L-carnitine reduces acute lung injury in experimental biliary obstruction

Surgical technique

All the procedures were performed aseptically. The rats were anesthetized using intramuscular injections of 25 mg/kg ketamine hydrochloride (Ketalar^®, Parke-Davis, Eczacibasi, Istanbul, Turkey) and 10 mg/kg xylazine (Rompun^®, Bayer, Leverkusen, Germany). After shaving the abdomen and preparing the site with 10% povidone-iodine solution, the abdomen was opened through a small upper midline incision. The common biliary duct was identified, mobilized, doubly ligated using 4-0 silk and divided. The control group animals had a similar incision followed by mobilization of the common biliary duct without ligation or division. All abdominal incisions were closed in 2 layers using 3-0 polyglactine (Vicryl^®; Ethicon, Woluwe, Belgium) and 4-0 polypropylene (Prolene^®; Ethicon, Woluwe, Belgium). After peritoneal saline (2cc) was injected for fluid resuscitation, the abdominal incisions were closed. All groups had access to unlimited consumption of rat chow and tap water until the sampling process.

In all groups, 10 days after the laparotomy, sternotomy was performed under general anesthesia. For biochemical analysis, 5 cc of blood was taken from the aorta before the sacrifice of each rat. The rats were sacrificed by inducing cardiac arrest through bloodletting from the inferior vena cava. After sacrifice, the lungs were extracted. The lung tissue samples taken for pathology were fixed with 10% formalin and the lung tissue for biochemical analysis was put in phosphate buffer.

Biochemical evaluation. Preparation of tissue homogenates

After the procedure, the lung tissues were put in glass tubes and filled with pH 7.4 phosphate buffers. The lung tissues were stored at -80°C, then removed and homogenized before testing (PCV Kinematice Status Homogenizator).

Determination of malondialdehyde (MDA) levels

The analysis of lipid peroxidation was carried out as described with a minor modification.11 The reaction mixture was prepared by adding 250 µL homogenate into 2 mL reaction solution (15% trichloroacetic acid: 0.375% thiobarbituric acid: 0.25 N HCl, 1:1:1, w/v) and heated at 100°C for 15 minute. The mixture was cooled to room temperature, centrifuged (10.000 g for 10 min) and the absorbance of the supernatant was recorded at 532 nm. 1,1,3,3-tetramethoxypropane was used as MDA standard. Malondialdehyde results were expressed as nmol/mg protein in the homogenate.

Determination of glutathione peroxidase (GSH-Px) levels

The formation of 5-thio-2-nitrobenzoate (TNB) was followed spectrophotometrically at 412 nm.12 The amount of GSH in the extract was determined as nmol/mg protein utilizing a commercial GSH-Px as the standard.

Measurement of catalase (CAT) activity

Catalase activity was measured at 37°C by following the rate of disappearance of hydrogen peroxide (H₂O₂ at 240 nm [e240 = 40 M⁻¹ cm⁻¹]).13 One unit of catalase activity was defined as the amount of enzyme catalyzing the degradation of 1 µmol of H₂O₂ permin at 37°C and specific activity corresponding to transformation of substrate (in µmol) (H₂O₂) per min per mg protein.

Measurement of superoxide dismutase (SOD) activity

The activity of SOD was assessed according to the method of Minami and Yoshikawa.14 In brief, this method depends on computing the difference between auto-oxidation of pyrogallol alone and in the presence of a cytosolic fraction that contains the enzyme. Enzymatic activity was expressed as ug/gm of tissue.

Measurement of myeloperoxidase (MPO) activity

Measurement of myeloperoxidase activity in the rat lungs was measured according to a previously described method. Lung samples were homogenized with a homogenizer using 6 mL of a homogenization buffer (50 mmol/L phosphate, pH 6.0). The homogenate was then sonicated and centrifuged at 4.4.500g for 30 minutes at 4°C. Measurement of myeloperoxidase activity in the supernatants was reacted with H₂O₂ in the presence of o-dianisidine (Sigma). The change in optical density at 460 nm was determined over one minute at 25°C in a spectrophotometer (DU-54; Beckman, Irvine, CA, USA).15

The rat C-reactive protein (CRP) enzyme-linked immunosorbent assay (ELISA) is based on a solid phase ELISA. The rat CRP of serum was performed using a commercial kit (Life Diagnostics, Inc., Westchester, PA, USA), according to the manufacturer’s instructions. Total bilirubin, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) levels were measured as parameters indicative of hepatic function with an autoanalyser (Olympus AU640, Tokyo, Japan).

Histopathological evaluation

The lung tissues fixed in 10% formalin for 24 hours were embedded in paraffin. Tissue sections cut at 5 µm were stained with hematoxylin & eosin (H&E) and Masson’s trichrome methods. Samples were examined using a Leica DFC280 light microscope and a Leica Q Win Image Analysis system (Leica Micros Imaging Solutions Ltd., Cambridge, UK). Assessment of tissue alterations in 20 different fields for each section was conducted by an experienced histologist who was unaware of the treatment. The results were classified into 4 grades that were described by Özdülger et al.16 Grade 1: Normal lung histology, Grade 2: Mild neutrophil infiltration of leukocytes, Grade 3: Moderate neutrophil leukocyte infiltration, perivascular edema formation, the partial destruction of lung structure, and Grade 4: Intensive neutrophil leukocyte infiltration, which defines complete destruction in the pulmonary structure.

Statistical analysis

The data was then entered and analyzed using the Statistical Package for Social Sciences version 18 (SPSS Inc, Chicago, IL, USA). The Kruskal-Wallis test was used for the comparison of the biochemical values of TNF-α, IL-6, IL-8, MDA, MPO, GSH-Px, and CRP with independent samples. As a result of the Kruskal-Wallis test, the Mann-Whitney U test was used to examine the differences between groups, and the Chi-square test was used for comparisons of the histopathological examination of lung tissue on day 10. A value of p<0.05 was accepted as statistically significant.