Evaluation of autonomic neuropathy in newly diagnosed and chronic type 2 diabetic patients: A retrospective cross-sectional study

Introduction: Diabetic autonomic neuropathy is one of the most important complications of diabetes mellitus (DM) that ultimately occurs in most patients. The purpose of this study was to screen and diagnose latent cases of autonomic neuropathy among patients with a history of DM for over 10 years, and recently diagnosed diabetic patients. Methods: This cross-sectional study was performed on 104 patients, consisting of 52 type 2 DM (T2DM) patients diagnosed in the last 6 months; and 52 T2DM patients with a history of DM for more than 10 years) referred to outpatient endocrine clinic of Imam Reza Medical Center in Tabriz University of Medical Sciences between 2015-2016. Blood pressure, resting heart rate and corrected QT interval were evaluated according to standard methods. The history of gastrointestinal and urinary tract neuropathy was extracted from patients’ history and physical

asymptomatic and is usually not included in the initial evaluation and follow-up of diabetic patients. 12 There is no reliable data in Iran on the prevalence of autonomic neuropathy among newly diagnosed T2DM patients. This study was designed to screen and diagnose latent cases of autonomic neuropathy in newly diagnosed diabetic patients and compare it with those with a history of more than ten years of T2DM.

Patients
This retrospective cross-sectional study was performed on 104 diabetic patients, including 52 T2DM patients diagnosed in the last 6 months and 52 patients with a history of T2DM for more than 10 years, referred to outpatient endocrinology clinic of Imam Reza hospital in Tabriz University of Medical Sciences. Cochrane formula with the power of 95% and a significance level of less than 0.05 was used for sample size calculation, based on the 7% prevalence of T2DM. 13 The study lasted for one year from April 1, 2015 to April 1, 2016.
The inclusion criteria were age >18 years, and having the ability to perform the tests correctly. Cases with ischemic heart, brain, renal or respiratory diseases, and malignancy; and the patients under insulin and alpha and beta blocker agents, and alcohol or caffeine users were excluded from the study.
After selecting patients, they were asked to avoid any intense physical activity before the tests and wait sitting on the chair for at least ten minutes. Then, they were taken to the examination room and were asked to lie in supine position on the bed and breathe in with normal rhythms and avoid moving on and speaking during the tests. Blood pressure measured from the left arm, and electrocardiogram (ECG) was recorded using bipolar limb leads. Data collection was performed after a minimum of 5 minutes of rest in lying position.

Determination of the average resting heart rate
The average heart rate after 5 minutes of completing rest in lying position was determined based on the records of electrocardiogram on lead II. Heart rates more than 100 beats/minute were considered as abnormal without correction for age and sex. In addition, the changes in heart rates were estimated through standing up position based on a ratio of 15 to 30. 14 The length of two consecutive sinusoidal waves at 15 and 30 beats after standing up was determined and their ratio was reported.

Determination of the corrected QT interval
For this purpose, the Q and T wavelengths were measured in the electrocardiogram on lead II and calculated by the Bazett's formula (QTc= QT/√RR). QTc, QT and RR represent the corrected QT, duration of QT interval and duration of RR interval, respectively. 15 The QT intervals >440 milliseconds were considered as abnormal. 16 Orthostatic hypotension test Two minutes after changing from lying to standing position, the systolic and diastolic blood pressures were measured and then compared to systolic and diastolic blood pressures in lying position. A reduction >20 mmHg in systolic pressure and 10 mmHg in diastolic pressure was considered as abnormal. 17 The test score was calculated for patients who fully completed all of five tests. For quantitative evaluation of cardiac autonomic neuropathy, the results of each test were scored between 0 (normal value) and 1 (abnormal value). Then, patients were allocated to one of the three groups including: without neuropathy (final sum of zero), intermediate (final sum of 1) and neuropathic (final sum of 2-5). 6,18,19 Evaluation of gastrointestinal and genitourinary neuropathy status Gastrointestinal and genitourinary neuropathies were determined through patients' medical history. Any compliant of autonomic system disorders such as benign paroxysmal positional vertigo, night sweats, sweating during eating, dry skin of the lower extremity, dysuria, urinary frequency, urinary incontinence, sexual problems, early satiety, reflux, diarrhea, constipation, and fecal incontinence were questioned from each patient. 20,21 Examining peripheral neuropathy Peripheral neuropathy was assessed by United Kingdom Screening Test. 18 This test includes the patient's symptoms (burning sensation, itching, pain, location of symptoms, timing of symptoms and how symptoms resolve) and clinical examination (achilles tendon reflex, vibration sensation and pain and temperature sensation).
Each of these cases is scored between 0-2. Patients were ranked according to the total score in one of the four groups including 0 to 2 points: Normal, 3 to 4 points: Mild polyneuropathy, 5to 6 points: Moderate polyneuropathy, 7 to 9 points: Severe polyneuropathy.

Statistical analysis
Data were analyzed using SPSS version 22 software. The mean values of the study groups were evaluated using the independent sample t test. The values of P < 0.05 were considered significant. The examining person and statistical analyzer were blinded to the newly diagnosed or chronic patient's groups.

Demographic characteristics of the studied patients
Of 104 patients under study, 54 (51.7%) were male and 50 (48.3%) were female. The mean age of the patients was 60.4 ± 17.2. Fifty-two subjects were newly diagnosed T2DM patients (less than 6 months) and 52 had a history of T2DM for more than ten years. As shown in Table 1 (28.7%) and smoking (26.5%) were the most frequent risk factors, respectively.

The comparison of heart rates in resting and standing positions in study groups
The mean resting heart rates in newly diagnosed and chronic T2DM patients did not show a significant difference between two groups (80±16.87 versus 84±12.54, P = 0.173, respectively). However, a significant difference was observed in the mean standing heart rates in mentioned groups (88±19.21 versus 96±15.74, P = 0.02, respectively) ( Figure 1).

The comparison of systolic blood pressure in resting and standing positions
The mean resting systolic blood pressure in newly diagnosed and chronic T2DM patients showed a significant statistical difference between groups (110±31.21 mm Hg versus 128±11.65 mm Hg, P < 0.0002, respectively). Moreover, similar results were also observed in comparison of the standing systolic blood pressure between mentioned groups (112±29.11 mm Hg versus 131±13.79 mm Hg, P < 0.0001, respectively) ( Figure 2).

The comparison of diastolic blood pressure in resting and standing positions
As it is presented in Figure 3, a significant difference was observed in resting diastolic blood pressure between newly diagnosed and chronic T2DM patients (71±21.37 mm Hg versus 80±21.43 mm Hg, P = 0.03, respectively). No significant statistical differences were observed in standing diastolic blood pressure between mentioned groups (75±57.61 mm Hg versus 84±11.34 mm Hg, P = 0.334, respectively) ( Figure 3).

Figure 1.
Resting and Standing heart rate in newly diagnosed and chronic type 2 diabetic patients. Data are presented as mean ± standard deviation. P < 0.05 was considered as statistically significant.

Figure 2.
Resting and standing systolic blood pressure in newly diagnosed and chronic type 2 diabetic patients. Data are presented as mean ± standard deviation. P < 0.05 was considered as statistically significant.

Figure 3.
Resting and standing diastolic blood pressure in newly diagnosed and chronic type 2 diabetic patients. Data are presented as mean ± standard deviation. P < 0.05 was considered as statistically significant.

The comparison of corrected QT interval in study groups
The mean QT intervals in newly diagnosed and chronic T2DM patients showed a significant difference (462±46.51 versus 482±17.72 ms/s, P = 0.106) (Figure 4).

The frequency of autonomic neuropathies in study groups
As shown in Table 2, of 52 newly diagnosed T2DM patients, 4, 2 and 5 subjects had cardiovascular autonomic impairment, gastrointestinal autonomic disorder, and genitourinary autonomic disorder, respectively. In contrast, of 52 patients with T2DM for more than 10 years, 16, 6 and 8 subjects had cardiovascular autonomic impairment, gastrointestinal autonomic disorder and genitourinary autonomic disorder, respectively.

The association between serum glucose and body mass index with frequency of autonomic neuropathy in study groups
Autonomic neuropathy was observed in 41 patients who were accounted for 39.42% of study population. A significant higher level of blood glucose was observed in patients with autonomic neuropathy in comparison to the patients without autonomic neuropathy (312±52.61 versus 131±24.7, P < 0.0001, respectively); indicating a significant association between the lack of blood glucose control and the presence of autonomic neuropathy in patients with T2DM. In addition, the mean level of body mass index (BMI) in patients with and without autonomic neuropathy were 33.3±5.3 and 28±4.7, P < 0.0001, respectively ( Figure 5).

Discussion
This study investigated the incidence of autonomic dysfunction in patients with T2DM for more than 10 years in comparison to the patients with newly diagnosed T2DM, using the non-invasive criteria of Ewing et al. 22 These tests are extensively used worldwide to examine the presence of cardiac autonomic neuropathy. Due to their simplicity, these tests are reliable and the results in the different studies have been reproducible. Ewing et al 22 considered the presence of autonomic cardiac neuropathy with abnormal results in any one of these tests, but we performed this study based on recommendations of the reference book. 19 In this study, we investigated the prevalence of microvascular complications (neuropathies) in diabetic patients with autonomic diabetic neuropathy. We separated patients based on the number of years since the onset of diabetes. A similar study conducted on 53 diabetic patients in Turkey, indicated similar results with our study. In this study, cardiovascular autonomic neuropathy was considered as an impairment in one of the tests. Based on patients' impairment rates in the tests, they were divided into three groups of mild, moderate, and severe cardiovascular autonomic neuropathies. Although, there was no relationship between severity of cardiovascular autonomic neuropathy and BMI in our patients, it was also observed that a large proportion of patients with heart cardiovascular autonomic neuropathy have other microvascular complications of DM. On the other hand, diabetic autonomic neuropathy has a high prevalence  Comparison of blood glucose levels and body mass index (BMI) between patients with (n=41) or without (n=63) neuropathy. Data are presented as mean ± standard deviation. P < 0.05 was considered as statistically significant. Gastrointestinal autonomic disorder 2 (3.84) 6 (11.55) Genitourinary autonomic disorder 5 (9.61) 8 (15.38) in comparison to other microvascular complications of DM. 14 In our study groups, the incidence of cardiovascular autonomic neuropathy was about 19%; while the incidence of cardiovascular autonomic neuropathy in a study conducted by Valensi et al 9 in the French Research Center of Diabetes yielded a 24.5% incidence; although, they accordingly included the type 1 diabetic patients in their study. Given the fact that resting heart rate measurement is a simple and fast test and is known as the first symptom of cardiovascular autonomic neuropathy in many studies; it can be a warning sign in patients for other complications of DM, including sensory neuropathy and nephropathy. 9 In study of Ko et al, 20 26 studies, the frequency of gastrointestinal symptoms in diabetic patients were estimated as 15%-25%, and 35%, respectively. Different sample sizes may be the cause of difference in findings of the mentioned studies with the others; since those two studies also included type 1 diabetic patients. Among the findings in the current study, the symptoms of gastrointestinal autonomic dysfunction were lower than that of previous studies, and there were no significant differences between recently diagnosed and chronic diabetic patients. In a study by Papatheodoridis and Karamanolis, 21 700 adults from the general population of Greece were investigated, of whom 53% presented at least one gastrointestinal complaint. In Sweden, Agréus et al 27 examined the incidence of gastrointestinal symptoms on 1290 normal individuals, of whom 54% indicated a minimal gastrointestinal complaint. Since, no study investigated the prevalence of gastrointestinal disorders in Iranian population, it cannot be concluded that the prevalence of gastrointestinal symptoms in diabetic patients in the present study is higher than the normal population. Small sample size of the studied population was one of the main limitations of the present study. Additionally, correlation analysis between albuminuria and nephropathy with cardiovascular autonomic neuropathy seems to be an important issue which was not evaluated in this study.

Conclusion
According to the findings of current study, the most common autonomic disorder in newly diagnosed T2DM patients and patients with a history of T2DM for more than 10 years was cardiovascular autonomic impairment which was more common in patients with longer history of diabetes. However, the comparison of gastrointestinal autonomic and genitourinary autonomic disorders in newly diagnosed diabetic patients and patients with a history of T2DM for more than 10 years had no significant differences. The overall incidence of gastrointestinal autonomic and genitourinary autonomic disorders was lower than the incidence of cardiovascular autonomic impairment.

What is current knowledge?
• Diabetic autonomic neuropathy is one of the most important complications of DM which occurs in the early stages in most asymptomatic patients. Despite the negative effects of autonomic neuropathy on diabetic patients' survival and quality of life, this complication is less studied compared to other complications of DM.

What is new here?
• There is no reliable data in Iran on the prevalence of autonomic neuropathy among newly diagnosed T2DM patients. This study was designed to screen and diagnose latent cases of autonomic neuropathy in newly diagnosed diabetic patients and compare it with those with a history of more than ten years of T2DM.