The Activity of Serum 8-Iso-Prostaglandin F 2 α as Oxidative Stress Marker in Patients with Diabetes Mellitus Type 2 and Associated Dyslipidemic Hyperglycemia

Background: Oxidative stress has been closely linked to the incidence of diabetic complications. Therefore, the aim of this research article was to study hyperglycemia and abnormal lipid profile in diabetic patient type 2 and its correlation with oxidative stress development as measured by 8-iso-PGF2α and 8-OHdG. Methods: Fifty (50) patients confirmed type 2 diabetes mellitus and eighty (80) non-diabetic control individuals were included in this study. All individuals were tested for blood glucose, lipid profile, 8-iso-PGF2α and 8-OHdG HdG. Results: The age of diabetic patients was observed to be ≥40 yrs in 96% and diabetes was frequently detected in female than in male patients (76% vs. 24%, p < 0.0001). Mean serum lipids were elevated in diabetic patients compared with control individuals (p < 0.0001) except in HDL-C, a significant decrease was recorded (p = 0.04). Serum 8-iso-PGF2α and 8-OHdG were elevated significantly in diabetic patients compared with non-diabetic control and a significant correlation was recorded between them (r = 0.6, p < 0.0001). 8-iso-PGF2α was associated with Age (r = 0.394, p < 0.0001), FBG (0.553, p < 0.0001), LDL-C (r = 0.2, p = 0.023), TG (r = 0.176, p = 0.045) and TC (r = 0.2, p = 0.02). Also, 8-OHdG was associated with age (r = 0.558, p < 0.0001), FBG (r = 0.67, p < 0.0001), LDL-C (r = 0.28, p = 0.001), TG (r = 0.358, p < 0.0001) and TC (r = 0.33, p < 0.0001). Age, FBG, HbA1c, LDL-C, TG and TC showed a significant linear regression with 8-isoPGF2α and 8-OHdG recording its role as significant predictors for the elevation of 8-iso-PGF2α and 8-OHdG. Therefore, hyperglycemia with oxidative stress development may play a role for dyslipidemia and diabetic complications. Conclusion: DiHow to cite this paper: Mukhtar, M.H., El-Emshaty, H.M., Alamodi, H.S. and Nasif, W.A. (2016) The Activity of Serum 8-Iso-Prostaglandin F2α as Oxidative Stress Marker in Patients with Diabetes Mellitus Type 2 and Associated Dyslipidemic Hyperglycemia. Journal of Diabetes Mellitus, 6, 318-332. http://dx.doi.org/10.4236/jdm.2016.64033 Received: November 7, 2016 Accepted: November 26, 2016 Published: November 29, 2016 Copyright © 2016 by authors and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/ Open Access M. H. Mukhtar et al. 319 abetic patient’s type 2 has a higher rate of abnormal serum lipids and correlates significantly with lipid peroxidation and oxidized DNA bases as measured by 8-isoPGF2α and 8-OHdG. Therefore, 8-iso-PGF2α and 8-OHdG could be used as oxidative biomarkers for evaluating diabetic patients with early prediction of its complications and cancer development.

abetic patient's type 2 has a higher rate of abnormal serum lipids and correlates significantly with lipid peroxidation and oxidized DNA bases as measured by 8-iso-PGF2α and 8-OHdG.Therefore, 8-iso-PGF2α and 8-OHdG could be used as oxidative biomarkers for evaluating diabetic patients with early prediction of its complications and cancer development.

Introduction
Diabetes mellitus is a group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion and insulin action or both [1].In type 2 diabetes, the cause is a combination of resistance to insulin action and an inadequate compensatory insulin secretory response.
Insulin resistance and type 2 diabetes are associated with a clustering of interrelated plasma lipid and lipoprotein abnormalities, which include reduced HDL cholesterol, a predominance of small dense LDL particles, and elevated triglyceride levels.Each of these dyslipidemic features is associated with an increased risk of cardiovascular disease [2].Insulin resistance has striking effects on lipoprotein size and subclass particle concentrations for VLDL, LDL and HDL [3] [4].
Oxidative stress due to overproduction of reactive oxygen species (ROS) has important role in prevention, initiation, and progression of chronic diseases from early childhood [5].The considerable reactive properties of ROS cause oxidative damage to lipids, deoxyribonucleic acid (DNA), and proteins.Recent evidence suggests that ROS could have a role in the development of hypertension, dyslipidemia, diabetes mellitus, and atherosclerosis [6].Oxidative stress status in diabetes could be clearly demonstrated by the increase of some specific biomarkers such as lipid hydroperoxides, DNA adducts and protein carbonyls [7].
Reactive oxygen species (ROS) can interact with DNA to produce damage including single-and double-stranded DNA breaks, deletions and nucleoside modifications.Among purine and pyrimidine base, guanine is more susceptible to oxidation.The hydroxyl radical can attack to C-8 position guanine and generate an oxidation product, 8-hydroxy deoxyguanine (8-OHdG) [8].8-OHdG, the oxidized form of the nucleoside 2'-deoxyguanosine present in DNA, is one of the most reliable and abundant markers of DNA damage because it reflects generalized cellular oxidative stress; it may be a risk factor for cancer, atherosclerosis and DM [6] [8].
Lipid peroxidation is most often measured using malondialdehyde (MDA) and 8-isoprostaglandin F2α (8-iso-PGF2α) [9] [10] [11].Isoprostanes are stable products of arachidonic acid peroxidation due to free radical activity and reliable biomarkers for oxidative stress, which are suitable for measuring lipid peroxidation in place of malondialdehyde (MDA) [12].Isoprostanes, including 8-iso-PGF2α are stable in biological fluids and easily detectable as well as not being affected by diet and modulated by endogenous antioxidants [13].
The measurement of bioactive F2-IsoPs levels offers a unique noninvasive analytical tool to study the role of free radicals in physiology, oxidative stress-related diseases, and acute or chronic inflammatory conditions [14].Elevated F2-IsoP levels have been associated with multiple chronic conditions which commonly have been interpreted as an etiological link between oxidative stress and the disease; specifically, type 2 diabetes and its risk factors, such as obesity, impaired glucose tolerance, and insulin resistance [15].
Therefore, the objective of this research article was to study hyperglycemia and abnormal lipid profile in diabetic patients and its correlation with oxidative stress development as detected by 8-iso-PGF2α and 8-OHdG.All participants underwent a clinical examination and a questionnaire including medical and family history.The exclusion criteria were for those with past history of type-1 diabetes, cancer, cardiovascular or renal diseases.Blood samples were obtained following at least 8 hours fasting period according to the American Diabetes Association criteria; only patients with fasting blood glucose above 126 mg/dl or postprandial two hours after meal above 200 mg/dl were included [16].Blood samples were collected for blood glucose analysis and serum was immediately separated by centrifugation at 3000 rpm for 10 min and stored at 4˚C until processed for lipid analysis and oxidative biomarkers (8-OHdG and 8-iso-PGF2α).
Serum LDL-C was calculated according to the computational formula of Friedewald et al. [17] [LDL = TC-HDL-TC/5.0mg/dl].Castelli index [18] was used for determining the ratio between total cholesterol and HDL-C.

Serum 8-Iso-PGF2α
8-iso-PGF2α was measured by direct 8-iso-PGF2α ELISA kit (Enzo Iife Sciences Inc., Switzerland).The direct 8-iso-PGF2α ELISA kit is a competitive immunoassay for the quantitative determination of 8-iso-PGF2α in biological fluids [21].The kit uses a polyclonal antibody to 8-iso-PGF2α to bind in a competitive manner with 8-iso-PGF2α in the sample or 8-iso-PGF2α covalently attached to alkaline phosphatase.After incubation, the excess reagents were washed away and substrate was added.The generated yellow color was read at 405 nm and the intensity of the bound yellow color is inversely proportional to the concentration of 8-iso-PGF2α in either standard or sample.The measured optical density is used to calculate the concentration of 8-iso-PGF2α from the calibration curve.

Serum 8-OHdG
Serum 8-OHdG was measured using an ELISA kit (Cloud-clone Corp., Assembled by USCo life science inc.USA).This assay employs the competitive inhibition enzyme immunoassay technique.Pre-coated microplate with 8-OHdG monoclonal antibody used for competitive inhibition reaction between biotin-labeled 8-OHdG and unlabeled 8-OHdG.After incubation, the unbound conjugate is washed off with subsequent addition of avidin conjugated horse radish peroxidase (HRP) followed by substrate solution.
The intensity of the developed color is reversely proportional to the concentration of 8-OHdG in the sample.The detection range was from 74.07 to 6,000 pg/ml.

Statistical Analysis
Data was analyzed using SPSS (version 17, Sydney, NSW, Australia).Statistical analysis was performed using independent sample test for parametric variables and Chi-square for categorical variables.Relationship between variables was detected by Pearson's correlation coefficient and linear regression analysis was performed to analyze 8-OHdG and 8-iso-PGF2α as dependent variables.Quantitative data was expressed as mean ± SD and qualitative data as frequencies and percentages.
Multiple linear regressions of 8-iso-PGF2α and 8-OHdG with various independent variables (Table 5) showed that Age, FBG, HbA1c, LDL-C, TC and TG were recorded as significant predictors for the elevation of 8-OHdG and 8-iso-PGF2α.

Discussion
Diabetes is a major source of morbidity, mortality and economic cost to society [22].
Despite well-controlled blood glucose, diabetic complications still inevitably take place via several mechanisms including excessive generation of free radicals in patients who suffer from diabetes mellitus (DM) [7].Therefore, this research article was conducted to study hyperglycemia and abnormal lipid profile in diabetic patients and its correlation with oxidative stress development as measured by 8-iso-PGF2α and 8-OHdG.
The of developing type 2 diabetes increases with age, obesity, and lack of physical activity [23].The age of diabetic patients in our study was observed to be ≥40 yrs in 96% proving that age plays a significant role in the risk of developing type 2 DM especially after 40 yrs [24].However, it has been observed that insulin secretion declines with advancing age, and this decline may be accelerated by genetic factors [25].
Diabetes is a disease characterized by poor glycemic control [26].Hyperglycemia generates reactive oxygen species (ROS), which in turn cause damage to the cells in many ways [27] [28].OS can simultaneously result damage in biomolecules including lipids, proteins, nucleic acids and carbohydrates [29].Lipids are reported as one of the primary targets of ROS [30].Isoprostanes are a recently discovered group of prostaglandin isomers.Results of previous studies suggest that they can be used as oxidative stress markers, because in a number of cardiovascular, pulmonary and neurological diseases, their levels in biological samples considerably increase [31].
Jelinek et al. [32] demonstrated a significant increase in 8-iso-PGF2α in the IFG group and supports the findings that, 8-iso-PGF2α was increased following an oral glucose tolerance in individuals with no diabetes but with either IFG or impaired glucose tolerance [33].8-iso-PGF2α was elevated in the sera of diabetic patients (2719.38 ± 1864.68 pg/ml) in current study compared with non-diabetic control (951.45 ± 669.44 pg/ml, p < 0.0001).Similar results were recorded by Gopaul et al. [34], that, the plasma levels of 8-iso-PGF2α in non-insulin dependent diabetes mellitus (NI-DDM, diabetes type 2) were higher (N = 39, 0.49 -2.16 nM) than in the control group (N = 16, 0.02 -0.63 nM) and by Davi et al. [35] in urine samples, that 8-iso-PGF2α in patients with (419 ± 208 pg/mg creatinine) were significantly higher (p = 0.0001) than in age-matched healthy subjects (208 ± 92 pg/mg creatinine).
A clinical study performed by Bandeira and coworkers [36] aimed at characterizing blood oxidative stress in diabetic patients, reported a significant higher lipid peroxidation which showed a close relationship with high glucose levels as observed by the fasting glucose and HbA1c levels.This observation are in consistent with our findings in diabetic patients, that higher lipid peroxidation as measured by 8-iso-PGF2α showed a close relationship with FBG (r = 0.553, p < 0.0001) and HbA1c (r = 0.6, p < 0.0001).
Therefore, increased lipid peroxidation presents a close relationship with the high glycemic levels and oxidative stress in diabetes mellitus [36] [37].
Changes in serum lipids have been demonstrated in the IFG stage and are associated with oxidation of arachidonic acid to 8-iso-PGF2α [38] [39].Changes in serum lipids was demonstrated in diabetic patients of our study and a significant association was recorded with 8-iso-PGF2α.Serum lipids showed significant elevation in diabetic patients (p < 0.0001) but in HDL-C, a significant decrease (p = 0.04) was recorded confirming other reports [32] demonstrated a significant reduction in both total cholesterol and HDL-C which explains the increased 8-iso-PGF2α as HDL-C caries 8-iso-PGF2α [10].
Like the oxidation of lipids and proteins, the oxidation of DNA reveals information on the overall state of the system being investigated.DNA oxidation is of particular concern for mitotic tissues, where increases in DNA mutations are believed to increase risk for cancer development [40].Oxidized DNA as measured by 8-OHdG in our study was elevated significantly (p < 0.0001) in diabetic patients compared with non-diabetic control referring to the risk in DNA oxidative damage and cancer development.Therefore, data collected by measurement of oxidized DNA bases in serum or excreted in urine, most notably 8-OHdG have served as a biomarker for carcinogenesis [41].
8-OHdG has been suggested to serve as a new sensitive biomarker of the in vivo oxidative DNA damage in diabetes [42].In current study, 8-OHdG in diabetic patients was associated with hyperglycemia and abnormal serum lipids.LDL showed significant elevation in diabetic patients and the correlation with 8-OHdG was highly significant (r = 0.28, p = 0.001) on contrary with other reports [43].Lower HDL-C was detected in diabetic patients more frequently than in non-diabetic control (p = 0.04) but negative correlation with 8-OHdG was detected insignificant consisting with the finding of Taskinen [44] and on contrary with the conclusion of Abdel-Aal et al. [45] that lower HDL-C showed significant negative correlation with 8-OHdG.Reduced HDL-C in diabetic subjects result from higher catabolic rate of HDL-C with normal activity of cholesterol ester transfer protein and hepatic lipase in insulin resistance conditions [45].
The good correlation between 8-OHdG and isoprostanes in diabetic patients was well documented by Harman et al. [46] and in our report (r = 0.6, p < 0.0001).However, some researchers [47] reported the elevated levels of 8-OHdG and 8-iso-PGF2α in the diabetics, although these studies have not shown the association between them.The association of 8-iso-PGF2α and 8-OHdG with hyperglycemia (FBG and HBA1c), elevated LDL-C and triglycerides in diabetic patients of our study was confirmed by other studies [43] [48] reflecting a generalized cellular oxidative stress which may be a risk factor for diabetic complication and cancer development as suggested by Brownlee [49] that hyperglycemia-induced overproduction of superoxide, plays a critical role in the biochemical abnormalities leading to vascular disease and diabetic complications.
Therefore, serum 8-OHdG and 8-iso-PGF2α could be used for early prediction of diabetic complications and cancer development as urinary 8-OHdG and 8-iso-PGF2α excretions have been measured in many studies because they are associated with cancer development and advancement of diabetes and atherosclerosis [50].

Conclusion
Diabetic patient's type 2 has a higher rate of abnormal lipid profile and correlates significantly with oxidative stress as measured by 8-iso-PGF2α and 8-OHdG.Therefore, 8-iso-PGF2α and 8-OHdG could be used as oxidative biomarkers for evaluating diabetic patients with early prediction for its complications and cancer development.

Limitations
 Although this is an attempt to study oxidative biomarkers that might be of significance in progression of diabetic patients, only a limited number of cases were investigated here. Since the prognosis of diabetes type 2 remains poor and identification of useful molecular prognostic markers for diabetic complications is required, follow up of diabetic patients must be studied in further study. Other risk factors like obesity might be of significance in the progression of diabetic complications and must be studied in further study.

Figure 1 .
Figure 1.Correlation between Fasting blood glucose (FBG) and HbA1c with Low density lipoprotein-Cholesterol (LDL-C) and Triglyceride (TG) in all participants.

Table 1 .
General characteristics and biochemical markers of all participants in the study group.

Table 2 .
Abnormal serum lipids among all participants in the study group.

Table 3 .
Incidence of 8-iso-PGF2α and 8-OHdG in all participants of the study group.