All the reagents and chemicals used in these experiments were analytical grade of highest purity. All organic solvents were HPLC grade. 1,1,3,3-tetraethoxypropane (TEP), trichloroacetic acid (TCA), 2-thiobarbituric acid (TBA) and dinitrophenylhydrazine (DNPH) were obtained from Sigma- Aldrich Com Ltd. Acetonitrile was purchased from Labscan (Dublin, Ireland).
Experimental animals:
Guinea pigs weighing 200–400 g (n: 10) were
anesthetized with pentobarbital. Kidney, liver and brain were removed and washed with cold NaCl 0.9% and immediately placed in liquid nitrogen. Then tissues were frozen at -70ºC until use.
Blood:
Blood was collected by venipuncture into EDTA from 10 healthy male and female volunteers (18-30 years, non smokers). After centrifugation (400ºg 10 min at 4ºC) plasma was immediately frozen on dry ice and stored at -70ºC.
Sample preparation for total MDA in HPLC:
Tissue preparation:
500 mg tissue was homogenized by Vetra-Turrax in a volume of 1.15% KCl, 24000 rpm/min 7. For alkaline hydrolysis of protein bound MDA, 200 µL 6 M NaOH was added to 1 ml homogenenate in an eppendorf cup and the sample was incubated in a 600C water bath for 45 min. An aliquot of 1 ml was diluted with an equal volume of acetonitrile to precipate proteins. The resulting suspension was then vortex mixed for 30 s and centrifuged at 15000g for 10 min. The upper clear supernatant (0.25 ml) was transferred to a 2 ml eppendorf cup, mixed with 25 µL DNPH solution (5mM in 2 M HCl, pH = 0.09) and incubated for 10 min at room temperature. After derivatisation, the samples were filtered through a 0.2 µm filter. Aliquots of 20 µL were injected into HPLC system 5,7,8,10-11.
Protein levels of tissues were measured by Lowry method 12.
Plasma preparation:
The same procedure for the tissues was applied to plasma. 50 µL of 6 M NaOH was added to 0.250 ml plasma and incubated in a 600C water bath for 45 min. The hydrolyzed sample was acidified with 0.125 ml of 35% (v/v) perchloric acid. After centrifugation, 0.250 ml supernatant was mixed with 25 µL DNPH solution and incubated for 10 min. A 20 µL volume of the reaction mixture was directly injected onto HPLC system 5.
Preparation of the standard curve:
MDA standard was prepared by dissolving 25 µL 1,1,3,3 tetraethoxypropane (TEP) in 100 ml of water to give a 1 mM stock solution. Working standard was prepared by hydrolysis of 1 ml TEP stock solution in 50 ml 1% sulfuric acid and incubation for 2 h at room temperature. The resulting MDA standard of 20 nmol/ml was further diluted with 1% sulfuric acid to yield the final concentration of 10.5, 2.5, 1.25 and 0.625 nmol/ml to get the standard curve for the estimation of total MDA 5. 0.250 ml of standards were mixed with 25 µL DNPH solution and incubated for 10 min. A 20 µL volume of the reaction mixture was directly injected onto HPLC system by filtered through a 0.2 µm filter or unfiltered 5. Plasma samples were not filtered therefore their results were evaluated with unfiltered curve.
HPLC analysis:
The samples were analyzed on an Agilent HP 1100 series HPLC apparatus (USA). The analytical column ODS 2 C18 (5 µm particle size, 125 µ4 mm). In our experiment, analytical column was 5 µm pore size spherisorb ODS-2 C18 reserve phase column (125µ4 mm) the retention time of the MDAhydra zone averaged 6.0 min for daily runs.
The mobile phase was acetonitrile-distilled water (38: 62, v/v) containing 0.2 % (v/v) acetic acid. HPLC apparatus was isocratic a condition at a flow rate 1 ml/min and UV detector was set at 310 nm. MDA peaks were determined according to its retention time and confirmed by spiking with added exogenous standard. Concentrations of MDA were calculated from standard curve prepared from 1,1,3,3 tetraethoxypropane and expressed as nmol/ml for the plasma and nmol/mg protein for the tissues 2,5.
Thiobarbituric acid method:
Plasma TBA:
MDA level of the plasma was measured by the following procedure according to Tomotsu et al. 0.5 plasma was shaken with 2.5 ml of 20% trichloroacetic acid (TCA) in a 10 ml centrifuge tube. 1ml of 0.6 % TBA was added to the mixture, shaken, and warmed for 30 min in a boiling water bath followed by rapid cooling. Then it was shaken into a 4 ml of nbutyl- alcohol layer in a separation tube and MDA content in the plasma was determined from the absorbance at 535 and 520 nm by spectrophotometer against butanol. The standards of 5, 10, 20 nmol/ml TEP were used. The results were expressed as nmol/ml plasma 13.
Tissue TBA:
The level of MDA was determined in the tissue samples homogenized in a ratio of 1/10 in 1.15 % (w/v) cold KCl solution, by the aid of TBA method reported by Uchiyama and Mihara 14. The results were evaluated from the standard curve (2.5, 5, 10, 20 nmol/ml) and expressed as nmol/mg protein.
Statistical analysis:
Statistical analyses were performed using a software program (SPSS 11.5 for windows, Chicago, IL, USA). Plasma and tissue MDA levels of HPLC and TBA methods were compared by Mann-Whitney U test. For tests of significance a p value of less than 0.05 was considered to be significant. The results were presented as mean ±SD.
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