Erythrocyte caspase-3 activation and oxidative imbalance in erythrocytes and in plasma of type 2 diabetic patients.

An increased oxidative stress and a decreased life span of erythrocytes (RBCs) are reported in patients with diabetes. Aim of this study was to assess in RBCs from patients with type 2 diabetes whether downstream effector mechanisms of apoptosis, such as activation of caspase-3, is operative, and whether an iron-related oxidative imbalance, occurring inside RBCs and in plasma, could be involved in caspase-3 activation. In 26 patients with type 2 diabetes and in 12 healthy subjects, oxidative stress was evaluated by means of different markers; non-protein-bound iron, methemoglobin and glutathione were determined in RBCs, and non-protein-bound iron was also determined in plasma. Erythrocyte caspase-3 activation was evaluated by an immunosorbent enzyme assay. Arterial hypertension, demographic and standard biochemical data were also evaluated. The results show, for the first time, that type 2 diabetic RBCs put into motion caspase-3 activation, which is significantly higher than in control RBCs. Such an effector mechanism of "eryptosis" was positively correlated to blood glucose levels and to the increased plasma NPBI level. Caspase-3 activation was also positively correlated to occurrence of arterial hypertension. The results suggest that an extracellular oxidative milieu can be responsible for erythrocyte caspase-3 activation in patients with type 2 diabetes. In turn, caspase-3 activation can be envisaged as a novel mechanism which, by impairing the maintenance of erythrocyte shape and function, might contribute to the shortened life span of RBCs from patients with type 2 diabetes and to hemorheological disorders observed in these patients.

Magnetic resonance imaging in the evaluation of the gastric emptying and antral motion: feasibility and reproducibility of a fast not invasive technique.

PURPOSE: The aim of this study was to evaluate reproducibility of a fast MRI protocol to measure gastric emptying and motility of the gastric antrum. METHODS AND MATERIALS: Gastric emptying and antral speed were measured in 12 type 1 diabetic patients (mean age 43 years) and 9 healthy volunteers (mean age 31 years). Subjects, fasting from 6h, were evaluated in supine position using a 1.5T MR scanner and a eight-channels phased-array body coil after ingestion of 400 ml of a vanillas pudding mixed with 5 ml of Gd-DTPA. Axial 3D T1w sequence at 0 and 30 min for volume evaluation and cine-steady state acquisition every 5 min for a total time of 30 min for gastric wave speed assessing were acquired. Two blinded observers extrapolated T(1/2) from gastric volume assessment and speed of gastric waves. RESULTS: All the patients tolerated the examination. The T(1/2) cut-off was of 115 min with an accuracy in differentiate controls from diabetics of 96% (95% CI 0.766-0.992; p<0.001), while the antral speed cut-off was of 0.15 mm/s with an accuracy of 87% (95% CI 0.628-0.977; p<0.001). The inter-observer agreement for the volumes at time 0 and 30 min was respectively 0.983 (95% CI 0.9628-0.9929; p<0.001) and 0.9933 (95% CI 0.9847-0.9971; p<0.001) with an agreement of 0.9918 (95% CI 0.9853-0.9954; p<0.001), while for antral speed it was of 0.935 (95% CI 0.9097-0.9528; p<0.001). CONCLUSIONS: MRI is a reproducible technique for the evaluation of gastric emptying and antral motility.

Oxidative stress, erythrocyte ageing and plasma non-protein-bound iron in diabetic patients.

Increased oxidative stress and decreased life span of erythrocytes (RBCs) are repeatedly reported in diabetes. In the aim to elucidate the mechanism of the latter, i.e. the events leading to erythrocyte ageing, this study determined in RBCs from diabetic patients iron release in a free desferrioxamine-chelatable form (DCI), methemoglobin (MetHb) formation, binding of autologous IgG to membrane proteins and in plasma non-protein-bound iron (NPBI), F(2)-Isoprostanes (F(2)-IsoPs) and advanced oxidation protein products (AOPP). DCI and MetHb were higher in diabetic RBCs than in controls and autologous IgG binding occurred in a much higher percentage of diabetic patients than controls. A significant correlation between DCI and IgG binding was found in diabetic RBCs. Plasma NPBI, esterified F(2)-IsoPs and AOPP were higher in diabetic patients and a significant correlation was found between plasma NPBI and intra-erythrocyte DCI. The increased DCI and autologous IgG binding appear to be important factors in the accelerated removal of RBCs from the blood stream in diabetes and the increase in plasma NPBI could play an important role in the increased oxidative stress.

Fractal analysis of monocytes in diabetes.

Fractal dimension of pericellular membrane of monocytes was evaluated in diabetic patients and in control subjects. Monocytes were collected from normal healthy volunteers (n = 6) and from diabetic (type 1 and type 2) patients (n = 9). Monocytes from healthy volunteers were also stimulated in vitro with the ionophore A23187 or with the oligopeptide FMLP. Monocytes, obtained by Ficoll-Hypaque, were examined with a Philips 300 transmission electron microscope. The cell contour was extracted, resized to a standard dimension and converted to a single pixel outline. Box-counting method was then applied to determine the fractal dimension. Fractal dimensions of monocytes appeared statistically increased in diabetic patients (type 1 and type 2), compared with sex- and age-matched controls (p < 0.01, p < 0.01). The mechanism underlying the observed increased complexity of pericellular membrane may be explained by the in vivo activation of the circulating monocyte in diabetes. In effect, fractal analysis of stimulated in vitro monocytes showed a significant increase of complexity of pericellular membrane, compared with their controls (p < 0.001). Our approach was able to assess and quantitatively evaluate in diabetic patients morphological modifications of the monocyte linked to its activation, offering new parameters useful to follow the effects of therapeutical procedures.