Recruitment and Imaging
Eighty-two adult subjects with diagnosed T2DM were recruited from the Diabetes Mellitus Outpatient Clinic of the 3rd Department of Internal Medicine, situated at Papageorgiou General Hospital, Thessaloniki, Greece, and divided into two groups according to the presence or absence of DPN. The DPN group was additionally subdivided according to the presence of active ulcers that were located below the ankle join. The inclusion criteria were: type T2DM, according to ADA and IDF criteria , and age >18 years. The exclusion criteria were: history of myocardial infraction, stroke, coronary revascularization or cardiac bypass, active liver disease, any chronic renal disease, any autoimmune disease, HIV infection, malignancy, primary neurologic disorders (previous spinal injury, a history of lumbar or cervical discopathy, carpal tunnel syndrome, alcoholism, inherited neuropathy), vitamin B9 or B12 deficiency, concomitant use of glucocorticoid, isoniazid or metronidazole. The Ethics Committee of the Papageorgiou General Hospital approved the study’s protocol and patients gave their informed consent prior to participation in accordance with the principles of the Declaration of Helsinki as revised in 2008.
MI was diagnosed via electrocardiographically gated Technetium-99 m (Tc-99 m) sestamibi single-photon emission computed tomographic (SPECT) images, a method attributing added value to non-gated SPECT perfusion images. The Summed Stress Score (SSS) which represents the extent and severity of a perfusion abnormalities was obtained and considered abnormal when ≥4 . Additionally, the percentage of ischemia was estimated for each patient, as described in the European Association of Nuclear Medicine guidelines . Patients were instructed to fast for a minimum of 4 h and withhold beta-blockers, calcium channel blockers, nitrates, methyl-xanthene containing drugs and caffeinated foods and beverages for at least 24–48 h prior to the examination . DPN was evaluated with the neuropathy disability score (NDS) by testing the sensations of pain, touch, cold, and vibration in both legs of each patient and assigning a score according to the level of impaired sensation. An NDS score ≥5 was indicative of DPN . Additionally, the group without DPN was determined based on a negative 10-g monofilament test . The ratio of the longest electrocardiographic RR interval between the 28th and 32nd beats after standing to the shortest interval between the 13th and 17th beats (RR ratio) and orthostatic hypotension were used as markers of CAN. One observer calculated the RR ratio. Abnormal autonomic function was defined as a loss of heart rate variability with an RR ratio of less than 1.04, postural hypotension with a fall in systolic blood pressure of 20 mmHg or more, or both . Peripheral arterial disease was diagnosed with the ankle-brachial index (ABI), with a technique that has been previously described, and the results were considered pathological when the values were <9 or >1.3 .
Continuous data were expressed as mean ± standard deviation or as the median (interquartile range) when lacking normality in distribution. Between-group comparisons were analyzed by one-way ANOVA. Post hoc comparisons were performed using the Tukey test. When application conditions were not met, nonparametric tests (Kruskal–Wallis and Mann–Whitney) were used. Categorical data were expressed as frequencies and percentages and then compared with the Chi-squared test. Spearman’s correlations were used to assess the relationship between SSS and other continuous variables. We subsequently included all variables that correlated with a p value <0.15 in a multiple linear regression model to identify predictors of SSS. In order to assess the performance of using the NDS to classify patients with ischemia, receiver-operating characteristic (ROC) curve was plotted and the area under the ROC curve (AUC) with its confidence interval was computed. p values <0.05 were considered as significant. Statistical analysis was performed using SPSS software (v.22.0; IBM, Armonk, NY, USA).