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Original Article
Lipid Peroxides in Patients with Inflammatory Bowel Disease Mohammed A. Alzoghaibi, Ibrahim A. Al Mofleh*, Abdulrahman M. Al-Jebreen*
Departments of Physiology and *Medicine, Gastroenterology Unit College of Medicine, King Saud University, Riyadh, Saudi Arabia
Background/Aims: In this study, we aimed to determine the levels of malondialdehyde (MDA) in patients with inflammatory bowel disease (IBD) to investigate its contribution to tissue injury in IBD. Materials and Methods: Forty-two patients with IBD (24 cases of Crohn’s disease and 18 cases of ulcerative colitis) and 38 matched healthy subjects (control group) were considered for study. MDA levels were quantified by the measurement of thiobarbituric acid reactive substances. Results: Plasma MDA levels of Crohn’s disease patients were significantly higher than the control group, but not higher than the ulcerative colitis patients. Plasma MDA levels of patients with ulcerative colitis were higher than the control group but not significant. Conclusion: Increased levels of plasma MDA in IBD is an important indication of oxidative stress. Patients with Crohn’s disease are more susceptible to oxidative stress than patients with ulcerative colitis.
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Address: Dr. Mohammed Alzoghaibi, College of Medicine, Department of Physiology, King Saud University, P.O. Box 7805, Riyadh 11472, Saudi Arabia. E-mail: zzoghaibi@hotmail. com
ABSTRACT
Key Words: Inflammatory bowel disease, lipid peroxides, malondialdehyde, oxidative stress
Received 01.05.2007, Accepted 22.07.2007 The Saudi Journal of Gastroenterology 2007 13(4): 187-90
Inflammatory bowel disease (IBD), Crohn’s disease and ulcerative colitis are debilitating illnesses of the bowel characterized by chronic inflammation of unknown etiology.[1] While the etiology is unknown, three main factors are assumed to play a role in the formation and progression of these diseases: (1) immune system dysfunction, (2) oxidant stress and (3) poor diet. Oxidant stress could be a major factor in IBD. Often the first attack is preceded by a stressful period in the life of the patient and flare-ups are often precipitated by emotional or physical stress. Emotional or physical stress can result in oxidative stress.[2] The state of oxidative stress may exist when there is an imbalance between the levels of antioxidant and reactive oxygen species (ROS).
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Oxygen-free radicals and lipid peroxides (oxidative stress) are highly reactive and damaging compounds.[3] Oxidative stress could be a major contributing factor to the tissue injury and fibrosis that characterize IBD. It is present in IBD as it is evident by the increased production of ROS, decreased antioxidant levels (e.g., β-carotene and vitamins C and E) and decreased antioxidant enzymes in the intestinal mucosa.[4-9]
In most of the recent studies, it has been demonstrated that inflammatory lesions of gut mucosa and smooth muscle cells have more accumulation of granulocytes than other layers.[10-15] Those activated cells release many inflammatory mediators such as toxic oxygen metabolites, lysosomal enzymes and arachidonic acid metabolites.[15,16-21] In turn, superoxide anion radicals (•O2), hydrogen peroxide (H2O2) and hydroxyl radicals (•OH), secreted by neutrophils and other phagocytes accumulate within inflammatory lesions,
thereby causing the impairment of the cellular membrane and cell death by leading lipid peroxidation.[22] The previous studies have contradictory results in reporting the levels of oxidative stress in IBD patients. Several studies have shown no difference in the levels of oxidative stress between IBD patients and normal subjects.[13,14] Other studies have shown a significant difference in the levels of oxidative stress between the IBD patients and the normal group. In this study, we therefore wanted to explore the exact levels of oxidative stress by measuring MDA – the break-down product of lipid peroxides – in the plasma of IBD patients.
MATERIALS AND METHODS Study population
Our study group comprised 42 patients (nonsmokers) with severe IBD (24 cases of Crohn’s disease and 18 cases of ulcerative colitis), who presented at the gastrointestinal unit of King Khalid University Hospital (KKUH), Riyadh, Saudi Arabia and 38 healthy subjects (control group) with a mean age of 29.5 ± 5.7 years and a body mass index (BMI) of 24.5 ± 3.4 and who were not on medications. Table 1 shows the demographic characteristics of the patients with Crohn’s disease and ulcerative colitis, who were under study. IBD patients were not on medications that could affect the oxidant activity. Blood samples were collected in the outpatient clinic and immediately transferred to the laboratory for the test of lipid peroxides. The study was approved by the Ethics Committee in our institute and an informed consent was obtained from all the participants. The Saudi Journal of Gastroenterology
187 Volume 13, Number 4 Ramadan 1428 H October 2007 CMYK 187
[Downloaded free from http://www.saudijgastro.com on Wednesday, September 28, 2016, IP: 91.150.169.99] Alzoghaibi, et al.
Control subjects Crohn’s disease patients Ulcerative colitis
N 38 24
Age 29.5 ± 5.7 24.8 ± 8.8
BMI (kg/m2) 24.5 ± 3.4 25.5 ± 14.8
MDA (µmol/L) 8.7 ± 0.19 11.3 ± 0.9
18
34.7 ± 10
23.2 ± 5
10.3 ± 0.2
* 12 10 8 6 4 2
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BMI - Body mass index, MDA - Malondialdehyde
14
MDA umol/mL
Table 1: Demographic characteristics and plasma malondialdehyde levels of patients and control subjects. Data given in the table as mean ± standard error of the mean
Lipid peroxide assay
Lipid peroxides were estimated by an improved analysis of thiobarbituric acid reactive substances (TBARSs), which primarily reflects malondialdehyde (MDA), in human fluids as described previously.[23] In the presence of heat and acid, MDA reacts with thiobarbituric acid (TBA) to form a red pigment with a peak wavelength (λmax) of 530 nm. Reagents were obtained from Sigma Chemical (St. Louis, MO). Tetramethoxypropane was used to generate MDA to obtain the standard curve in the range from 0.1 µM to 5.0 µM. Blood serum (200 µL) was mixed with 0.2 M phosphoric acid (200 µL), 5 mM butylated hydroxytoluene (25 µL) and 0.11 M TBA (25 µL). The mixture was incubated at 90°C for 45 min and then placed on ice for a very short duration in order to stop the reaction. MDA was extracted by the addition of n-butanol (500 µL). The absorptions at 530 nm and 570 nm were measured using a 96-well plate reader (Specta Microplate Autoreader, Tecan, Research Triangle Park, NC).
Statistical analysis
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Data are expressed as mean ± standard error of the mean (SEM) values. Comparisons between the control subjects, Crohn’s disease and ulcerative colitis patients were made using ANOVA and results were considered to be statistically significant when P was < 0.05. We used post hoc test (Bonferroni test) to detect significance. Standard statistical software (SPSS, version 12; Chicago, IL) was used for the analyses.
RESULTS Table 1 shows the demographic characteristics of the studied subjects. There was no statistical difference either in age or BMI between the three study groups.
Release of reactive oxygen species
TBARS levels differed significantly between the control subjects and Crohn’s disease patients (8.7 ± 0.19 µmol/mL vs 11.3 ± 0.9 µmol/mL, respectively, P
