ISSN: 1301-2193 E-ISSN: 1308-9846
  • Turkish Journal of
    Endocrinology and Metabolism

Abstract
Objective:
In this study it is aimed to investigate the oxygen-dependent bactericidal activity of leukocytes in patients with diabetes mellitus type 2.
Materials and Methods: All the data obtained from 28 patients with diabetes mellitus were compared with 31 demographically matched control individuals. Leukocytes of patients with diabetes mellitus and healthy control group were separated from blood samples for determination of oxygen-dependent antimicrobial activity enzymes including superoxide dismutase (SOD), glutathione reductase (GSH-R), gluthatione peroxidase (GSH-Px) and catalase. As lipid peroxidation products, thiobarbituric acid reactive substances (TBARS) were measured. 
Results: SOD, GSH-R, and catalase activities of patients with type 2 diabetes were not significantly different from control group (p>0.05). GSH-Px levels of patients with diabetes mellitus were significantly lower than control group (0.012±0.08 U/mg.pr versus 0.057±0.046 U/mg.pr, p<0.05). Measurements of TBARS were not different between patients with diabetes mellitus and control group (5.49±1.31 mmol/l versus 5.53±1.45 mmol/l, p=0.905).
Conclusions: It is difficult to suggest that in patients with diabetes mellitus the tendency to some known infections is due to alteration in oxygen-dependent bactericidal activity of leukocytes. Turk Jem 2009; 13: 20-4
Key words: Oxygen dependent bactericidal activity, tip 2 diabetes mellitus

Özet
Amaç:
Bu çalışmada tip 2 diyabetik hastalarda lökositlerde oksijen bağımlı baktesididal aktivitenin etkilenip etkilenmediğini araştırdık.
Gereç ve Yöntemler: Çalışmaya 28 diyabetik hasta ve demografik açıdan benzer 31 normal birey alınmıştır. Çalışmaya alınan bireylerden alınan kan örneklerinden lökositler ayrıştırılmış ve oksijen bağımlı antimikrobiyal aktivitede rol oynayan enzimlerden superoxide dismutaz (SOD), glutathione reduktaz (GSH-R), gluthatione peroxidaz (GSH-Px), katalaz ile lipid peroxidation ürünü olarak, thiobarbiturik asid reaktive substans (TBARS) ölçülmüştür.
Bulgular: Tip 2 diyabetiklerde lökositlerde SOD, GSH-R, and katalaz aktiviteleri kontrol grubundan farksız bulunmuştur (p>0.05). GSH-Px düzeyleri ise diyabetiklerde kontrol grubuna göre daha düşük bulunmuştur (0.012±0.08 U/mg.pr'e karşı 0.057±0.046 U/mg.pr, p<0.05). TBARS düzeyleri ise diyabetik grupta kontrol grubundan farksız bulunmuştur (5.49±1.31 mmol/l karşı 5.53±1.45 mmol/l, p=0.905).
Sonuç: Diyabetik bireylerin bilinen bazı enfeksiyonlara karşı yatkın olmasının lökositlerdeki oksijen bağımlı bakterisidal aktivite ile ilişkili olduğunu söylemek zordur. Türk Jem 2009; 13: 20-4
Anahtar kelimeler: Oksije bağımlı bakterisidal aktivite, tip 2 diyabet


Introduction

Diabetes mellitus is a disorder characterized by altered glucose tolerance and impaired lipid and carbohydrate metabolisms.
It is estimated that up to 8% of adult population  has diabetes mellitus and the total  number of people with diabetes mellitus is supposed to rise approximately from 191 million in 2005 to 330 million in 2025 (1,2).
Although the advent of new medical technologies and intensive medical management with insulin analogues or oral antidiabetic drugs significantly improved the outcome of complications caused by diabetes, it is still an important problem to handle the late onset diabetic complications which cause important morbidity and mortality. In earlier studies, it was suggested that infections played a role in increased morbidity and mortality related to diabetes mellitus (3,4). Unfortunately, most of these studies could not show any strong evidence for this idea (6-9). On the other hand, increased incidences of some infections were clearly shown among patients with diabetes mellitus (10-17).
Immunological studies conducted on individuals with diabetes mellitus revealed a substantial data that at least some of host defense mechanisms have weakened due to hyperglycemia (18,19). Among those, leukocyte chemotactic activity (20-22), leukocyte mobilization and adhesion defects (23-25), leukocyte phagocytosis and opsonization defects (26-28) and diminished bactericidal activity are frequently mentioned (29,30).
In addition to general defects of immunity, decreased immune response to infections due to microvascular or macrovascular dysfunction, urinary colonization due to retention of urine in bladder secondary to autonomous neuropathy, better growth of bacteria in urine with high glucose and some other non-immunologic or anatomic factors can also be suggested as major factors that sensitize patients with diabetes mellitus to infections (31-33).
In this study, it is aimed to investigate whether oxygen-dependent antimicrobial mechanisms are affected in individuals with diabetes mellitus. As indicators of oxygen-dependent bactericidal activity SOD, GSH-Px, GSH-R and CAT were studied, and thiobarbituric acid reactive substances (TBARS) were studied as indicators of free radical production. All the data obtained from patients with diabetes mellitus were compared with demographically matched control individuals.

Materials and Methods

The study protocol included 28 patients with diabetes mellitus and demographically matched 31 control individuals. Diabetic ketoacidosis, diabetic nephropathy, systemic infection, smoking, use of any drug that has a known effect on leukocytes (steroids, cancer drugs, immunosuppressors, interferons, etc.) or antioxidant drug use (melatonin, carnitine, vitamin drugs, Ginkgo biloba extract, allopurinol, etc.) were the criteria for exclusion from the study. For the measurement of biochemical parameters, from the individuals in patient and control groups venous blood samples were drawn after overnight fasting and were transferred into plastic tubes containing acid-citrate-dextrose (ACD) in order to separate leukocytes from other blood components e.g. erythrocytes, by dextran sedimentation method.
Method for preparation of leukocytes
Ten milliliters of anticoagulated blood collected in ACD, as the usual source of white blood cells, was placed into a 15-ml plastic centrifuge tube. Two ml of dextran solution in sodium chloride 5 g/dl, was added and mixed. The mixture was allowed to stand for 45 min. for sedimentation of cells. The supernatant was drawn off and discharged into another plastic centrifuge tube. At 4 °C, the tubes were centrifuged at 500xg for 10 min. The supernatant was drawn off and discarded (34).
Button of white blood cells was resuspended in 1 ml of cold sodium chloride, 0.9 g/dl. The cells were shock treated by adding 3 ml of ice-cold distilled water and mixed gently for 45 s, and by immediately adding 3 ml of cold sodium chloride, 1.8 g/dl and mixing, they were centrifuged at 500xg for 10 min. The supernatants were drawn off and discarded. The steps in stage 2 were repeated for second shock treatment. The specimens were stored at -80 °C until the enzyme assays were performed. The protein concentrations of each sample were determined (35).
Determination of protein, parameters of antioxidative system and lipid peroxidation.
Frozen specimens were allowed to thaw at room temperature and then the specimens were completely suspended by sonication, three bursts of 10 s each in a sonicator cup filled with ice water. Protein concentrations in specimens were determined by the method of Lowry (36). TBARS were measured using an established method after treatment with thiobarbituric acid (37). The samples, treated with an acid (TCA/TBA/HCl) solution to precipitate protein, were heated to produce a colored product, and were centrifuged. The absorbances were measured at 535 nm. The standard curve was constructed with malondialdehyde (MDA) that was generated by acid treatment of 1,1,3,3- tetrametoxypropane.
The specimens were incubated in 60 mM sodium-potassium phosphate buffer, pH 7.4, containing 65 mmol/ml H2O2 at 37 °C for 60 s for determination of CAT activities (38). The enzymatic reaction was stopped with 32.4 mM ammonium molybdate and the yellow complex of molybdate and H2O2 was measured at 405 nm, and the activities were calculated using the absorbances of the samples as well as the three different blank solutions.
Activities of GSH-R were calculated, without addition of FAD, by measuring the decrease in absorbance at 340 nm, due to oxidation of NADPH to NADP. GSH-R was determined in an assay in 0.1 M potassium phosphate buffer, pH 7.4, containing 80 mM EDTA, 7.5 mM GSSG, 2 mM NADPH (35).
Superoxide radicals generated by the employment of xanthine and xanthine oxidase react with 2-[4-iodophenyl]-3-[4-nitrophenol]-5-phenyltetrazolium chloride (INT) to form red formazan dye (39). The samples and the standards were treated with mixed substrate (0.05mM xanthine, 0.025 mM INT, and 80 U/L xanthine oxidase). The SOD activity was then measured by the degree of inhibition of this reaction.
GSH-Px catalyses the oxidation of reduced glutathione (4 mM) by cumene hydroperoxide (0.18 mM) (40). In the presence of GSH-R and NADPH (0.28 mM) the oxidized glutathione is immediately converted to the reduced form with a concomitant oxidation of NADPH to NADP+. The decreases in absorbance at 340 nm were measured and GSH-Px activities were calculated.
Statistical analysis
Data collected in the study were analyzed using SPSS (Statistical Package for Social Sciences, version 12.0) for Windows. Results were expressed as mean±SE. Student t and Mann-Whitney U tests were used for evaluation of parametric and nonparametric data, respectively. A p value of 0.05 was considered significant.
Several parameters (oxidative and anti-oxidative) were studied together to eliminate any deviation in any one of the parameters.  Since the studies may give variable results, we used TBARS which seems to have consistent and reproducible results for the calculation of power. The calculated power of the study was 80%.

Results

Demographic and biochemical characteristics of patients with diabetes mellitus and control group are summarized in Table 1. Patients in diabetes mellitus group included 13 female and 15 male patients with a mean age of 50.78±6.20 years. Control group included 15 females and 16 males with a mean age of 50.35±5.56 years. The mean disease duration of patients with diabetes mellitus was 6.7±3.9 years. Body mass index  (BMI) of patients with diabetes and control group was 28.21±3.37 kg/m2 and 28.38±3.42 kg/m2, respectively. Mean overnight fasting blood glucose of patients with diabetes was 178±62 mg/dl, while it was 88±7 mg/dl in control group. Glycosylated hemoglobin levels of patients with diabetes and control groups were 8.11±2.05% and 5.06±0.19%, respectively.
Mean SOD levels of patients with diabetes mellitus and control group were found to be 2.14±1.31 U/mg.pr and 2.48±1.19 U/mg.pr, respectively (p=0.289). Mean GSH-Px levels of patients with diabetes mellitus and control group were found to be 0.012±0.08 U/mg.pr and 0.057±0.046 U/mg.pr, respectively (p<0.01), (Figure 1, 2, 3, 4, 5) and mean GSH-R level of patients with diabetes mellitus was 1.92±1.84 U/mg.pr, while it was 2.33±1.41 U/mg.pr for control individuals (p=0.335). Catalase enzyme measurements of patients with diabetes mellitus revealed a mean value of 0.20±0.13 U/mg.pr, while it was 0.26±0.12 U/mg.pr for control group (p=0.096). Mean TBARS levels of patients with diabetes mellitus was 5.49±1.31 mmol/l and it was 5.53±1.45 mmol/l for control group (p=0.905).

Discussion

Intracellular killing process of leukocytes is conducted by oxidative or non-oxidative mechanisms. While some of the microorganism are killed with non-oxidative mechanisms, some other microorganism such as Staphylococcus aureus, Escherichia coli, Serratia marcescens, Candida albicans, Aspergillus, Plasmodium, Leishmania are killed by oxidative  mechanisms which are also called “respiratory burst” (41-43). Respiratory burst is mainly characterized by a rapid increase in oxygen uptake and abrupt ROS production. ROS include reactive species such as superoxide anion, hydrogen peroxide, hydroxyl radicals and hypochlorous acid. Toxic oxygen products released after respiratory burst kill microorganisms shortly after phagocytosis (41). As an important point, in vivo biological activities of these free radicals are controlled by enzymes SOD, GSH-Px, GSH-R and CAT. Changes in the levels of these enzymes directly affect the levels of free radicals in leukocytes with the oxygen-dependent antimicrobial capacity of the cell. There is still little evidence concerning the scavenging potential of the circulating leukocytes of diabetic patients, especially regarding the well-documented impairment of immune system in diabetes mellitus and its proposed role in the development of diabetic complications. In order to be able to demonstrate the possible differences in terms of oxidative antimicrobial capacity of leukocytes between patients with diabetes mellitus and healthy individuals, the activities of those enzymes were measured in the present study. Except GSH-Px levels, we did not find a significant difference between patients with diabetes mellitus and healthy controls in terms of the levels of these enzymes that could directly affect the levels of free radicals in leukocytes with the oxygen-dependent antimicrobial capacity of the cell. 
Some previous studies found SOD levels to be decreased among patients with diabetes mellitus (44,45). It was suggested that at high concentrations, glucose reacted with superoxide and as a result, there was a decline in the total amount of superoxide,,a natural substrate for SOD. As an adaptation mechanism, when the substrate levels were decreased, levels of SOD also decreased in the cell. However, it was not certainly reported in which conditions and at what concentrations glucose acted as an oxidant or an antioxidant. On the other hand, the activity of SOD was also suggested to be controlled by non-enzymatic mechanisms and therefore no change in SOD levels among patients with diabetes mellitus was also reported (46). In agreement with this hypothesis, in the present study we did not find any difference in terms of SOD levels between patients with diabetes mellitus and healthy individuals (2.14±1.31 U/mg.pr vs. 2.48±1.19 U/mg. pr, p=0.289).
For its activity, GSH-Px needs glutathione as a coenzyme and since its levels are known to decrease in patients with diabetes mellitus, levels of the enzyme may also be expected to decrease (44). GSH-Px is already shown to be susceptible to non-enzymatic glycation, and it may be inactivated under conditions of severe oxidative stress (47,48). Inactivation of GSH-Px by peroxynitrite and inactivation of GSH-Px in erythrocytes obtained from patients with type 2 diabetes mellitus when incubated with H2O2 were also shown previously (49,50). In our study, GSH-Px levels decreased accordingly, which could be considered as a sign of increased oxidative stress, although some other studies reported opposite findings (46). GSH-R is responsible for the reduction of oxidized glutathione and needs NADPH as a cofactor. In patients with diabetes mellitus, due to increased NADPH poliol pathway and decreased regeneration of NADPH by pentose phosphate pathway the enzyme might be less effective. However, in our study we could not find any difference in terms of GSH-R levels between patients with diabetes mellitus and healthy individuals (1.92±1.84 U/mg.pr  vs 2.33±1.41 U/mg.pr, p=0.335). In diabetes mellitus patients, CAT activities were previously shown to be unchanged (44). Similarly, in our study, catalase activity of patients with diabetes mellitus was found to be unaffected either. Additionally, we did not find any difference between patients with diabetes mellitus and healthy individuals in terms of TBARS levels, which are considered to be the important end products of the reaction between peroxi radicals and thiobarbituric acid (44).
In conclusion, our data suggest that enzyme levels which control the oxidative capacity of leukocytes, except GSH-Px, are not changed in patients with diabetes mellitus and it is difficult to suggest that the tendency to some known infections among patients with diabetes mellitus is mainly due to change in oxidative antimicrobial mechanisms.
Acknowledgements
This work was supported by the Research Fund of The University of Istanbul (Project number: T- 883/17072000).

Address for Correspondence: Mustafa Kanat, MD, Abant Izzet Baysal University, Izzet Baysal Medical School, Department of Internal Medicine, Bolu, Turkey Phone: +90 374 253 46 56 E-mail: mustafa.kanat@gmail.com Recevied: 07.07.2009 Accepted: 04.08.2009

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