الفهرس 
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Abstract
Chronic obstructive pulmonary disease (COPD) is a condition that makes it difficult to move air into and out of a person’s lungs. It is characterized by expiratory airflow obstruction and hyperinflation, and is becoming the most prevalent respiratory disease in the world. Tobacco smoking is the greatest risk factor for the development of COPD, although approximately 85% of smokers do not develop clinically relevant airflow obstruction. Several epidemiological studies have suggested that genetic factors are likely to have a role in determining an individual’s susceptibility to COPD. Polymorphisms in several genes have been shown to be involved in the development of COPD and one such candidate is the vitamin D binding protein (VDBP) gene. An important factor in investigating the role of VDBP is the variation in allele frequency between populations.
Recently, research has found that vitamin D may play a role in multiple chronic diseases such as cancer, autoimmune diseases, infections, and cardiovascular disorders. Vitamin D may also have a role in several diseases involving the respiratory system. In some clinical studies, lower levels of vitamin D, measured as plasma 25-hydroxyvitamin D [25 (OH) D], have been associated with lower lung function and faster lung function decline, but the results have been conflicting. Antioxidants are substances that may protect cells from the damage caused by unstable molecules known as free radicals.
Antioxidants interact with and stabilize free radicals and may prevent some of the damage free radicals might otherwise cause. Free radical damage may lead to cancer. An antioxidant is a molecule capable of slowing or preventing the oxidation of other molecules. Oxidation is a chemical reaction that transfers electrons from a substance to an oxidizing agent. Oxidation reactions can produce free radicals, which start chain reactions that damage cells. Antioxidants terminate these chain reactions by removing free radical intermediates, and inhibit other oxidation reactions. They do this by being oxidized themselves, so antioxidants are often reducing agents such as thiols, ascorbic acid (vitamin C), or polyphenols. There is evidence to suggest an imbalance between oxidants and antioxidants in the lungs and the blood in smokers as well as in patients with COPD.
Aims of the study
1- Evaluate any relationship that may exist between VDBP allele frequency and the risk for COPD in the Egyptian population.
2- Evaluate the evidence of the effect of vitamin D on COPD.
3- Examine the role of altered levels of oxidant–antioxidants in disease severity of COPD and correlate it with the degree of airflow obstruction in the Egyptian population.
Subjects and methods
The present study, included 120 subjects, 80 patients diagnosed as COPD and 40 healthy volunteers (20 smokers and 20 nonsmokers). Genomic DNA was extracted from 5 ml of frozen whole blood using a DNA extraction kit (Nucleon Bacc II, Tepnel Life Sciences, UK) according to the manufacturer’s protocol.
Primers for the polymerase chain reaction (PCR) were designed from the published VDBP gene sequence, and the VDBP gene was amplified using a commercially available PCR reaction master mix, according to the manufacturer’s instructions (thermo Fisher scientific company). The PCR product was genotyped by restriction fragment length polymorphism analysis, involving overnight restriction enzyme digestion at 37 °C with the Eco T14 and Hae III restriction enzymes (thermo Fisher scientific company). The digested fragments were resolved on 3% agarose gels, stained with ethidium bromide, and observed under ultraviolet light. Plasma [25(OH) D] was measured by Enzyme Linked Immunosorbent Assay (DiaSorin, Stillwater, Minnesota, USA) in all study participants. A quantitative colorimetric and spectrophotometric determination of reduced glutathione (GSH), glutathione peroxidase activity (GSH-Px), superoxide dismutase activity (SOD) in erythrocytes, plasma catalase activity (CAT), serum lipid peroxides (LP) and nitric oxide (NO).
The results obtained could be summarized as follow:
I- Results of Vitamin D Binding Protein Gene Polymorphism
1- The distribution of the six major genotypes of VDBP in the patients and the control groups are significantly different.
• The 1S-1S genotype with a significant difference between the patients and the healthy nonsmoking group and between healthy nonsmokers and healthy smokers.
• The 1F-1S genotype with a significant difference between the patients and the healthy nonsmoking group and between healthy nonsmokers and healthy smokers.
• The 1F-1F genotype is significantly different between the patients and the healthy nonsmoking group.
• Also we found 2-2 genotype with a significant difference between the patient and the healthy nonsmoking group and between healthy nonsmokers and healthy smokers.
• Regarding 1S-2 and 1F-2 genotypes, we did not find any significant difference between the patient and the healthy nonsmoking group and between healthy nonsmokers and healthy smokers.
• Nonsignificant statistical differences were observed between patients and healthy smokers regarding all six major genotypes.
2-Alleles frequencies of the genotypes
• The frequency of allele 1S is significantly different between patient group and healthy nonsmoking group and between healthy nonsmokers and healthy smokers.
• The frequency of allele 1F is significantly different between patient group and healthy nonsmoking group and between healthy nonsmokers and healthy smokers.
• While the frequency of allele 2 is significantly different between patient group and healthy nonsmoking group.
• Nonsignificant statistical differences were observed between patients and healthy smokers regarding all alleles.
3-Genotype frequencies according to the stages of COPD patients: we found that there is a significant association between the COPD stages and VDBP genotype or allele frequencies as follows:
• The 1S-1S genotype is significantly different between severe patients and mild patients, and between mild and moderate patient.
• The 1F-1S genotype is significantly different between severe patients and mild patients, and between mild and moderate patient.
• The 1F-1F genotype is significantly different between mild and moderate patients.
• The 1S-2 genotype is significantly different between severe patients and mild patients, and between mild and moderate patient.
• The 1F-2 genotype is significantly different between mild and moderate patients.
• The 2-2 genotype is significantly different between severe and mild patients.
II-Results of Vitamin D
• The mean concentration of [25(OH) D], was significantly higher in the control groups (smokers and nonsmokers) than in the severe COPD group.
• The mean concentration of [25(OH) D], was significantly higher in the control groups (smokers and nonsmokers) than in the moderate COPD group.
• The mean concentration of [25(OH) D], was not significantly higher in the control groups (smokers and nonsmokers) than in the mild COPD group.
III-Results of Oxidant –Antioxidant
• The mean concentration of nitric Oxide (NO) was significantly higher in the control subjects (smokers and nonsmokers) compared with COPD group.
• Also the mean concentration of nitric oxide (NO) was significantly higher in control nonsmokers group, compared to control smokers group.
• The mean concentration of lipid peroxide (LP) was significantly higher in COPD patients, compared with control subjects (smokers and nonsmokers).
• The mean concentration of glutathione (GSH) was significantly higher in the control subjects (smokers and nonsmokers) than in COPD patients.
• There is no significant difference in the concentration of glutathione peroxidase (GSH-Px) in all study participants (COPD patients, control smoker, control nonsmokers).
• The mean concentration of catalase (CAT) was significantly higher in control nonsmokers than in COPD patients and control smokers.
• The mean concentration of superoxide dismutase (SOD) was significantly higher in the control subjects (smokers and nonsmokers) than in the COPD patients. Also the mean concentration of superoxide dismutase (SOD) was significantly higher in control nonsmokers group than in the control smokers group.
Conclusion
1- VDBP polymor¬phisms might be associated with COPD in Egyptian population.
2- The results show a significant association of lower plasma [25(OH) D] levels with a higher risk of COPD.
3- These results support the hypothesis that an oxidant–antioxidant imbalance, associated with oxidative stress in COPD patients, plays an important role in the progression of disease severity, also these results revealed the presence of an oxidative presence in smokers and in subjects with COPD and that the imbalance may be important in the pathogenesis of this disease. Nitric oxide (NO) metabolism was not increased in patients with chronic obstructive pulmonary disease compared to healthy subjects. It has been reported that GSH plays a major role in pulmonary antioxidant protection.