Paracetamol Inhibits Copper Ion–Induced, Azo Compound–Initiated, and Mononuclear Cell–Mediated Oxidative Modification of LDL

Materials

Na¹²⁵I was purchased from Du Pont–New England Nuclear. DMEM, Ham’s F-10, and gentamicin were obtained from Bio-Whittaker. Heat-inactivated fetal calf serum, PMA, BHT, SOD (specific activity, 6.3 U/μg), and catalase (specific activity, 2.2 U/μg) were purchased from Sigma Chemical Co. AAPH and AMVN were from Polysciences Inc. Tissue culture dishes were supplied by Costar. Dynabeads M-450 anti–glycophorin A were purchased from Dynal AS, whereas Polymorphprep was delivered from Nycomed Pharma AS. BCA protein assay was obtained from Pierce Laboratories Inc, and a kit for determination of lipid peroxides was purchased from Kamiya Biomedical Company. Agarose gels (Paragon lipoprotein electrophoresis) were purchased from Beckman Instruments Inc.

Scavenging of DPPH Radical

Scavenging of DPPH free radicals was measured as described previously.¹⁸ The DPPH radical has a deep violet color due to its unpaired electron, and radical scavenging can be followed spectrophotometrically by the loss of absorbance at 517 nm as the pale yellow nonradical form is produced. DPPH was dissolved in methanol to obtain a concentration of approximately 45 μg/mL, corresponding to an absorption of approximately 1.3 at 517 nm. Pyrogallol, a potent radical scavenger, was dissolved in methanol to a concentration of 2% and used as a control for 100% scavenging. The decline in radical concentration in the presence of paracetamol or sodium salicylate was determined by adding 50 μL test substance, dissolved to indicated concentrations in DMSO or methanol, respectively, to 3 mL DPPH solution, and the absorbance at 517 nm was continuously monitored for 15 minutes in a Shimadzu UV-160A spectrophotometer. Separate experiments showed that the absorbance of DPPH dissolved in methanol and DMSO (300:50, vol/vol) or methanol was stable for at least 15 minutes. Percent radical scavenging was calculated as 100×(A₀−A_t)/(A₀−A_p), where A₀ is the initial absorbance of DPPH, and A_t and A_p are the absorbances after 15 minutes with test solutions and with pyrogallol solutions, respectively.

Isolation and Labeling of LDL

LDL was isolated from freshly prepared plasma obtained from healthy volunteers by sequential ultracentrifugation in a Centrikon T-2060 ultracentrifuge in the density range 1.019 to 1.063 g/mL in a TFT 45.6 rotor at 43 000 rpm for 24 hours at 10°C.¹⁹ The final preparations were dialyzed extensively against 0.15 mol/L NaCl, 20 mmol/L sodium phosphate, and 2 mmol/L EDTA (PBS), pH 7.4, at 4°C. The purity of the LDL preparations was evaluated with agarose gel electrophoresis, using the Beckman Paragon system. LDL was labeled with ¹²⁵I-tyramine cellobiose.²⁰ The final preparation was dialyzed extensively against PBS. More than 95% of the radioactivity was precipitated by 10% (wt/vol) trichloroacetic acid. The final specific activity of ¹²⁵I-labeled LDL was 1309 counts per minute (cpm)/ng. Before use, the ¹²⁵I-labeled LDL was diluted with unlabeled LDL to a specific activity of 40 cpm/ng.

LDL was stored in the presence of EDTA (2 mmol/L) under N₂ at 4°C and used within 1 to 2 weeks. Protein concentrations were determined by BCA protein assay. Intra-assay coefficient of variation was 2.9% (n=18).

Peripheral Blood Mononuclear Cells

The cells were isolated from citrated, freshly collected blood from healthy volunteers. The blood (5 mL) was immediately layered over 5 mL Polymorphprep. After centrifugation (480g, 1600 rpm) for 30 minutes at 22°C in a swing-out rotor, two leukocyte bands were visible. The top band, consisting of mononuclear cells, was removed and washed with 15 mL Ham’s F-10 (2000 rpm for 10 minutes) and resuspended in 1 mL medium. Contaminating erythrocytes were removed by addition of 100 μL suspension of Dynabeads M-450 anti–glycophorin A (4×10⁸ particles/mL). The Dynabeads were allowed to adhere to the erythrocytes for 30 minutes before they were removed using a magnet. The erythrocyte-free mononuclear cell fraction was counted in a Coulter counter, centrifuged (2000 rpm for 10 minutes), and resuspended in Ham’s F-10 with 2.5% gentamicin. The viability was >97% as determined by exclusion of trypan blue stain, and the purity was >90% as determined by May-Grünwald and Giemsa staining. Typically, the mononuclear cell preparation consisted of 87.4±9.5% lymphocytes and 12.6±9.5% monocytes (n=4).

Cu²⁺-Induced Oxidation of LDL

Labeled and unlabeled LDLs were subjected to Cu²⁺-induced oxidative modification.²¹ To remove EDTA before oxidation, the LDL was dialyzed extensively against EDTA-free PBS (pH 7.4) at 4°C. LDL (100 μg LDL protein/mL) was incubated at 37°C for 0, 1, 6, and 24 hours in the presence of 5 μmol/L CuSO₄ and paracetamol (200, 300, and 400 μmol/L in EDTA-free PBS). Aliquots were removed, and the oxidation was stopped by refrigeration (4°C) and addition of EDTA (200 μmol/L final concentration) and BHT (40 μmol/L final concentration). These aliquots were assayed for lipid peroxides, relative electrophoretic mobility, and metabolism by macrophages (see below).

Kinetics of Cu²⁺-Induced Oxidation of LDL

The kinetics of Cu²⁺-induced oxidation of LDL was followed by determining the changes in absorbance at 234 nm in a Shimadzu UV-160A spectrophotometer with six cuvette positions.²² LDL (25 μg/mL) was incubated at 37°C in the presence of Cu²⁺ (final concentration, 1.67 μmol/L). To obtain similar ratios for the test drug and LDL as described above for Cu²⁺-induced oxidation, paracetamol concentrations in the range of 10 to 100 μmol/L were tested. The increase in the absorbance was measured every 5 minutes, up to 195 minutes. From this analysis the lag time (in minutes) for the formation of conjugated dienes, the formation rate (nmol/mg LDL protein per minute), and the maximum amount of conjugated dienes formed (nmol/mg LDL protein) were calculated using a molar extinction coefficient of E_234nm=2.52×10⁴ mol/L⁻¹×cm⁻¹.²³ The intra-assay coefficients of variation were 4.0%, 5.2%, and 2.0% for lag time, formation rate, and maximum amount of conjugated dienes formed, respectively (n=6).

Azo Compound–Initiated Oxidation of LDL

Unlabeled LDL was dialyzed extensively against EDTA-free PBS (pH 7.4) at 4°C and immediately subjected to oxidation initiated by AAPH or AMVN, which are water- and lipid-soluble azo compounds that thermally decompose to produce peroxyl radicals at constant rates within the water and lipid phases, respectively.^{24 25} The oxidation of LDL (100 μg/mL) was carried out at 37°C for 6 or 24 hours in the presence of AAPH or AMVN, respectively (final concentrations, 4 mmol/L or 500 μmol/L, dissolved in PBS or methanol, respectively), in the absence or presence of paracetamol (100 and 200 μmol/L). Oxidation was stopped as described for Cu²⁺-induced oxidation. LDL was assayed for lipid peroxides and electrophoretic mobility in agarose gel (see below).

Cell-Mediated Oxidation of LDL

Unlabeled LDL was dialyzed extensively against EDTA-free PBS (pH 7.4) at 4°C and immediately subjected to cell-mediated oxidation by freshly isolated peripheral blood mononuclear cells.^{26 27} The cells were seeded in 24-well tissue culture plates at a density of 2×10⁶ cells/mL. All experiments were performed in Ham’s F-10 with gentamicin in a total volume of 250 μL/well. The cells were preincubated for 30 minutes at 37°C (in a 95% air and 5% CO₂ atmosphere), in the absence or presence of paracetamol (100 and 400 μmol/L dissolved in Ham’s F-10), sodium salicylate (800, 2000, and 4000 μmol/L dissolved in Ham’s F-10), or α-tocopherol (10 μmol/L dissolved in ethanol; final ethanol concentration, 0.5% vol/vol). LDL (100 μg/mL) was added, together with PMA (100 ng/mL) and Cu²⁺ (final concentration, 5 μmol/L); oxidation was carried out for 6 hours at 37°C (in a 95% air, and 5% CO₂ atmosphere) and was stopped as described for Cu²⁺-induced oxidation. In experiments including SOD (10 and 100 μg/mL) or catalase (50 μg/mL), the enzymes were added just prior to oxidation.^{26 27} SOD and catalase were heat-inactivated in a water bath at 100°C for 60 minutes. Microscopic examination of the cells was performed after the preincubation period and at the end of the study. LDL in the medium was assayed for lipid peroxides and electrophoretic mobility in agarose gel (see below).

Extent of Modification of Cu²⁺-, Azo-, and Cell-Modified LDL

Statistical Analysis

Results are presented as mean±SD (n≥3) or mean and range (n=2). The Mann-Whitney nonparametric test was used for calculation of statistical significance of differences between LDL oxidatively modified in the presence or absence of paracetamol or sodium salicylate. The level of significant differences was set at P<.05.

Scavenging of DPPH Radical

The radical scavenging capacities of paracetamol and sodium salicylate were studied by the DPPH test. Paracetamol showed radical scavenger activity at all the concentrations tested (Fig 2⇓). After 15 minutes of incubation, 50% scavenging was obtained at a paracetamol concentration of 135 μmol/L. In contrast, sodium salicylate at concentrations of 25, 50, 100, 200, 800, 2000, and 4000 μmol/L showed no radical scavenging capacity (data not shown). The effect of paracetamol on LDL oxidation was then studied in Cu²⁺-induced, azo compound–initiated, and mononuclear cell–mediated oxidation systems, whereas the effect of sodium salicylate was tested in the cellular oxidation system only.

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Figure 2.

Line graphs show DPPH radical scavenging as a function of time (A) and paracetamol concentration (B). Concentrations were DPPH solution 45 μg/mL (♦); pyrogallol 2% (▪); and paracetamol 25 (▵), 50 (×), 100 (∗), 200 (•), and 400 (+) μmol/L. Percent radical scavenging was calculated after 15 minutes (B). The data show mean values of duplicates from one of two representative experiments.

Cu²⁺-Induced Oxidation of LDL

LDL was subjected to Cu²⁺-induced lipid peroxidation. The continuous monitoring of optical density at 234 nm showed striking differences in the formation of conjugated dienes formed in the absence and presence of paracetamol (Fig 3⇓). Thus, the average lag time for conjugated dienes increased from 45 minutes (range, 38 to 55 min) in the absence of paracetamol to 68 minutes (range, 57 to 80 min) in the presence of 100 μmol/L paracetamol. The maximum rate of formation of conjugated dienes was reduced by 67%, from 11.7 nmol/mg per minute (range, 8.1 to 13.6 nmol/mg per minute) in the absence of paracetamol to 4.0 nmol/mg per minute (range, 1.2 to 6.8 nmol/mg per minute) in the presence of 100 μmol/L paracetamol. Similarly, the maximum amount of conjugated dienes formed was reduced by 58%, from 610 nmol/mg (range, 575 to 651 nmol/mg) in the absence of paracetamol to 264 nmol/mg (range, 132 to 395 nmol/mg) in the presence of 100 μmol/L paracetamol. There were linear correlations between the paracetamol concentrations tested and the lag times (r=.95), rate of oxidation (r=−.92), and amounts of conjugated dienes formed (r=−.91), suggesting a concentration-dependent inhibition of LDL oxidation.

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Figure 3.

Line graph shows formation of conjugated dienes during oxidation of LDL in the absence or presence of paracetamol. LDL (25 μg/mL) was incubated at 37°C in PBS containing 1.67 μmol/L Cu²⁺ and 0 (○), 10 (•), 25 (▪), 50 (×), and 100 μmol/L (▾) paracetamol. Changes in absorbance at 234 nm were measured every 5 minutes for 195 minutes. The data show results from one of two representative experiments.

The amount of lipid peroxides in LDL increased more than 50-fold during 6 hours of oxidation in the absence of paracetamol (from 15±15 nmol/mg LDL before oxidation to 842±226 nmol/mg LDL; n=3; Fig 4A⇓). The presence of paracetamol in concentrations of 200, 300, and 400 μmol/L reduced the content of lipid peroxides formed during 1 hour to 79±23%, 63±9%, and 57±8% respectively, of that of LDL oxidized in the absence of the drug (n=3; P<.05 for 300 and 400 μmol/L versus no addition; Fig 4A⇓). After 6 hours of oxidation the lipid peroxide amounts were 89±4%, 85±2%, and 83±3%, respectively, of that of LDL oxidized in the absence of the drug (n=3; P<.05).

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Figure 4.

Line graphs show formation of lipid peroxides in LDL during Cu²⁺-induced oxidation (A) and uptake of Cu²⁺-oxidized LDL by J774 macrophages (B). LDL (100 μg/mL) was oxidized by incubation at 37°C with 5 μmol/L Cu²⁺ in PBS in the presence of 0 (○), 200 (×), 300 (♦), and 400 (▾) μmol/L paracetamol for the indicated periods of time. Uptake of oxidized LDL (10 μg/mL; specific activity, 40 cpm/ng) by macrophages was determined after incubation for 5 hours at 37°C. Each point represents the mean of duplicates (lipid peroxides) or triplicates (macrophage uptake) from one of three representative experiments.

Cu²⁺-induced oxidation of LDL for 24 hours increased the relative electrophoretic mobility from 1.0 before oxidation to 5.4±0.7 (n=3), indicating a considerable modification in the protein moiety of LDL in the absence of paracetamol. Paracetamol at final concentrations of 200, 300, and 400 μmol/L significantly reduced the relative electrophoretic mobility (to 88±2%, 90±2%, and 84±4%, respectively, of that of LDL oxidized in the absence of the drug; n=3; P<.05 versus without addition of paracetamol).

The uptake of LDL by J774 macrophages increased markedly with oxidation for 24 hours (Fig 4B⇑). Thus, approximately eightfold more oxidized LDL than native LDL was taken up by the macrophages (4.7±0.8 versus 0.6±0.1 μg/mg cell protein; n=3), indicating that LDL was modified to a form recognized by the scavenger oxidized-LDL receptor in this cell type. LDL oxidized in the presence of paracetamol (final concentrations of 200, 300, and 400 μmol/L) showed reduced uptake by macrophages compared with LDL oxidized for 24 hours in the absence of the drug (87±11%, 71±16%, and 78±16%, respectively; n=3; P<.05 versus without addition of paracetamol). Taken together, the data show that paracetamol may protect LDL against Cu²⁺-induced oxidative modification.

To test whether paracetamol inhibited the oxidative modification of LDL by chelating copper ions, the absorbance spectrum of paracetamol (200 μmol/L) in the absence and presence of Cu²⁺ (5 μmol/L) was measured. The wavelength spectra were identical (data not shown), suggesting that paracetamol exerts its protective effect by mechanisms other than simple metal chelation.

Azo Compound–Initiated Oxidation of LDL

To further examine the mechanism by which paracetamol inhibited the oxidative modification of LDL, LDL was subjected to metal ion–independent oxidation for 6 and 24 hours, initiated by the water- and lipid-soluble azo compounds AAPH and AMVN. The azo compounds induce oxidation by a temperature-dependent generation of peroxyl radicals, independent of preformed lipid peroxides in the LDL particles.²⁵ When AAPH was used to initiate oxidation, 100 μmol/L paracetamol inhibited LDL oxidation, as indicated by a reduction in the content of lipid peroxides of 70% (P<.05; Fig 5A⇓) and in the relative electrophoretic mobility by 34% (P<.05; Fig 5B⇓). Paracetamol at 200 μmol/L showed similar effects, suggesting that paracetamol was able to scavenge the aqueous peroxyl radicals generated. Paracetamol was less active in scavenging peroxyl radicals generated in the lipid phase. Thus, 200 μmol/L paracetamol significantly reduced the lipid peroxides by 30% (282±35 versus 193±35 nmol/mg LDL; n=3), and the relative electrophoretic mobility by 12% (1.4±0.1 versus 1.3±0.2; n=3).

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Figure 5.

Bar graphs show amount of lipid peroxides (A) and relative electrophoretic mobility (B) in LDL during azo compound–induced oxidation. LDL (100 μg/mL) was incubated at 37°C with 4 mmol/L AAPH in PBS in the absence (Control) or presence of 100 or 200 μmol/L paracetamol for 6 hours. Results are mean±SD from four separate experiments.

Cell-Mediated Oxidation of LDL

To examine the effect of paracetamol in a more physiological system, human peripheral blood mononuclear cells were used to mediate the modification of LDL. Also, it was of interest to determine whether sodium salicylate was able to protect LDL from oxidative modification in a cellular system. Freshly isolated mononuclear cells were preincubated with paracetamol or sodium salicylate for 30 minutes prior to cell-mediated oxidation of LDL for 6 hours. In the absence of the drugs, the amount of lipid peroxides formed during cell-mediated oxidation of LDL was significantly higher than that formed during Cu²⁺-induced oxidation in Ham’s F-10 without cells (854±142 versus 493±209 nmol/mg LDL; n=6; P<.04). Relative electrophoretic mobility of cellularly oxidized LDL was 2.6±0.5% versus 1.4±0.2% for Cu²⁺-oxidized LDL in cell-free dishes (n=6; P<.005). By preincubating the cells with 100 μmol/L paracetamol, the amount of lipid peroxides formed was reduced by 69% (P<.01; Fig 6A⇓). Similarly, paracetamol reduced the relative electrophoretic mobility of oxidized LDL by 38% (P<.01; Fig 6B⇓). Paracetamol at 400 μmol/L showed similar effects (Fig 6A⇓ and 6B⇓). In comparison, 10 μmol/L α-tocopherol reduced the lipid peroxides formed in LDL during 6 hours of oxidation and the relative electrophoretic mobility by 52±28% and 65±7%, respectively (n=3; P<.04 and P<.02 versus no addition).

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Figure 6.

Bar graphs show amount of lipid peroxides (A) and relative electrophoretic mobility of LDL (B) after cell-mediated oxidation of LDL. Peripheral blood mononuclear cells were preincubated for 30 minutes in the absence (Control) or presence of paracetamol (100 or 400 μmol/L) or salicylate (800 μmol/L) prior to oxidation of LDL (100 μg/mL) for 6 hours. Results are mean±SD from six separate experiments with triplicates.

In contrast to paracetamol, no effect of sodium salicylate (800 μmol/L) was observed (871±168 versus 854±142 nmol lipid peroxides/mg in the absence of the drug; relative electrophoretic mobility 2.7±0.3% versus 2.6±0.5% in the absence of sodium salicylate; Fig 6A⇑ and 6B⇑). Similarly, the presence of sodium salicylate at a concentration of 4 mmol/L did not change the amount of lipid peroxides generated or the relative electrophoretic mobility (87±19% and 101±14%, respectively, versus no addition of sodium salicylate; n=5). Sodium salicylate at a concentration of 2 mmol/L slightly increased the generation of lipid peroxides and the relative electrophoretic mobility (117±14% and 114±10%, respectively, versus no addition of sodium salicylate; n=4; P<.05).

To examine the role of superoxide anion and hydrogen peroxide in mononuclear cell–mediated LDL oxidation, we evaluated the effects of SOD and catalase, which catalyze the breakdown of superoxide and hydrogen peroxide, respectively. SOD (10 and 100 μg/mL) and catalase (50 μg/mL) inhibited the formation of lipid peroxides in LDL by 88%, 92%, and 98%, respectively, whereas relative electrophoretic mobility was reduced by approximately 50% by both enzymes (Table 1⇓). Heat-inactivated SOD and catalase failed to block the lipid peroxidation of LDL, suggesting that the inhibitory effect was dependent on enzyme activity rather than nonspecific effects. However, in cell-free dishes SOD (10 and 100 μg/mL) significantly inhibited lipid peroxidation by 63% and 53%, respectively, whereas catalase completely blocked the LDL oxidation (data not shown), suggesting that the enzymes might bind copper ions. In separate experiments SOD (100 μg/mL) did not affect LDL oxidation in the presence of paracetamol (100 μmol/L), as indicated by the content of lipid peroxides and by the relative electrophoretic mobility in agarose gels (Table 2⇓). However, in the presence of 800 μmol/L salicylate, SOD inhibited the formation of lipid peroxides by 47% and reduced the relative electrophoretic mobility by 36%. Taken together, the results show that paracetamol, but not sodium salicylate, is able to protect LDL against peripheral blood mononuclear cell–mediated oxidative modification and that superoxide anion and hydrogen peroxide might be involved in the reaction pathway.