Abstract
Cardiovascular autonomic neuropathy (CAN) is one of the most clinically significant complications of diabetes mellitus. Even though many ethological factors have been attributed for the pathogenesis of this disease no attempts were made to correlate DNA damage as a causative factor. Hence the present study was undertaken to asses the extent of somatic DNA damages by cytokinesis-block micronuclei assay (CBMN). An attempt is also being made to correlate the habits and/or risk factors and socioeconomic status with CAN. The CBMN frequency of 46 patients suffering from autonomic neuropathy was compared with that of 25 healthy age and sex matched controls. All the subjects were suffering from type 2 diabetes for at least 8 years and have varying degrees of coronary artery diseases. The mean CBMN frequency of the patients was statistically higher than that of the healthy control subjects (P < 0.05). The CBMN frequency was found to be significantly altered in CAN patients who where physical inactivity and smoking. A significant correlation could also be observed between CAN and smoking, diabetes mellitus, hypertension, dyslipidemia, abdominal obesity, and physical activity.
Keywords: Autonomic neuropathy, Cardiovascular autonomic neuropathy (CAN), DNA damage, Cytokinesis-block micronuclei (CBMN) assay, Coronary artery diseases (CAD)
Introduction
Cardiovascular autonomic neuropathy (CAN) is one of the most overlooked of all serious complications of diabetes. This encompasses damage to the autonomic nerve fibers that innervate the heart and blood vessels, resulting in abnormalities in heart rate control and vascular dynamics [1]. Cardiovascular autonomic neuropathy (CAN) may carry an increased risk of morbidity and mortality [2]. CAN impair the ability to conduct activities of daily living, lowers quality of life, and increases the risk of death. It also accounts for a large portion of the cost of care [3]. Cardiovascular autonomic neuropathy occurs in about 17% of patients with type 1 diabetes and 22% of those with type 2. An additional 9% of type 1 patients and 12% of type 2 patients have borderline dysfunction [4].
The pathophysiology of the autonomic neuropathy depends on the etiology of each particular type like hyperglycemia, increased oxidative stress, autoimmune factors etc. These may range from genetic disorders with specific gene defects to metabolic disorders with accumulation of toxins and to autoimmune disorders with identifiable autoantibodies.
DNA damage, as evidenced by DNA adducts and oxidative DNA damage has been observed in vascular tissues. Higher levels of DNA adducts in vascular tissues than in other tissues have been reported [5]. An increased level of micronuclei has been shown to be marker of chromosome damage. The cytokinesis-block micronuclei assay is a comprehensive system for measuring DNA damage, cytostasis and cytotoxicity. DNA damage events are scored specifically in once-divided binucleated (BN) cells and include (a) micronuclei (MNi)—a biomarker of chromosome breakage and/or whole chromosome loss, (b) nucleoplasmic bridges (NPBs)—a biomarker of DNA misrepair and/or telomere end-fusions, and (c) nuclear buds (NBUDs)—a biomarker of elimination of amplified DNA and/or DNA repair complexes [6].
No serious attempts were made earlier to correlate somatic DNA damage with CAN. Hence present study was undertaken to quantify the extent of somatic DNA damages by cytokinesis block micronuclei (CBMN) assay in subjects suffering with Diabetic autonomic neuropathy. An attempt is also being made to asses the extend of DNA damage in patients with risk factors associated with cardio vascular diseases.
Materials and Methods
Forty-six subjects suffering from autonomic neuropathy formed the study groups. All the subjects were suffering from type 2 diabetes for at least 8 years and have varying degrees of coronary artery diseases. Twenty-five healthy age and sex matched control subjects were selected. Detailed anthropometric, socio-economic, demographic and other relevant clinical information were recorded using proforma. These subjects were referred from Hridaylaya Institute of Preventive Cardiology, Tiruvananthapuram and General Hospital, Tiruvananthapuram to Genetika, centre for Advanced Genetic Studies.
Three ml blood was collected aseptically in heparinized vacuutainers and used for lymphocyte separation and CBMN assay. Two ml of lymphoprep (pharmacia) added to a 10 ml centrifuged tube and overlaid 3 ml of blood sample to the tube and centrifuged at 1,000 rpm for 10 min. Drawn off the lymphocyte layer and transferred to a 10 ml tube. Suspended the cell pellet in RPMI 1640 medium and centrifuged for 10 min. Removed the supernatant and repeated the above step. Peripheral lymphocyte culture was performed as described by Moorhead et al. [7]. The CBMN test was done using the cytochalasin B technique described by Fenech [6]. The lymphocytes were cultured in sterile bottles using RPMI 1640 medium containing 15% fetal calf serum. Lymphocyte cultures were prepared for each subject. Each culture contained 2.0 × 106 cells in 5 ml RPMI 1640 supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin, 10% fetal bovine serum and 1% phytohemagglutinin. At 44 h after initiation, cells were blocked in cytokinesis by adding cytochalasin B (Sigma, St. Louis, MO; final concentration, 4 μg/ml). The total incubation time for all cultures was 72 h. After incubation, the cells were fixed in 3:1 methanol/glacial acetic acid, dropped onto clean microscopic slides, air-dried, and stained with Giemsa stain. For each sample, 1,000 binucleated cells were scored at 100× magnification. The numbers of micronuclei per 1,000 binucleated cells were recorded.
Statistical Analysis
‘t’ Test was performed using SPSS for comparing the CBMN frequencies of the study subjects and the control subjects. Analysis of variance (ANOVA) was done to compare the CBMN values with and without various risk/life-style factors. Association between various risk/lifestyle factors and socio-economic variables and CAN is analyzed using chi-square test. The contribution of various risk/life-style factors for cardiovascular autonomic neuropathy was studied by Logistic regression analysis.
Results
Anthropometric and Socio-economic data of the patients and control subjects is given in Table 1. Somatic DNA damage in CAN patients at different age groups compared to that of normal control subjects are given in Table 2. The age of the study subjects ranged from 32 to 72 years with mean age of 55.2 years whereas the age of the control subjects ranged from 25 to 60 years with a mean age of 50.5 years. The mean CBMN frequency of the patients was statistically higher than that of the healthy control subjects (P < 0.05). 73.9% of study subjects were males. The mean CBMN frequency for female patients was 13.5 whereas in males it was 15.82. The difference was statistically significant (P < 0.05).
Table 1.
Anthropometric and socio-economic data of the patients and control subjects
| Body mass index | Abdominal circumference (Cm) | ||||||||
|---|---|---|---|---|---|---|---|---|---|
| Age group (years) | Sex | Patients | Control | Patients | Control | ||||
| LIG/MIG | HIG | LIG/MIG | HIG | LIG/MIG | HIG | LIG/MIG | HIG | ||
| 31–40 | Male | 27.48 ± 2.98 (n = 2) | Nil | 24.16 ± 3.13 (n = 5) | Nil | 98 ± 5.29 (n = 2) | Nil | 88.2 ± 5.45 (n = 5) | Nil |
| Female | Nil | Nil | 25.32 ± 3.05 (n = 3) | Nil | Nil | Nil | 80.67 ± 6.55 (n = 3) | Nil | |
| 41–50 | Male | 25.24 ± 4.33 (n = 8) | 29.64 ± 3.55 (n = 3) | 23.42 ± 2.96 (n = 6) | 24.07 ± 2.31 (n = 2) | 95.62 ± 8.07 (n = 8) | 100 ± 5.99 (n = 3) | 85.5 ± 5.88 (n = 6) | 90 ± 2.83 (n = 2) |
| Female | 22.55 ± 4.39 (n = 2) | Nil | 22.04 ± 4.97 (n = 2) | Nil | 83 ± 8.88 (n = 2) | Nil | 81.5 ± 6.68 (n = 2) | Nil | |
| 51–60 | Male | 27.38 ± 4.25 (n = 8) | 23.48 ± 4.29 (n = 2) | 25.53 ± 3.04 (n = 3) | 26.67 ± 3.07 (n = 2) | 96.5 ± 6.79 (n = 8) | 97 ± 8.81 (n = 2) | 89.33 ± 6.03 (n = 3) | 90 ± 6.03 (n = 2) |
| Female | 25.35 ± 4.42 (n = 6) | 32.51 ± 0 (n = 1) | Nil | Nil | 96.17 ± 7.99 (n = 6) | 102 ± 0 (n = 1) | Nil | Nil | |
| >60 | Male | 25.32 ± 4.21 (n = 6) | 26.48 ± 4.78 (n = 5) | 31.39 ± 3.58 (n = 2) | Nil | 91.17 ± 7.99 (n = 6) | 94.85 ± 10.5 (n = 5) | 89 ± 5.98 (n = 2) | Nil |
| Female | 30.24 ± 4.38 (n = 3) | Nil | Nil | Nil | 102 ± 9.23 (n = 3) | Nil | Nil | Nil | |
All values are mean ± SD. n Number of subjects; LIG/MIG low income/middle income group; HIG high income group
Since there is no clear demarcation of urban/rural area in Kerala, there is no relevance in classifying the subjects on this basis
Table 2.
Distribution of CBMN frequency with age
| Subjects | Age range | All ages | t | P | |||
|---|---|---|---|---|---|---|---|
| 31–40 | 41–50 | 51–60 | >60 | ||||
| CBMN frequency of study subjects | 13.5 ± 2.08 (n = 2) | 14.82 ± 2.34 (n = 13) | 15.43 ± 2.32 (n = 17) | 15.77 ± 2.32 (n = 14) | 15.21 ± 2.3 (n = 46) | 9.328 | 0.001 |
| CBMN frequency of controls | 9.86 ± 1.2 (n = 6) | 10.8 ± 1.23 (n = 10) | 11.0 ± 1.26 (n = 7) | 11.0 ± 1.16 (n = 2) | 10.6 ± 1.22 (n = 25) | ||
Values are mean ± SD. Compared to the control group the CBMN frequency was higher in the patients at all age groups (P < 0.05)
The CBMN frequency of patients and control subjects with various risk factors and life style pattern are given in Table 3. Of the major risk factors like diabetes, hypertension, dyslipidemia, abdominal obesity, smoking and alcoholism only smoking showed significant contribution for the increase in CBMN frequency in patients. Other life style factors such as physical activity, diet, socioeconomic status and area of residence were found to influence the CBMN frequency among patients. But significant alteration could be observed only in patients who are physically inactive. This study also revealed increased level of somatic damages in subjects having more than one risk factor.
Table 3.
Distribution of CBMN frequencies with risk/life-style factors in patients and control subjects
| Risk/life style factors | Control (n = 25) | Study subjects (n = 46) | ||||
|---|---|---|---|---|---|---|
| Number | CBMN frequency | Number | CBMN frequency | t | P | |
| Alcoholism | ||||||
| Yes | 3 | 12.66 ± 1.3 | 6 | 16.83 ± 2.27 | 1.899 | 0.064 |
| No | 22 | 10.31 ± 1.22 | 40 | 14.97 ± 2.29 | ||
| Smoking | ||||||
| Yes | 3 | 12.66 ± 1.31 | 27 | 15.74 ± 2.30 | 1.867 | 0.049 |
| No | 22 | 10.31 ± 1.22 | 19 | 14.47 ± 2.31 | ||
| Diabetes | ||||||
| Yes | 0 | 0 | 46 | 15.21 ± 2.9 | – | – |
| No | 25 | 10.6 ± 1.22 | 0 | 0 | ||
| Hypertension | ||||||
| Yes | 2 | 11.0 ± 1.16 | 36 | 16.1 ± 2.29 | −1.386 | 0.173 |
| No | 23 | 10.56 ± 1.22 | 10 | 14.97 ± 2.29 | ||
| Dyslipidemia | ||||||
| Yes | 0 | 0 | 39 | 15.48 ± 2.29 | 1.935 | 0.048 |
| No | 25 | 10.6 ± 1.2 | 7 | 13.71 ± 2.39 | ||
| Abdominal obesity | ||||||
| High | 2 | 12.0 ± 1.2 | 22 | 15.54 ± 2.29 | −0.999 | 0.323 |
| Normal | 23 | 10.47 ± 1.22 | 24 | 14.86 ± 2.23 | ||
| Physical activity | ||||||
| Sedentary | 16 | 10.5 ± 1.22 | 31 | 16.2 ± 2.30 | −2.091 | 0.042 |
| Non-sedentary | 9 | 10.46 ± 1.21 | 15 | 14.47 ± 2.29 | ||
| Diet | ||||||
| Vegetarian | 0 | 0 | 1 | 13.0 ± 0 | 0.343 | 0.335 |
| Non-vegetarian | 25 | 10.6 ± 1.22 | 45 | 15.26 ± 2.3 | ||
| Socio-economic status | ||||||
| High | 4 | 9.5 ± 1.26 | 11 | 15.72 ± 2.34 | 2.399 | 0.184 |
| Middle/low | 21 | 10.8 ± 1.2 | 35 | 15.05 ± 2.32 | ||
| Area of residence | ||||||
| Urban | 17 | 10.5 ± 1.2 | 28 | 15.46 ± 2.3 | −1.121 | 0.086 |
| Rural | 8 | 10.46 ± 1.22 | 18 | 14.83 ± 2.33 | ||
| Family h/o CAD | ||||||
| Yes | 0 | 0 | 4 | 16.00 ± 2.646 | 1.143 | 0.259 |
| No | 25 | 10.6 ± 1.22 | 42 | 15.03 ± 2.205 | ||
Values are mean ± SD. Comparison of the values is made only in patients with and without risk factors
Contribution of life style/risk factors for cardiovascular autonomic neuropathy is given in Table 4. Subjects above the age of 50 have an increased chance of developing diabetic autonomic neuropathy than subjects below the age of 50 (Odd’s ratio = 3.674; Confidence interval = 1.321–10.222). Smoking is another risk factor for developing CAN (Odd’s ratio = 10.035 (Confidence interval = 2.618–38.459). Subjects with family history of CAD have 100% occurence of CAN whereas subjects without family history of CAD have 59.7%. This observed difference is statistically significant (P < 0.05). Subjects with increased abdominal obesity have 10.542 times higher risk than subjects without abdominal obesity for developing CAN. Subjects with hypertension have 41.4 times more risk for developing CAN. Logistic regression analysis was performed for the following risk factors vis hypertension, smoking, abdominal obesity and age > 50 showed significant correlation with cardiovascular autonomic neuropathy and the results were given in the Table 5.
Table 4.
Contribution of life style/risk factors for cardiovascular autonomic neuropathy
| Life style/risk factors | Case | Control | Total | χ2 | P |
|---|---|---|---|---|---|
| N (%) | N (%) | N (%) | |||
| Occupation | |||||
| Sedentary | 31 (55.4%) | 25 (44.6%) | 56 (100.0%) | 10.336 | P < 0.001 |
| Non-sedentary | 15 (100.0%) | 0 (0%) | 15 (100.0%) | ||
| Hypertension | |||||
| Yes | 36 (94.7%) | 2 (5.3%) | 38 (100.0%) | 32.143 |
P = .000 Odd’s ratio = 41.4 |
| No | 10 (30.3%) | 23 (69.7%) | 33 (100.0%) | ||
| Diabetes | |||||
| Yes | 46 (100.0%) | 0 (0%) | 46 (100.0%) | 71.000 | P = .000 |
| No | 0 (0%) | 25 (100.0%) | 25 (100.0%) | ||
| Dyslipidemia | |||||
| Yes | 39 (100.0%) | 0 (0%) | 39 (100.0%) | 47.028 | P = .000 |
| No | 7 (21.9%) | 25 (78.1%) | 32 (100.0%) | ||
| Abdominal obesity | |||||
| Yes | 22 (91.7%) | 2 (8.3%) | 24 (100.0%) | 11.481 |
P = .001 Odd’s ratio = 10.542 |
| No | 24 (51.1%) | 23 (48.9%) | 47 (100.0%) | ||
| Socio-economic status | |||||
| Low | 19 (100.0%) | 0 (0%) | 19 (100.0%) | 18.335 | P = .000 |
| Medium | 16 (43.2%) | 21 (56.8%) | 37 (100.0%) | ||
| High | 11 (73.3%) | 4 (26.7%) | 15 (100.0%) | ||
| Family h/o CAD | |||||
| Yes | 9 (100.0%) | 0 (0%) | 9 (100.0%) | 5.601 | P = .018 |
| No | 37 (59.7%) | 25 (40.3%) | 62 (100.0%) | ||
| Smoking | |||||
| Yes | 26 (89.7%) | 3 (10.3%) | 29 (100.0%) | 13.880 |
P = .000 Odd’s ratio = 10.035 |
| No | 20 (46.3%) | 22 (53.7%) | 41 (100.0%) | ||
| Age | |||||
| Above 50 | 31 (77.5%) | 9 (22.5%) | 40 (100.0%) | 6.489 |
P = .011 Odd’s ratio = 3.674 |
| Up to 50 | 15 (48.4%) | 16 (51.6%) | 31 (100.0%) |
Table 5.
Logistic regression analysis showing correlation between risk factors and cardiovascular autonomic neuropathy
| Risk factors | B | SE | Wald | df | Sig. | Exp (B) |
|---|---|---|---|---|---|---|
| Hypertension | 3.786 | 1.047 | 13.074 | 1 | .000 | 44.077 |
| Smoking | 2.752 | .996 | 7.636 | 1 | .006 | 15.667 |
| Abdominal obesity | 2.747 | 1.116 | 6.062 | 1 | .014 | 15.590 |
| Age > 50 | .198 | .908 | .047 | 1 | .828 | 1.219 |
| Constant | −16.397 | 4.022 | 16.621 | 1 | .000 | .000 |
Variable(s) entered on step 1: hypertension, smoking, abdominal obesity, age > 50
Discussion
Diabetic neuropathies are the common in cause of incapacitation, morbidity, devastating complications and premature death [8]. These neuropathies comprise of clinical changes in peripheral nerves, autonomic nerves and central nervous system with diffuse or focal damage/regeneration. Previous population-based studies have reported prevalence rates for polyneuropathy ranging from 8 to 54% in type 1 diabetic patients and from 13 to 46% in type 2 diabetic patients [9]. In an earlier study by Fedele et al. [10] the male to female ratio was almost 1:1 whereas in the present study 73.9% were males. These differences may be due the high prevalence alcoholism, smoking among males compared to females in this area. The prevalence of CAN in the present study was found to be increasing with progression of age. Vinik et al. [11] reported a direct correlation between CAN with age and duration of diabetes mellitus. We observed an increased chance of developing cardio autonomic neuropathy in subjects above the age of 50.
Tesfaye et al. [12] reported that, the incidence of neuropathy is associated with potentially modifiable cardiovascular risk factors including a raised triglyceride level, body-mass index, smoking, and hypertension. Higher levels of triglyceride, blood pressure, insulin, weight, and lower HDL cholesterol are associated with both diabetes and macrovascular disease, the latter showing evidence of beginning prior to the onset of the former [13]. Forrest et al. [14] observed hypertension was a strong risk factor for neuropathy in young patients with type 1 diabetes. Dyslipidemia leads to high levels of oxidized LDLs that may injure dorsal root ganglia neurons and contribute to the development of diabetic neuropathy [15]. The present study is in well agreement with the previous reports, as we also found a strong correlation between CAN and various risk factors like diabetes, hypertension, dyslipidemia, abdominal obesity and smoking.
Autonomic dysfunction is very common in patients with diabetes. It has been suggested that autonomic dysfunction can be detected in at least 40% of the patients by formal autonomic nervous system testing [16]. The prevalence of neuropathy was 50% in those with diabetes for 25–29 years and 72% in those with diabetes for >30 years [17]. The development of cardiovascular autonomic dysfunction was independently associated with microvascular complications and glycemic control status in patients with type 2 diabetes [18]. In the present study all the patients with CAN were diabetic for a minimum period of 8 years prior to the study. This prolonged illness may be the reason for the onset of CAN in these patients.
DNA damage is a form of cell stress and injury that has been implicated in the pathogenesis of many neurological disorders [19]. DNA damage is caused by multiple endogenous and exogenous factors such as oxidative stress, age, smoking, hypertension, hyperlipidemia and diabetes mellitus [20]. Andreassi [21] reported that diabetes is a major determinant of somatic DNA instability in patients with CAD. DNA damage might represent an additional pathogenetic dimension and a possible therapeutic target in the still challenging management of coronary artery disease concerning diabetics. Diabetes accelerates the accumulation of the somatic mutation in mitochondrial DNA, which could possibly be a new marker for estimating the duration of diabetes [22]. Somatic damages are detected via chromatin loss from the nucleus leading to micronuclei in the cytoplasm of the cell. MN is scored by Cytokinesis Block Micronuclei Assay developed by Fenech [6]. The purpose of present study was to evaluate the extent of somatic damage by using Cytokinesis Block Micronuclei Assay in patients with cardio autonomic diabetic neuropathy. The CBMN frequency in patients was found to be altered with life style and risk factors like smoking, alcoholism, diabetes mellitus, hypertension, dyslipidemia, abdominal obesity, physical activity, diet, socioeconomic status and area of residence.
A strong correlation between CBMN frequencies and cardio autonomic neuropathy was observed in this study. The higher the number of risk factors higher will be the chance of developing CAN. This may be attributed to the increased DNA damage with risk factors mainly smoking which may cause an increase in blood pressure. Subjects with risk factors like family history of CAD, increased abdominal obesity, hypertension and age above 50 years, have profound influence in developing cardiac autonomic neuropathy. Life style modification with diet and exercise, maintaining blood pressure at normal level, lowering serum lipids and blood sugar and avoiding tobacco and alcohol will reduce the risk of CAD and autonomic neuropathy.
Acknowledgments
The financial support given by the Kerala State Council for Science, Technology and Environment is gratefully acknowledged.
Contributor Information
A. Supriya Simon, Email: supriyasimon_a@yahoo.co.in
D. Dinesh Roy, Email: dineshroyd@rediffmail.com
References
- 1.Vinik AI, Ziegler D. Diabetic cardiovascular autonomic neuropathy. Circulation. 2007;115:387–397. doi: 10.1161/CIRCULATIONAHA.106.634949. [DOI] [PubMed] [Google Scholar]
- 2.Astrup AS, Tarnow L, Rossing P, Hansen BV, Hilsted J, Parving HH. Cardiac autonomic neuropathy predicts cardiovascular morbidity and mortality in type 1 diabetic patients with diabetic nephropathy. Diabetes Care. 2006;29(2):334–339. doi: 10.2337/diacare.29.02.06.dc05-1242. [DOI] [PubMed] [Google Scholar]
- 3.Vinik AI, Erbas T. Recognizing and treating diabetic autonomic neuropathy. Clevel Clin J Med. 2001;68:928–944. doi: 10.3949/ccjm.68.11.928. [DOI] [PubMed] [Google Scholar]
- 4.Ziegler D, Gries FA, Muhlen H, Rathmann W, Spuler M, Lessmann F. Prevalence and clinical correlates of cardiovascular autonomic and peripheral diabetic neuropathy in patients attending diabetes center. The DiaCAN Multicenter Study Group. Diabetes Metab. 1993;19:143–151. [PubMed] [Google Scholar]
- 5.Izzotti A, Flora S, Petrilli GL, Gallagher J, Rojas M, Alexandrov K, et al. Cancer biomarkers in human atherosclerosis lesions: detection of DNA adducts. Cancer Epidemiol Biol Prev. 1995;4:105–110. [PubMed] [Google Scholar]
- 6.Fenech M. The cytokinesis-block micronucleus technique: a detailed description of the method and its application to genotoxicity studies in human populations. Mutat Res. 1993;285:35–44. doi: 10.1016/0027-5107(93)90049-l. [DOI] [PubMed] [Google Scholar]
- 7.Moorhead PS, Nowell PC, Mellman WJ, Battips DM, Hungerford DA. Chromosome preparations of leukocytes cultured from human peripheral blood. Exp Cell Res. 1960;20:613–616. doi: 10.1016/0014-4827(60)90138-5. [DOI] [PubMed] [Google Scholar]
- 8.Krendel DA, Younger DS. Autoimmune diabetic neuropathy. Neurol Clin. 1997;15:959–972. doi: 10.1016/S0733-8619(05)70358-3. [DOI] [PubMed] [Google Scholar]
- 9.Koopman RJ, Mainous AG, Liszka HA, Colwell JA, Slate EH, Carnemolla MA, et al. Evidence of nephropathy and peripheral neuropathy in US adults with undiagnosed diabetes. Ann Fam Med. 2006;4:427–432. doi: 10.1370/afm.577. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Fedele D, Comi G, Coscelli C, Cucinotta D, Feldman EL, Ghirlanda G, et al. A multicenter study on the prevalence of diabetic neuropathy in Italy. Italian Diabetic Neuropathy Committee. Diabetes Care. 1997;20:836–843. doi: 10.2337/diacare.20.5.836. [DOI] [PubMed] [Google Scholar]
- 11.Vinik AI, Holland MT, Le Beau JM, Liuzzi FJ, Stansberry KB, Colen LB. Diabetic neuropathies. Diabetes Care. 1992;15:1926–1975. doi: 10.2337/diacare.15.12.1926. [DOI] [PubMed] [Google Scholar]
- 12.Tesfaye S, Chaturvedi N, Eaton SEM, Ward JD, Manes C, Tirgoviste C, The EURODIAB Prospective Complications Study Group et al. Vascular risk factors and diabetic neuropathy. N Engl J Med. 2005;352:341–350. doi: 10.1056/NEJMoa032782. [DOI] [PubMed] [Google Scholar]
- 13.Haffner SM, Stern MP, Hazuda HP, Mitchell BD, Patterson JK. Cardiovascular risk factors in confirmed prediabetic individuals. Does the clock for coronary heart disease start ticking before the onset of clinical diabetes? JAMA. 1990;263:2893–98. doi: 10.1001/jama.263.21.2893. [DOI] [PubMed] [Google Scholar]
- 14.Forrest KY, Maser RE, Pambianco G, Becker DJ, Orchard TJ. Hypertension as a risk factor for diabetic neuropathy: a prospective study. Diabetes. 1997;46:665–670. doi: 10.2337/diabetes.46.4.665. [DOI] [PubMed] [Google Scholar]
- 15.Vincent AM, Hayes JM, McLean LL, Giri AV, Pennathur S, Feldman EL. Dyslipidemia-induced neuropathy in mice. The role of oxLDL/LOX-1. Diabetes. 2009;58:2376–2385. doi: 10.2337/db09-0047. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Ewing DJ, Martyn CN, Young RJ, Clarke BF. The value of cardiovascular autonomic function tests: a 10 year experience in diabetics. Diabetes Care. 1985;8:482–491. doi: 10.2337/diacare.8.5.491. [DOI] [PubMed] [Google Scholar]
- 17.Orchard TJ, Dorman JS, Maser RE, Becker DJ, Ellis D, LaPorte RE, et al. Factors associated with avoidance of severe complications after 25 yr of IDDM. Pittsburgh Epidemiology of Diabetes Complications Study I. Diabetes Care. 1990;13:741–747. doi: 10.2337/diacare.13.7.741. [DOI] [PubMed] [Google Scholar]
- 18.Hyun Ko S, Park SA, Cho JH, Song KH, Yoon KH, Cha BY, et al. Progression of cardiovascular autonomic dysfunction in patients with type 2 diabetes. Diabetes Care. 2008;31:1832–1836. doi: 10.2337/dc08-0682. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Martin LJ. DNA damage and repair: relevance to mechanisms of neurodegeneration. J Neuropathol Exp Neurol. 2008;67(5):377–387. doi: 10.1097/NEN.0b013e31816ff780. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Andreassi MG. Coronary atherosclerosis and somatic mutations: an overview of the contributive factors for oxidative DNA damage. Mutat Res. 2003;543:67–86. doi: 10.1016/S1383-5742(02)00089-3. [DOI] [PubMed] [Google Scholar]
- 21.Andreassi MG, Botto N, Simi S, Casella M, Manfredi S, Lucarelli M, et al. Diabetes and chronic nitrate therapy as co-determinants of somatic DNA damage in patients with coronary artery disease. J Mol Med. 2005;83:279–286. doi: 10.1007/s00109-005-0634-8. [DOI] [PubMed] [Google Scholar]
- 22.Nomiyama T, Tanaka Y, Hattori N, Nishimaki K, Nagasaka K, Kawamori R, et al. Accumulation of somatic mutation in mitochondrial DNA extracted from peripheral blood cell. Diabetologia. 2002;45:1577–1583. doi: 10.1007/s00125-002-0893-7. [DOI] [PubMed] [Google Scholar]