The effects of simvastatin and fenofibrate on malondialdehyde and reduced glutathione concentrations in the plasma, liver, and brain of normolipidaemic and hyperlipidaemic rats

Simvastatin and fenofibrate were chosen for this investigation, because both agents cross the blood-brain barrier (31, 32). Simvastatin (CAS-79902-63-9) (Lipex^®, Merck Sharp & Dohme, Haarlem, The Netherlands) (simvastatin) and fenofibrate (CAS-49562-28-9) (Tricor^®, Recipharm Fontaine, Fontaine-lès-Dijon, France) (fenofibrate) tablets were suspended in 5 mL/kg saline and administered daily (at 9.00–10.00 a.m.) by gavage. Our choice of fenofibrate doses of 30 and 50 mg/kg/day and of simvastatin doses of 10 and 50 mg/kg/day is based on literature data (33–36) from different experiments on rats.

This study included male 10-week-old normolipidaemic (N=72) and hyperlipidaemic rats (N=21). The normolipidaemic rats were the Wistar strain and weighed 250–300 g (University of Zagreb School of Medicine, Department of Pharmacology, Zagreb, Croatia). The hyperlipidaemic rats were the HsdOla: Zucker-Lepr fa-fa strain (Harlan Laboratories srl, Udine, Italy) and weighed 270–300 g. The animals were kept in macrolone cages at controlled room temperature (22 °C) and 12:12 h day/night cycles and had free access to standard pellet diet. Animal handling and treatment followed the national (37) and European (38) guidelines and recommendations for the use of laboratory animals. The experiments were approved by the ethics committee of the University of Zagreb School of Medicine (approval No. 380-59-10106-23-111/33).

The experiment lasted 21 days (3 weeks). Normolipidaemic rats were randomly divided into four experimental (N=10) and four control groups (N=8). The first experimental group was receiving simvastatin 10 mg/kg/day and the second simvastatin 50 mg/kg/day. The third experimental group was receiving fenofibrate 30 mg/kg/day and the fourth fenofibrate 50 mg/kg/day. Corresponding control groups were given saline (0.9 % NaCl solution) 5 mL/kg b.w. daily by gavage.

Hyperlipidaemic Zucker rats were divided in two experimental and one control group. The first experimental group (N=7) was receiving simvastatin 50 mg/kg/day and the second (N=8) fenofibrate 30 mg/kg/day. The control group (N=6) was receiving saline as described above.

On day 22 of the study, after an overnight fast of 12 h, all rats were sacrificed under diethyl ether anaesthesia. Blood samples were taken by cardiac puncture and frozen at -20 °C immediately after sampling. Liver and brain tissues were also taken immediately after sacrifice. Liver was rinsed with saline in situ via the superior vena cava to remove blood. The brain was also rinsed with saline. The whole brain and a part of the liver were homogenised in saline in the 1:4 ratio and centrifuged at 3500 g for 20 min. The separated supernatants were frozen at -80 °C.

The samples were prepared by pipetting 100 µL of plasma and 500 µL of precipitation reagent into labelled, light-protected vials, which were vortexed and then centrifuged at 8125 g for 5 min. The obtained supernatant (500 µL) was then transferred to a labelled derivatisation vial and added 100 µL of derivatisation reagent. The vials were capped, briefly vortexed, and incubated at 95 °C for 60 min. Followed abrupt cooling under a stream of cold water, and then we added 500 µL of neutralisation buffer to the cooled mixture and injected 20 µL of the prepared sample into the column.

Plasma MDA concentrations were determined with a high performance liquid chromatograph (HPLC) (Nexera, Shimadzu Corporation, Kyoto, Japan) using a commercially available kit (Cat. No. 67000, Chromsystems Instruments and Chemicals GmbH, Gräfelfing, Germany), according to the manufacturer’s instructions. Sample preparation was based on protein precipitation followed by derivatisation. The HPLC system consisted of a fluorescent detector (RF-20AXS), isocratic pump (LC-20ADXR), column thermostat (CTO-20A), autoinjector (SIL-30AC), degasser (DGU-20A5), and control unit (CBM-20A). Chromatographic conditions were as follows: isocratic mobile phase flow 1.0 mL/min; pressure ~125 bar; wavelength 515 nm (excitation) and 553 nm (emission); column temperature 25 °C; analysis time 5 min; injection volume 20 µL.

Data from the detector were collected and processed with the Shimadzu LabSolutions software, and the measured plasma MDA concentrations are expressed in micromoles per litre (µmol/L).

Brain and liver tissue MDA concentrations were measured using a modified method described by Drury et al. (39), which is based on MDA binding to 2-thiobarbituric acid (TBA) to form a pink compound that absorbs light at the wavelength of 538 nm. For the standard calibration curve we used 1,1,3,3-tetraethoxy propane. Further oxidation was prevented by adding butylated hydroxytoluene (BHT).

The sample solution contained 50 µL of the brain or liver tissue supernatant, 5 µL of BHT (0.2 %, w/v), 750 µL of phosphoric acid (1 %, v/v), 250 µL of TBA (0.6 %, w/v), and 445 µL of ultrapure water (18 MΩ; obtained from a Thermo Scientific Smart2Pure 3 UV/UF water purification system, Thermo Fisher Scientific Inc., Waltham, MA, USA). We stirred it and incubated in a boiling water bath for 30 min, then stopped the reaction by cooling in water, and then injected 20 µL of the prepared solution into a HPLC consisting of a degasser, isocratic pump, thermostated column space, and UV detector (LC-20AD, Shimadzu). The pre-column and analytical column were C-18 reverse phases (LiChrospher, Merck, Darmstadt, Germany) with 5 µm particles. The dimensions of the pre-column were 4.0 × 4.0, and of the columns 4.0 x 125.0 mm.

Mobile phase consisted of 50 mmol/L KH₂PO₄ and methanol (60:40, v/v, pH 6.8). The flow rate was 1 mL/min. MDA was measured with a UV detector at 532 nm and column temperature was 32 °C. Under these conditions, the MDA retention time was 2.5 min. Its brain and liver tissue concentrations are expressed as micromoles per litre (µmol/L).

Rat plasma, brain, and liver GSH concentrations were measured according to the Ellman’s spectrophotometric method (40), which is based on glutathione (thiol) coupling with 5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB). In this rapid reaction, a disulphide bond is cleaved to form 2-nitro-5-thiobenzoate (NTB), which is ionised to NTB^2- and whose yellow colour is read at 412 nm. We added 100 µL of trichloroacetic acid (TCA, 5 % solution) to 300 µL of supernatant and first stirred the mixture with a shaker and then centrifuged it at 10,000 g for 10 min. Reaction mixtures consisted of 850 µL of phosphate buffer and 50 µL of DTNB added to100 µL H₂O for the blank sample or to 100 µL standards for standard samples or to 100 µL of tissue or plasma supernatant and were measured at 412 nm using a Cecil 9000 spectrophotometer (Cecil Instruments Limited, Cambridge, UK). All measurements were done in triplicate. The concentration of GSH was calculated from the calibration diagram and is expressed as micrograms per millilitre (µg/mL).

All data were analysed with the GraphPAD Prism software, version 6 (GraphPAD Software, San Diego, CA, USA). For normolipidaemic rats, the results are shown as arithmetic means ± standard deviations (x̄±SD) and p-values. For hyperlipidaemic rats the results are shown as arithmetic means ± standard deviations and p-values of Dunnett’s multiple comparison test, which is considered optimal for comparison of different drug doses with a single control group. All applied tests were two-way. P values of less than 0.05 are considered statistically significant (41).

Table 1 shows MDA and GSH concentrations in the plasma, liver, and brain of normolipidaemic rats receiving either of the two simvastatin doses. Compared to control, only the higher dose (50 mg/kg/day) significantly lowered MDA concentrations (20 %; p=0.044) in the plasma, neither dose lowered it significantly in the liver, and only the lower dose (10 mg/kg/day) decreased it significantly in the brain 59 % (p<0.001).

GSH concentrations increased significantly (63 %; p=0.002) only in the liver of rats receiving the lower simvastatin dose (10 mg/kg/day).

Table 2 shows MDA and GSH concentrations in the plasma, liver, and brain of normolipidaemic rats receiving either fenofibrate dose (30 or 50 mg/kg/day). Both significantly lowered MDA concentrations in the plasma [39 % (p=0.017) and 42 % (p<0.001), respectively], but neither changed them significantly in the liver or brain.

The higher fenofibrate dose significantly increased plasma GSH (31 %; p=0.03), and both doses significantly lowered it in the liver [54 % (p=0.013) and 60 % (p<0.001), respectively]. Neither dose significantly affected brain GSH concentrations.

Table 3 shows plasma, liver, and brain MDA and GSH concentrations in hyperlipidaemic rats receiving either the higher simvastatin (50 mg/kg/day) or lower fenofibrate dose (30 mg/kg/day). Compared to control, simvastatin did not significantly affect MDA concentrations in any of the organs, but fenofibrate significantly lowered them in the plasma (33 %; p=0.009) and increased them in the brain (39 %; p=0.032). It did not change MDA concentrations in the liver.

As for GSH concentrations, both drugs had a significant (lowering) effect only in the liver (45 % and 80 % vs control, respectively).