophoretic Mobility Measurement of Lipid Peroxides Comparing Methods for Detecting... Analysis of 8-OH-dG Scavenging of DPPH Radical Wavelength Spectra Measurement The Effect of TTA... Interactions Between LDL and... Results Discussion References

 
Oxidation of LDL was initiated by either copper ions or the azo-compounds, AAPH and AMVN, being water- and lipid-soluble, respectively. Azo compounds thermally decompose to peroxyl radicals at constant rates in the water or lipid phases.^25,26 The oxidation of LDL (50 µg/mL) was carried out at 37°C for different time intervals in the presence of 5 µmol/L copper ions, or for 6 hours in the presence of 4 mmol/L AAPH or 1 mmol/L AMVN, (final concentration, dissolved in PBS or methanol, respectively), in the absence or presence of TTA. The reaction was arrested after appropriate time intervals by cooling to 4°C and by addition of EDTA (200 µmol/L final concentration) and BHT (40 µmol/L final concentration). Aliquots were assayed by agarose gel electrophoresis (Paragon, Beckman Instruments, Inc.) in 0.05 mol/L barbital buffer, pH 8.6, and stained with sudan black B. REM was calculated as the mobility of oxidized LDL relative to that of native LDL. The intra-assay coefficient of variation was 1.5% (n=9 copper-oxidized LDL).

	Measurement of Lipid Peroxides

Top Abstract Introduction Methods Synthesis of... LDL Preparation and Storage... Copper Ion–Induced LDL... Preincubation Electrophoretic Mobility Measurement of Lipid Peroxides Comparing Methods for Detecting... Analysis of 8-OH-dG Scavenging of DPPH Radical Wavelength Spectra Measurement The Effect of TTA... Interactions Between LDL and... Results Discussion References

 
The same aliquots analyzed for electrophoretic mobility were analyzed for their lipid peroxides content by a colorimetric method in which hemoglobin catalyzes the reaction of hydroperoxides with the methylene blue derivative, forming an equimolar concentration of methylene blue with absorbance maximum at 675 nm. The assay was performed using a kit purchased from Kamiya Biomedical Company. The intra-assay coefficient of variation was 2.0% (n=8 copper-oxidized LDL).

	Comparing Methods for Detecting Oxidation of LDL

Top Abstract Introduction Methods Synthesis of... LDL Preparation and Storage... Copper Ion–Induced LDL... Preincubation Electrophoretic Mobility Measurement of Lipid Peroxides Comparing Methods for Detecting... Analysis of 8-OH-dG Scavenging of DPPH Radical Wavelength Spectra Measurement The Effect of TTA... Interactions Between LDL and... Results Discussion References

 
The oxidation of 25 µg/mL LDL protein by 10 µmol/L copper ions was monitored over time by the three methods; conjugated dienes, lipid peroxides production, and electrophoresis. Electrophoretic mobility was the first parameter to be affected followed by conjugated dienes and lipid peroxides (data not shown).

	Analysis of 8-OH-dG

Top Abstract Introduction Methods Synthesis of... LDL Preparation and Storage... Copper Ion–Induced LDL... Preincubation Electrophoretic Mobility Measurement of Lipid Peroxides Comparing Methods for Detecting... Analysis of 8-OH-dG Scavenging of DPPH Radical Wavelength Spectra Measurement The Effect of TTA... Interactions Between LDL and... Results Discussion References

 
Formation of 8-OH-dG was obtained as described elsewhere.²⁷ Briefly, 3 mmol/L ascorbic acid was added to 167 µmol/L 2-deoxyguanosine, in 100 mmol/L phosphate buffer of pH 8.1 at 37°C. The fatty acids tested were dissolved in 1% Triton X-100 and added at the indicated concentration simultaneously with the oxidative agent, and incubated for several time intervals as specified in each experiment. Separation of 8-OH-dG by high performance liquid chromatography (HPLC) was performed by using 4.6 x 150 mm Supelcosil C-8 column protected by a 2 x 20 mm Perisorb RP-18 precolumn in a HPLC system from Spectra-Physics. The mobile phase was 8% aqueous methanol containing 10 mmol/L NaH₂PO₄X2H₂O at a flow rate of 1 mL per minute. 8-OH-dG was detected using an electrochemical detector (Esa Coulochem 2) equipped with a 5011 high-sensitivity analytical cell. The potentials for the electrodes 1 and 2 were adjusted to 150 and 430 mV, respectively. A 5020 guard cell was set at a potential of 450 mV. The 8-OH-dG standard was a generous gift from Dr Aarsaether.

	Scavenging of DPPH Radical

Top Abstract Introduction Methods Synthesis of... LDL Preparation and Storage... Copper Ion–Induced LDL... Preincubation Electrophoretic Mobility Measurement of Lipid Peroxides Comparing Methods for Detecting... Analysis of 8-OH-dG Scavenging of DPPH Radical Wavelength Spectra Measurement The Effect of TTA... Interactions Between LDL and... Results Discussion References

 
Scavenging of 1.1-diphenyl-2-picrylhydrazyl (DPPH) free radicals was measured as described previously.²⁸ Briefly, a 0.03 mL of the stock solution of the tested compounds (0.1 mmol/L) was mixed with 3 mL of 0.1 mmol/L DPPH solution (in ethanol) in a cuvette and the time course of the optical density change was determined at 517 nm for 20 minutes.

	Wavelength Spectra Measurement

Top Abstract Introduction Methods Synthesis of... LDL Preparation and Storage... Copper Ion–Induced LDL... Preincubation Electrophoretic Mobility Measurement of Lipid Peroxides Comparing Methods for Detecting... Analysis of 8-OH-dG Scavenging of DPPH Radical Wavelength Spectra Measurement The Effect of TTA... Interactions Between LDL and... Results Discussion References

 
The UV and the visible spectra of copper ion in the presence and absence of TTA or palmitic acid, were measured as described²⁹ using Cary 1 Varian spectrophotometer. The pH was about 6, the copper salts and the various fatty acids were dissolved in ethanol and the experiment was run at room temperature. The absorbance curves of the visible (400 to 850 nm) and the UV (250 to 400 nm) wavelength ranges were recorded.

	The Effect of TTA on the Oxidation State of Copper

Top Abstract Introduction Methods Synthesis of... LDL Preparation and Storage... Copper Ion–Induced LDL... Preincubation Electrophoretic Mobility Measurement of Lipid Peroxides Comparing Methods for Detecting... Analysis of 8-OH-dG Scavenging of DPPH Radical Wavelength Spectra Measurement The Effect of TTA... Interactions Between LDL and... Results Discussion References

 
In order to check the oxidation state of the copper after mixing TTA and copper(II) to form a complex, color-specific redox reagents were used. Bathocuproine sulfonate that gives an orange-red color with copper(I), but no color with copper(II), was used as described elsewhere.³⁰ Briefly, 10 mL of 0.1 mol/L Cu(ClO₄)₂·6H₂O were prepared in water and slowly added to 10 mL of 0.2 mol/L solution of fatty acid in ethanol. The mixture was stirred for 30 minutes at room temperature. A precipitate appeared and the reaction mixture was filtered and the residue was washed with water and ethanol to remove unreacted copper(II) perchlorate and fatty acid respectively. The pale blue crystals were dried. The presence of copper(I) in the TTA-copper complex, was examined by adding 1 mL of 10 mmol/L bathocuproine sulfonate solved in 10% ammonium acetate as well as 1 mL water to 1 mL of ethanol solution (10 mL) containing 16.3 mg of the complex. As a control the same experiment was performed with either 2.9 mmol/L of Cu(ClO₄)₂.6H₂O, or 9.7 mg palmitic acid solved in ethanol (10 mL). Further, the experiments were repeated with 1 mL hydroxylamine · HCl (10%) instead of water. Hydroxylamine reduces copper(II) to copper(I).

	Interactions Between LDL and TTA

Top Abstract Introduction Methods Synthesis of... LDL Preparation and Storage... Copper Ion–Induced LDL... Preincubation Electrophoretic Mobility Measurement of Lipid Peroxides Comparing Methods for Detecting... Analysis of 8-OH-dG Scavenging of DPPH Radical Wavelength Spectra Measurement The Effect of TTA... Interactions Between LDL and... Results Discussion References

 
To test whether TTA can bind to the LDL particle in vitro, we added different concentrations of freshly prepared LDL to a constant concentration of radio labeled TTA (10 µmol/L final concentration). After 1 hour incubation at room temperature, 6% perichloric acid (final concentration) was added and the mixture was centrifuged at 12000 rpm for 12 minutes. The radioactivity in both the supernatant and the pellet was measured.

	Results

Top Abstract Introduction Methods Synthesis of... LDL Preparation and Storage... Copper Ion–Induced LDL... Preincubation Electrophoretic Mobility Measurement of Lipid Peroxides Comparing Methods for Detecting... Analysis of 8-OH-dG Scavenging of DPPH Radical Wavelength Spectra Measurement The Effect of TTA... Interactions Between LDL and... Results Discussion References

 
The Effect of TTA on Copper Ion Induced LDL Oxidation
Diene Production
The 3-thia fatty acids had different effect on the kinetics of LDL oxidation (Fig 1). Palmitic acid that was used as a control and 3-thia fatty acids of chain length from octylthioacetic acid to hexadecylthioacetic acid increased the lag time, but not as significant as TTA. Shorter and longer 3-thia fatty acids were less effective. TTA increased the lag time and the half time in a dose-dependent manner (Fig 2), whereas the rate of oxidation and maximal diene production remained unchanged compared to the control (Table 1). Palmitic acid had no significant effect on oxidative modification of LDL. -Tocopherol and reduced glutathione increased the lag time while addition of the polyunsaturated fatty acid, eicosapentaenoic acid (20:5 n-3) reduced the lag time (data not shown). No changes in the lag time of LDL oxidation were obtained by the addition of the sulfinyl or sulfonyl forms of TTA, tetradecyloxyacetic acid, and 3, 14-dithiahexadecanedioic acid (Table 1).

View larger version (22K): [in this window] [in a new window]   Figure 1. The effects of different chain length of 3-thia fatty acids on the diene absorbance at 234 nm. The oxidation was performed as described in "Methods" and 20 µmol/L of the fatty acid analogues were added. () no addition, () TTA, (x) palmitic acid, () octylthioacetic acid, () undecylthioacetic acid, () pentadecylthioacetic acid and () hexadecylthioacetic acid.

View larger version (13K): [in this window] [in a new window]   Figure 2. The effect of TTA concentration on the () lag time and () T1/2. The oxidation was performed as described in "Methods." Data were obtained from 5 healthy volunteers, the values represent mean±SD.

View this table: [in this window] [in a new window]   Table 1. Change in Percentage of Some Indexes of Copper Ion-Induced LDL Oxidation due to Addition of 20 µmol/L of Different Sulfur and Oxygen Substituted Fatty Acids

Lipid Peroxidation
TTA reduced the production of lipid peroxides originating from LDL oxidation, being almost blocked when 80 µmol/L of TTA was added and incubated for 1 or 2 hours (Fig 3). However, the antioxidant capacity of TTA was less effective in inhibiting lipid peroxide production after 3 hours.

View larger version (20K): [in this window] [in a new window]   Figure 3. The effect of various TTA concentrations on copper ion induced LDL oxidation as assessed by lipid peroxides production. () no addition, () 40 µmol/L, () 80 µmol/L. The oxidation time were: 0 (native LDL), 1, 2 and 3 hours. Data shown are from one representative experiment.

Electrophoretic Mobility
The changed electrophoretic mobility of LDL oxidized for various time intervals is shown in Fig 4. Addition of TTA reduced the REM of oxidized LDL in a dose-dependent manner. Indeed, the REM of the copper ion-treated LDL incubated for 1 and 2 hours in the presence of 80 µmol/L of TTA was similar to native LDL.

View larger version (56K): [in this window] [in a new window]   Figure 4. The effect of various TTA concentrations on copper ion induced LDL oxidation as assessed by agarose gel electrophoresis. Data shown are from one representative experiment.

To test the effect of TTA and palmitic acid on the electrophoresis method, increasing concentrations of TTA and palmitic acid (dissolved in KOH) were added to LDL (50 µg/ml) which had been oxidized for six hours by 4 mmol/L AAPH at 37°C. Addition of TTA at a concentration of more than 160 µmol/L, was found to increase the electrophoretic mobility, probably explained by an increase in the net negative charge caused by the sulfur atom. Palmitic acid up to 320 µmol/L, had no effect on the electrophoretic mobility (data not shown).

The Effect of TTA on Ascorbic Acid-Induced 2-Deoxyguanosine Modification
Fig 5 shows the effects of TTA on ascorbic acid-induced formation of 8-OH-dG from 2-deoxyguanosine incubated for different time intervals. This 3-thia fatty acid reduced in a dose-dependent manner the 8-OH-dG formation at all time intervals. More than 50% reduction in ascorbic acid-induced formation of 8-OH-dG was observed in the presence of 100 µmol/L TTA. No inhibition of 8-OH-dG generation was observed when the sulfonyl form of TTA or tetradecyloxyacetic acid was present (Table 2).

View larger version (17K): [in this window] [in a new window]   Figure 5. The effect of TTA concentration on the production of 8-OH-dG incubated for () 2 hours, () 2.5 hours and (x) 3 hours. Data are obtained from one representative experiment.

View this table: [in this window] [in a new window]   Table 2. Amount of 8-OH-dG Produced by Ascorbate After Incubation with 100 µmol/L of Different Fatty Acid Analogues for Different Time Intervals

Mechanisms of Action
Binding of Copper Ion to TTA
In order to verify the mechanisms by which TTA inhibits oxidation of the LDL, its effect on copper ion binding was tested. This was done by measuring the UV and the visible absorbance spectra of TTA in the presence and absence of copper ion. The results were compared with those obtained by adding palmitic acid that was used as a control. The visible absorbance spectra measure the d-d transition for copper from nondegenerated t2g-orbitals (which make the band very broad) to d_x^2-y2-orbitals where the separation increased with the stability of the complex, ie, the more stable the complex is, the smaller the _max.³¹ This means that copper in H₂O (_max 821 nm) are less stable than in palmitic acid (_max 814 nm), which again are less stable than in TTA (_max 794 nm) (Fig 6A).

View larger version (23K): [in this window] [in a new window]   Figure 6. The absorbance of (1) copper ions, (2) palmitic acid and copper ions, (3) TTA and copper ions and (4) TTA in ethanol scanned at (A) visible spectra, (B) ultraviolet spectra.

The UV spectra (Fig 6B) of the TTA-copper complex showed two main bands at 275 nm and 332 nm indicating that there are two different transitions in the TTA-copper complex. In comparison, the palmitic acid-copper complex had only a single band, at 275 nm. The common absorbance peak at 275 nm, is due to charge-transfer transition from oxygen nonbonding electrons to the antibonding (t2g*) orbitals of the copper ion. The band at 332 nm in the TTA-copper complex is assumed to be due to the charge-transfer transition of the sulfur nonbonding electrons to the antibonding (t2g*) of the copper ion.³² This suggests that in the palmitic acid-copper complex there is an oxygen-copper bond, while in the TTA-copper complex there are both sulfur-copper and oxygen-copper bonds. The shift of _max to longer wavelengths results from weakening the copper-ligand bond.³¹ Apparently, the copper-oxygen bond is stronger than the copper-sulfur one.

The Effect of TTA on the Oxidation State of Copper
Table 3 shows that neither palmitic acid nor TTA has any effect on the oxidation state of copper, ie, copper (II) was not reduced to copper (I).

View this table: [in this window] [in a new window]   Table 3. Absorbance at 479 nm of Different Solutions Tested for Copper(I) and Copper(II) with the Use of Bathocuproine Sulfonate

LDL Oxidation Initiated by Azo Compounds
To examine possible alternative mechanisms, LDL was subjected to copper ion-independent oxidation, initiated by the lipid and water soluble azo compounds AMVN and AAPH, respectively. The effect of TTA and palmitic acid on the AAPH and AMVN induced LDL oxidation as assessed by electrophoresis is shown in Table 4. With increased amounts of TTA, the REM of the LDL particle decreased. It should be emphasized that this method underscores the real effect due to the interference of TTA with the method (see above). Nevertheless, TTA decreased the REM as compared to palmitic acid. When AMVN was used, TTA inhibited LDL oxidation in a dose-dependent manner. 160 µmol/L TTA reduced the content of lipid peroxides by 84% and the REM by 28%. In comparison with AAPH as the oxidative agent, the lipid peroxides were reduced by 35% and the REM by 9% (Table 4).

View this table: [in this window] [in a new window]   Table 4. Effect of Different Concentrations of TTA and Palmitic Acid on Both the Relative Electrophoretic Mobility and the Lipid Peroxides Production of Both AMVN and AAPH-Induced LDL Oxidation for Six Hours

DPPH Radical Scavenging
We also tested the capacity of TTA to scavenge the DPPH free radical and compared the effect with that of probucol, a synthetic antioxidant, and -tocopherol, a natural antioxidant. As shown in Fig 7, TTA failed to scavenge the DPPH radicals.

View larger version (15K): [in this window] [in a new window]   Figure 7. The DPPH radical scavenging as a function of time. The absorbance was monitored at 517 nm for 20 minutes in the presence of (x) TTA, () -tocopherol and (o) probucol.

Interactions Between LDL and TTA
Radioactive ¹⁴C labeled TTA was incorporated into the LDL particle under the in vitro conditions (Fig 8). Preincubation of TTA with the LDL particle had almost the same preventive effect on the oxidizability of the LDL particle (Table 5) as when TTA was added simultaneously with copper ions (Table 1).

View larger version (16K): [in this window] [in a new window]   Figure 8. The interaction between C14 labeled TTA and different LDL protein concentrations. The values are ratio active counts in the pellet part. Data are shown as mean±SD (n=3).

View this table: [in this window] [in a new window]   Table 5. Effect of Preincubation of LDL and TTA Before Oxidation

	Discussion

Top Abstract Introduction Methods Synthesis of... LDL Preparation and Storage... Copper Ion–Induced LDL... Preincubation Electrophoretic Mobility Measurement of Lipid Peroxides Comparing Methods for Detecting... Analysis of 8-OH-dG Scavenging of DPPH Radical Wavelength Spectra Measurement The Effect of TTA... Interactions Between LDL and... Results Discussion References

 
This study presents evidence that besides being a hypolipidemic agent TTA also possesses antioxidant properties as assessed in two different experimental systems and by several methods of detecting molecular oxidative damage. This is of interest not only because delay of LDL-oxidation may be part of the anti-atherosclerotic mechanism of TTA, but also because inhibition of 8-OH-dG formation may indicate a more omnipotent antioxidant role for TTA.

TTA not only reduced the peroxidation of lipids in LDL, but obviously also protected the protein moiety of the LDL particle against oxidation as is evident from its beneficial effect on the electrophoretic mobility (Table 4).

Under the present experimental conditions TTA was incorporated into the LDL particle (Fig 8), and obviously very fast as deduced from the finding that the protection of LDL oxidation was almost the same whether TTA was preincubated with LDL before oxidation, or added simultaneously with the pro-oxidant. Our finding is supported by others who found TTA in the LDL fraction of the TTA treated hamsters.¹¹

Earlier, it has been reported that 2 hours after a single dose (150 mg/kg body weight) of TTA to rats, plasma concentration was about 100 µmol/L.³³ Thus, the TTA concentrations used in this study were kept well within a range which may be of biological relevance (Fig 2). This may also be true for using copper as a pro-oxidant, since its in vivo involvement in atherosclerosis is becoming more evident through several lines of studies.^34–36

An intriguing question concerns the mechanism by means of which TTA behaves as an antioxidant. TTA did not change the oxidation state of copper ions (Table 3), implying that it was kept in the copper (II) state. Therefore, the antioxidant effect of TTA might be due to attenuating the redox cycle between the copper (II) and copper (I) redox states. This cycle has been proposed as the mechanism of action by which copper confer its catalytic role, and that copper (I) rapidly forms the ultimate initiating radical, that is capable of abstracting a hydrogen atom from a polyunsaturated fatty acid.^22,37 Binding of copper on the other hand could not be the full explanation for several reasons. First, TTA was effective also when metal independent oxidants like azo compounds (Table 4), and ascorbate were used (Table 2). Second, the fact that copper induced LDL oxidation was delayed, and not prevented by TTA, contradicts that the effect of TTA could be explained only by the formation of an inactive TTA-copper complex. The introduction of an extended lag period into the oxidation process by TTA (Fig 1) is typical for an antioxidant that acts by radical scavenging. In contrast, metal ion-binding antioxidants are expected to give a constant inhibition through the reaction.³⁹ Moreover, the results from the experiments with the azo compounds, AMVN and AAPH, suggest that TTA was able to scavenge the peroxyl radicals, with higher capacity to scavenge the peroxyl radicals generated in the lipid phase than in the water phase (Table 4). This is consistent with our finding that TTA is incorporated into the LDL particle and most probably is located in the lipid phase. It seems plausible that TTA mainly incorporated in the lipid phase as radiolabeled TTA injected intravenously into rats was found in the triacylglycerol fraction of the LDL (data to be published).

Nevertheless, the change of the spectra of copper caused by TTA and palmitic acid, is consistent with the formation of a copper-fatty acid complex (Figs 6A & 6B). In agreement with this assumption is the finding of Balasubramanian et al³⁸ that nonesterified fatty acids such as palmitic acid, myristic acid and lauric acid inhibit iron-dependent lipid peroxidation.

The negative result obtained with TTA in the classical DPPH radical scavenging test (Fig 7), indicates that the conditions required by TTA to take part in redox reactions are different from that of -tocopherol and probucol. On the other hand, the substantial power of TTA to attenuate the effect of radicals generated by ascorbate and protect DNA-fragments from oxidation, is of considerable biological interest (Fig 5). If a prediction of the free radical reactions is to be made, one should consider the essential physico-chemical factors like the one electron reduction potential which is a key thermodynamic property,⁴⁰ and the rate constant for the reaction which is another important factor. Thus, it must be kept in mind that a reaction that is in principle thermodynamically possible may not be kinetically feasible, ie, the rate constant for the reaction is too small to be biologically significant.⁴¹ Given that the key elements involved in LDL oxidation are not totally identified and that considerable variation in the physico-chemical factors results from different incubation conditions when different compounds are questioned, it is not surprising to find that TTA behaves differently in different radical generating systems.

Although the data of the present study cannot be translated directly into the in vivo situation, one may point out that when rats were treated with TTA for 12 weeks, there was a significant increase in the plasma monounsaturated fatty acids. This was mainly accounted for by an increased level of oleic acid (18:1 n-9) and a decrease in the polyunsaturated fatty acids accompanied with reduced levels of linoleic acid (18:2 n-6).⁴² It has been reported that the LDL content of 18:2 n-6 strongly correlates with either the rate of oxidation or the extent of oxidation, the percent of 18:1 n-9 in LDL correlate well with the delay before LDL oxidation and the reduction in the content of polyunsaturated fatty acids in LDL particles will decrease the substrate being available to be oxidized.⁴³ The in vivo effects of TTA that have been reported in the above mentioned study affecting the lipid metabolism and thus, LDL lipid composition and structure indicate further antioxidant properties.

In summary, this study suggests that 3-thia fatty acids exert antioxidant properties with TTA being the most potent among them. It appears that charge and fatty acid chain length play an important role in altering the oxidative resistance that might be mediated through the lipid solubility. We have for the first time described intrinsic antioxidant properties of TTA that may be of biopharmacological interest both in the prevention of atherosclerosis and in the protection of DNA against oxidative damage. The underlying redox mechanism seems to be a combination of metal ion binding and radical scavenging. Further studies are in progress to clarify molecular mechanisms and biological relevance.

	Selected Abbreviations and Acronyms

 

TTA = Tetradecylthioacetic acid=CH3-CH2-(CH2)12-S-CH2-COOH Tetradecylsulfinylacetic acid = CH3-CH2-(CH2)12-SO-CH2-COOH Tetradecylsulfonylacetic acid = CH3-CH2-(CH2)12-SO2-CH2-COOH Tetradecyloxyacetic acid = CH3-CH2-(CH2)12-O-CH2-COOH 3, 14-Dioxyhexadecanedioic acid = HOOC-CH2-O-(CH2)10-O-CH2-COOH 3, 14-Dithiohexadecanedioic acid = HOOC-CH2-S-(CH2)10-S-CH2-COOH LDL = low-density lipoprotein ER = endoplasmic reticulum PBS = phosphate-buffered solution HPLC = high-performance liquid chromatography UV = ultraviolet REM = relative electrophoretic mobility

	Acknowledgments

 
The authors are grateful to Kari Williams and Hans Henriksen for excellent technical assistance. We are also grateful to Dr Niels Aarsaether (University of Bergen) for providing us with the 8-OH-dG. The work was supported by a grant from the University of Bergen.

Received December 12, 1996; accepted July 31, 1997.

	References

Top Abstract Introduction Methods Synthesis of... LDL Preparation and Storage... Copper Ion–Induced LDL... Preincubation Electrophoretic Mobility Measurement of Lipid Peroxides Comparing Methods for Detecting... Analysis of 8-OH-dG Scavenging of DPPH Radical Wavelength Spectra Measurement The Effect of TTA... Interactions Between LDL and... Results Discussion References

 
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