CARDIOVASCULAR AUTONOMIC NEUROPATHY: ALPHA-LIPOIC ACID, INSULIN RESISTANCE AND INFLAMMATORY PARAMETERS IN PATIENTS WITH TYPE 2 DIABETES MELLITUS

. This paper presents the results obtained during the study of possible alpha-lipoic acid (ALA) impact on the state of insulin resistance (IR), the content of some pro-and anti-inflammatory factors in patients with type 2 diabetes mellitus (T2DM) and cardiovascular autonomic neuropathy (CAN). Our study involved 33 persons with diagnosed T2DM and CAN, which were allocated to one of two groups. The control group (15 patients) received standard hypoglycemic treatment, whereas the intervention group (18 patients) received ALA 600 mg in film-coated tablets/q.d. in addition to standard therapy. Treatment period was 3 mos in duration. Levels of glycated hemoglobin A1c, glucose, immunoreactive insulin (IRI), leptin, interleukin (IL) IL-6 and IL-8, high sensitivity C-reactive protein (hs-CRP), tumor necrosis factor-alpha (TNF-alpha) in the blood were measured. TNF-alpha/IL-10 ratio and Homeostasis model assessment IR (HOMA-IR) were calculated. The significant reduction in glucose, IRI, leptin concentration and HOMA-IR parameters; decrease in the hs-CRP, TNF-alpha, IL-6 and IL-8 levels, TNF-alpha


Introduction
Diabetic cardiovascular autonomic neuropathy (CAN) is an independent risk factor for major cardiovascular events, development of heart arrhythmias, silent myocardial ischaemia, sudden cardiac death and cardiovascular mortality [1][2][3][4]. The impetus for this work was prompted by the hypothesis that physiological antioxidants, including alpha-lipoic acid (ALA), a powerful lipophilic "trap" of free radicals, promote the prevention of neurovascular disorders, provide the basis for the pathogenetically-based application of ALA in the treatment of diabetic angio-, neuropathy, in particular including diabetic CAN.
Our study focused on determining whether ALA has a potential impact on inflammation, glycemic control, insulin resistance (IR) parameters in patients with type 2 diabetes mellitus (T2DM) and CAN.

Material and methods
This study was designed as an open-label comparative controlled study. A total of 33 T2DM patients with CAN aged between 50-59 yrs, DM duration between 1-6 yrs, with glycated hemoglobin A1c (HbA1c) level of 7.030.13% and median body mass index (BMI) 27.1±0.32 kg/m 2 were enrolled. For pursoses of the study patients were allocated to two groups: the control group (15 patients) received standard hypoglycemic treatment, whereas the intrevention group (18 patients) received ALA (Thioctic Acid) 600 mg in film-coated tablets/q.d. in addition to standard therapy. Treatment period was 3 mos in duration. The age, sex distribution in the two groups was similar, as well as BMI and duration of DM, which made groups homogeneous as possible. The study was conducted for 2 yrs. All patients were examined before the start of the trial and after the end of the treatment period. All involved patients did not take ALA and was on stable regime of hypoglycemic and antihypertensive treatment for 6 mos. Cardiovascular autonomic reflex tests (CART's) are considered as the "gold" standard tests in the assessment of CAN. Screening for CAN, that included five CART's, timeand frequency-domain heart rate variability tests was performed for all enrolled patients. The obtained results were evaluated as follows: pathologicalas "1" scores, borderline values -as "0.5" scores, normal values -as "0" score. The individual scores were summed up and the median score for involved patients was 2.8±0.32 [5,6]. Glucose oxidase method was used for measurement of glucose concentration in the blood, while a highly sensitive method of ion-exchange liquid chromatography with D-10 analyzer and BIO-RAD reagents (USA) was used for HbA1c determination. Serum immunoreactive insulin (IRI) level was measured by using commercial kits from immunogen insulin immunoradiometric assay reagents (Czech Republic); interleukin (IL) IL-6, IL-8 and IL-10, tumor necrosis factor-alpha (TNF-alpha) -from Vector-Best (Russia); leptin -from Immunotech Leptin (Czech Republic) test kits; high sensitivity C-reactive protein (hs-CRP) -from diagnosisrelated group (USA). TNF-alpha/IL-10 ratio was calculated. For the calculation of Homeostasis model assessment IR (HOMA-IR) was used formula: fasting IRI (mcIU/mL) x fasting glucose (mmol/L)/22.5 [7].
To compare repeated measurements (before and after treatment) the Wilcoxon signed-rank test was used. A comparison of the obtained data in the two groups was carried out using the nonparametric Mann-Whitney test. All data are expressed as median (Me) and interquartile range (Q25-Q75). Statistical significance was set at p<0.05. All tests were performed using the ANOVA (MicroCal Origin v. 8.0) software.

Results
We found out that HbA1c of patients with T2DM and CAN was not statistically significantly influenced by the treatment (p>0.05). There were no differences between the groups in levels of preprandial blood glucose, IRI concentrations and HOMA-IR parameters at baseline. Baseline and post-treatment parameters of blood glucose, IRI concentrations and HOMA-IR parameters in patients with T2DM and definite stage of CAN are shown in the Table 1. As shown in the Table 1 Table 2.
There were no differences between the groups in leves of hs-CRP, leptin and TNF-alpha at baseline. As shown in the Table 2 after three months of ALA prescription to patients with T2DM and CAN a statistically significant reduction in TNF-alpha (p<0.001), leptin (p<0.05) and hs-CRP (p<0.05) levels was found.
Compared to the data, obtained in the control group, more pronounced decrease in TNF-alpha (p<0.05) and leptin (p<0.05) was found. Investigated parameters did not differ between baseline levels and after 3 mos in the control group (Table 2). Table 2 Baseline and post-treatment parameters of hs-CRP, leptin, TNF-alpha, IL-6, After 3 mos of treatment content of IL-6 (p<0.05), IL-8 (p<0.05) and TNFalpha/IL-10 ratio (p<0.01) was statistically significant lower compared with the pretreatment values and values, obtained in control group, namely IL-6 (p<0.05), IL-8 (p<0.05) and TNF-alpha/IL-10 ratio (p<0.05). In control group, no positive dynamic of the concentrations of studied parameters was found (p>0.05). IL-10 levels did not differ between two groups before and after treatment, showing no changes, compared with pretreatment values, in either group (Table 2).

ALA exibit direct free radical scavenging properties both in vitro and in vivo.
There is a strong link between hyperglycemia, oxidative stress (OS), increased production and decreased clearance of reactive oxygen species (ROS), low grade inflammation and development/progression of T2DM and its complications. OS has been suggested to contribute to defective nerve blood supply and endoneurial oxidative damage [8,9]. Some studies have demonstrated the ability of ALA to reduce expression of vascular cell adhesion molecule-1 (VCAM-1) and endothelial adhesion of human monocytes in vitro. Futhermore, ALA prevents the upregulation of intercellular adhesion molecule-1 and VCAM-1 in spinal cords and TNF-alpha stimulated cultured brain endothelial cells [10][11]. ALA is largely known as an antiinflammatory and antioxidant molecule, the effects of which are exerted through the modulation of nuclear factor kappa-light chain-enhancer of activated B cells (NF-kB). Different inflammatory cytokines, including IL-1β and IL-6, in different cell types and tissues, are modulated by NF-kB. IL-1β and IL-6 undergo DNA methylation-dependent modulation in neural models and pave the road to study of the epigenetic mechanisms triggered by ALA [9]. ALA may effectively reduce OS by preventing lipid peroxidation (LPO) and protein damage as other antioxidants significantly inhibit the ROS such as proxy nitrite, nitric oxide, hydroxyl radicals, superoxide anion in the membrane and aquatic environment. ALA has drawn considerable attention due to its properties to prevent/treat chronic complications of DM [12].
Some studies showed that treatment with ALA either i.v. or p.o. was associated with glucose disposal improvement in patients with T2DM [13]. Also, some other studies findings suggest improvement in HbA1c, LPO, antioxidant enzymes and inflammatory markers. ALA as a compound with strong antioxidant potential, has a function in decreasing damages caused by chronic low-grade inflammation and diabetic neuropathies [14][15][16]. The results of Irbesartan and Lipoic Acid in Endothelial Dysfunction (ISLAND) clinical trial showed that supplementation with ALA in daily dose of 300 mg for 4 wks was associated with the 15% significant decrease in serum IL-6 levels. As IL-6 is a recognized marker of inflammation in coronary atherosclerotic plaques, and regulates the expression of other inflammatory cytokines, such as TNF-alpha and IL-1, this finding may prove the antiinflammatory effects of ALA. Nevertheless, the body of evidence is currently too limited and could not be considered as conclusive, so futher investigations are needed [17,18].

Conclusion
Our study suggests that administration of ALA contributed to a statistically significant decrease of glucose, IRI concentrations, HOMA-IR parameters; TNFalpha, hs-CRP, IL-6 and IL-8, leptin levels and TNF-alpha/IL-10 ratio, but does not significantly affect the content of IL-10 content in patients with T2DM and definite CAN. The obtained data may indicate a decrease in the activity of the proinflammatory link of the immune response and allow us to consider ALA as a promising agent in the CAN complex treatment in patients with T2DM. Our study has several limitations. First, the sample size was small, second -short duration.
However, further studies with longer duration and larger population are needed to validate our findings and provide evidence for the hidden therapeutic potential of ALA.