The surface electromyographic evaluation of the Functional Reach in elderly subjects.

This study proposes a comprehensive assessment of myoelectric activity of the main muscles involved in the Functional Reach (FR) test, in 24 elderly subjects. A specific protocol for the surface electromyography (sEMG) signal acquisition during FR-test was developed. Results show that anterior muscles activate following a caudo-cranial order. Tibialis Anterior (TA) is the first to be activated (-18.0±16.3% of the FR-period), together with Rectus Femoris (-10.4±17.9%). Then, Rectus Abdominis (19.7±24.7%) and Sternocleidomastoideus (19.9±15.6%) activate after the FR-start. Hamstrings, Soleus, and L4-level Erectores Spinae (posterior muscles) activate after the FR-start in this order (11.4±16.8%, 17.7±16.6%, and 35.2±29.0%, respectively) and remain active until the movement end. The analysis of the kinematic strategies adopted by subjects revealed an association between TA-activation patterns and two kinematic strategies (hip/mixed strategy), quantified by an increase (p<0.05) of TA-activity duration in subjects adopting the hip strategy (89.9±34.5) vs. subjects adopting the mixed strategy (27.0±16.8). This suggests that TA sEMG activity could be able to discriminate among kinematic strategies, providing different information on balance control. Thus, the present analysis represents the first attempt to quantify the sEMG activity during FR-test in elderly subjects, providing an early contribution in building a reference frame for balance assessment in clinical context.

Muscle activation patterns related to diabetic neuropathy in elderly subjects: A Functional Reach Test study.

BACKGROUND: This study was designed to assess, in healthy elderly, non-neuropathic and neuropathic diabetic subjects, the activation patterns of the main muscles involved in the Functional Reach Test, a well-recognized method to identify elderly subjects at risk of balance impairments. METHODS: Surface electromyographic analysis of Sternocleidomastoideus, Rectus Abdominis, Erectores Spinae at L4 level, Rectus Femoris, Hamstrings, Tibialis Anterior and Soleus was performed in 10 healthy, 10 diabetic non-neuropathic and 10 diabetic neuropathic subjects. FINDINGS: Results showed that in every group the first motor is Tibialis Anterior, that is recruited before the start of the test. An earlier activation of Tibialis Anterior (P<0.05) was detected in diabetic neuropathic (ON at -24% of the test period), compared with healthy (-11%) and diabetic non-neuropathic (-13%) groups. A significant earlier activation of Sternocleidomastoideus and Rectus Abdominis was found in diabetic neuropathic group, only with respect to healthy subjects. No significant difference was found in Rectus Femoris, Soleus, Hamstrings an Erectores Spinae onset among the three groups. INTERPRETATION: Results suggest a trend of diabetic neuropathic patients in earlier anticipation of the activation of the anterior body-muscles. In particular, the earlier onset of Tibialis Anterior is likely to be performed to adjust the movement timing and to compensate for the delay in the recruitment of the motor units. This anticipation might be involved in the altered postural control with increased balance impairment detected in diabetic neuropathic patients, and thereby it might also be proposed as an index of neuropathy, evidenced in a simple and non-invasive manner.

Diabetes-related quality of life is enhanced by glycaemic improvement in older people.

AIMS: To investigate the validity and reliability of the Audit of Diabetes-Dependent Quality of Life instrument in older Italians with diabetes and to test the association of diabetes-related quality of life with glycaemic control over time. METHODS: A total of 558 outpatients with Type 2 diabetes from the Diabetic Unit of the Italian National Research Centre on Aging Hospital in Ancona were enrolled to complete questionnaires (Audit of Diabetes-Dependent Quality of Life-19 and the Short-Form-12), and to undergo clinical and biochemical testing at baseline and at 12 months of follow-up. The overall impact of diabetes using the average weighted impact score from the Audit of Diabetes-Dependent Quality of Life questionnaire was calculated. Participants were categorized according to this score as having either less or more negative diabetes-related quality of life. RESULTS: Participants had a mean ± SD age of 67.7 ± 9.2 years and 51.8% were male. Factor analysis and Cronbach's coefficient of internal consistency (Cronbach's α = 0.931) confirmed that the 19 domain-specific Audit of Diabetes-Dependent Quality of Life items could be combined into a single scale in this Italian population. The impact score correlated with the physical (r = 0.275; P < 0.001) and mental components (r = 0.291; P < 0.001) of the Short-Form-12 questionnaire. Significant differences were found according to diabetic complications in specific Audit of Diabetes-Dependent Quality of Life items and impact scores. Insulin use had a greater association with a more negative quality of life compared with other antidiabetic agents. A multivariate linear regression model with restricted linear spline application showed that the relationship between HbA1c and impact score was not linear and that the change in the impact score was associated with improved glycaemic control in those with a less negative diabetes-related quality of life at 12 months. CONCLUSIONS: The Audit of Diabetes-Dependent Quality of Life-19 is a valid tool for measuring the impact of diabetes on quality of life in older Italians. Perception of diabetes-related quality of life is associated with glycaemic control over time.

Assessment of the activation patterns of the muscles involved in the FR test in diabetic neuropathic patients.

This study was designed to assess, in elderly neuropathic diabetic (DN) patients, the activation patterns of the main muscles involved in the Functional Reach (FR) Test, a well-recognized method to identify elderly subjects at risk of recurrent falls. Surface electromyographic (sEMG) analysis of Sternocleidomastoideus (Scm), Rectus Abdominis (RAbd), Erectores Spinae at L4 level (L4), Rectus Femoris (RF), Hamstrings (Ham), Tibialis Anterior (TA) and Soleus (Sol) was performed to this aim. Results in DN patients are compared with a control group (CH) of healthy age-matched subjects. In DN patients, TA is identified as the first muscle to be recruited (ON at -34% of the FR-period) before the movement start, in order to initiate the body forward displacement. RF is the first muscle to be recruited after TA and, togheter with RAbd, showed a progressive earlier onset from CH group. Sol and Ham (ON after the FR-start), followed by L4, act mainly as tonic muscles, opposing the movement and preventing falls. Compared to the CH group, the DN subjects show an anticipatory recruitment (-34%±6%) of TA, showing a statistically significant difference (p<;0.05) in comparison to CH group, together with the Scm activation. Results suggest a trend of DN patients in anticipating the activation of the anterior muscles of the body. This is likely due to an attempt to compensate the neuropathy-related proprioception dysfunction and to adjust the movement timing. In conclusion, the present study shows that sEMG is a suitable tool to deepen the interpretation of the FR-test execution and proposes the earlier start of TA as a possible element to identify the presence of neuropathy in diabetic subjects.

Identification of peripheral neuropathy in type-2 diabetic subjects by static posturography and linear discriminant analysis.

BACKGROUND: An early diagnosis of peripheral neuropathy in diabetic patients is useful in order to slow down the progress of this complication. Nerve conduction tests are the gold standard for this diagnosis but they are challenging for the patients. This study examines whether it is possible to assess the presence of diabetic neuropathy at an early stage by static posturography tests. METHODS: Static posturography tests were performed on 37 type-2 diabetic subjects (25 neuropathic patients and 12 non-neuropathic control subjects). Each subject was tested twice under two visual conditions: open and closed eyes. Both "global" (classic) and "structural" (model-based) posturographic parameters (PP) were derived from centre-of-pressure trajectories. A total of 65 PP were computed but only five were selected, normalized and fed to a linear classifier based on linear discriminant analysis. RESULTS: This method correctly classified 86.5% of the patients. Five subjects were misclassified and only 2 false negatives out of 25 neuropathic subjects were erroneously diagnosed as control subjects. CONCLUSIONS: This paper shows that "global" and "structural" parameters derived by static posturography tests, and classic linear statistical approaches, can be used for the diagnosis of neuropathy provided PP are properly chosen and normalized.

Homocysteinylation of low-density lipoproteins (LDL) from subjects with Type 1 diabetes: effect on oxidative damage of human endothelial cells.

BACKGROUND: Homocysteine (Hcy) is an independent risk factor for cardiovascular disease (CVD). Individuals with Type 1 and Type 2 diabetes are more susceptible to the effects of homocysteine than non-diabetic subjects. The interaction between homocysteine-thiolactone (Hcy-thiolactone), a reactive product of Hcy, and low-density lipoproteins (LDL) induces the formation of homocystamide-LDL adducts (Hcy-LDL) and it has been suggested that homocysteinylation could increase atherogenicity of lipoproteins. AIM: The aim of the study was to compare the effect of in vitro homocysteinylation of LDL isolated from healthy control subjects (C-LDL) and from Type 1 diabetic patients (DM-LDL) and to investigate the effect of homocysteinylated LDL (Hcy-C-LDL and Hcy-DM-LDL) on peroxynitrite production of endothelial cells. METHODS: The in vitro homocysteinylation of LDL isolated from control (n = 12) and DM subjects (n = 12) was carried out by incubating lipoproteins with Hcy-thiolactone. The reaction was verified by quantifying the increase in sulphydryl groups (-SH groups) in Hcy-LDL with respect to control LDL. Control and homocysteinylated LDL were incubated with human aortic endothelial cells (HAEC) in culture. Peroxynitrite production in cells treated in different experimental conditions was assayed by a fluorimetric method. RESULTS: The increase in -SH groups after incubation with homocysteine was greater in LDL from diabetic subjects compared with LDL from control subjects (P < 0.001). In addition, peroxynitrite production from HAEC incubated with Hcy-LDL from diabetic patients was greater than after incubation with Hcy-LDL from control subjects and untreated LDL from diabetic patients (P < 0.001). CONCLUSIONS: These results show that LDL from diabetic patients is more susceptible to in vitro homocysteinylation than LDL from non-diabetic individuals and demonstrate that the compositional changes in Hcy-LDL from diabetic subjects have cytotoxic effects on human endothelial cells.

Protective effect of paraoxonase activity in high-density lipoproteins against erythrocyte membranes peroxidation: a comparison between healthy subjects and type 1 diabetic patients.

High-density lipoproteins (HDL) plays a key role in the protection against oxidative damage of lipoprotein and biological membranes. The aim of the present study was to investigate the relationship between the antioxidant role of HDL and the HDL-paraoxonase (PON) activity in healthy subjects and in type 1 diabetic patients. Moreover, the ability of HDL of controls and diabetic patients to protect and/or repair biological membranes from oxidative damage was studied. HDL were isolated from 31 type 1 diabetic patients and 31 sex- and age-matched healthy subjects and immediately used to evaluate lipid hydroperoxides and HDL-PON activity. Erythrocyte membranes obtained from healthy subjects were oxidized with 2,2-azo-bis(2-aminidinopropane)dihydrochloride and then incubated in the presence of HDL isolated from healthy or type 1 diabetic subjects, with measurements of membrane lipid hydroperoxides before and after the incubation. HDL from type 1 diabetic patients showed higher levels of lipid hydroperoxides and a lower activity of HDL-PON than healthy subjects. Moreover, HDL of type 1 diabetic patients protected less efficiently erythrocyte membranes against oxidative damage compared with HDL from healthy subjects. A negative correlation was found between HDL-PON activity and the levels of hydroperoxides of HDL, confirming the relationship between PON and lipid peroxidation and suggesting that subjects with low PON activity are more exposed to oxidative damage than subjects with high PON activity. The ability of HDL to protect erythrocyte membranes was positively correlated with HDL-PON activity and negatively correlated with the levels of lipid hydroperoxides of HDL of healthy subjects. These results confirm a linkage between PON activity and lipid peroxidation of lipoproteins and suggest that the ability of HDL to protect erythrocyte membranes might be related to the PON activity. It might be hypothesized that the decrease of PON activity in diabetic patients and the lower HDL protective action against membrane peroxidation could contribute to acceleration of arteriosclerosis in type 1 diabetes mellitus.

Gestational diabetes affects platelet behaviour through modified oxidative radical metabolism.

AIMS: Patients with Type 1 and Type 2 diabetes mellitus show altered platelet function including decreased nitric oxide synthase (NOS) activity and increased peroxynitrite production. Gestational diabetes mellitus (GDM) is a clinical condition which is ideal for evaluating short-term effects of impaired glucose metabolism, ruling out the possibility that the platelet abnormalities are a consequence of diabetic complications. The aim of the present work was to study NO metabolism in platelets from pregnant women with GDM. The production of peroxides was also studied as it is strongly involved in peroxynitrite formation. METHODS: Platelet NOS activity and peroxynitrite production, levels of hydroperoxides and thiobarbituric acid reactive substances (TBARS) in platelet membranes in the basal state and after in vitro peroxidative stress with phenylhydrazine were determined in 40 pregnant women with GDM, 40 healthy pregnant women (pregnant controls) of comparable age and gestational age, and 15 healthy non-pregnant women (controls). RESULTS: NOS activity was significantly increased in both groups of pregnant women compared with non-pregnant ones, and in GDM women compared with pregnant controls. Production of peroxynitrite was higher in GDM women than in pregnant controls, who also had significantly reduced production compared with non-pregnant women. Basal levels of peroxidation of the platelet membranes evaluated either by hydroperoxide content and TBARS levels or the susceptibility to peroxidation were increased in GDM patients in comparison with both control groups. CONCLUSIONS: We have shown a modification in platelet NO and peroxynitrite production and an increase in platelet indicators of oxidative stress in GDM women compared with healthy pregnant women which might be at the basis of a cellular dysfunction.

Glycated low density lipoproteins modify platelet properties: a compositional and functional study.

The interaction between low density lipoproteins (LDL) and platelets might play a central role in the development of atherosclerosis in diabetes. The aim of the present study was to investigate whether the glycation of LDL is associated with modifications of their physico-chemical and functional properties and to study the action of glycated LDL (glycLDL) on platelets. LDL and platelets were isolated from 15 healthy subjects. The content of thiobarbituric acid-reactive substances and the generalized polarization of the fluorescent probe Laurdan were determined in LDL glycated in vitro. Platelets were incubated with native LDL, GlycLDL, and minimally oxidized LDL, and the following parameters were evaluated: platelet aggregation, nitric oxide production, intracellular Ca(2+) concentrations, Na(+)/K(+)-adenosine triphosphatase (Na(+)/K(+)-ATPase), and Ca(2+)-ATPase activities. GlycLDL showed increased thiobarbituric acid-reactive substance levels, a red shift of the Laurdan emission maximum, and a decrease in generalized polarization, indicating a higher polarity and a reduced molecular order compared with native LDL. GlycLDL caused a significant increase in platelet nitric oxide production, intracellular Ca(2+) concentration, and aggregating response to ADP; an inhibition of the platelet membrane Na(+)/K(+)-ATPase activity; and a stimulation of Ca(2+)-ATPase activity. Minimally oxidized LDL did not cause statistically significant changes in the parameters studied. The present work demonstrates that glycation induces compositional and structural changes in LDL and suggests that an altered interaction between glycLDL and platelets might play a role in the vascular complications of diabetes.

Sialic acid content in erythrocyte membranes from pregnant women affected by gestational diabetes.

Sialic acid (SA) content, membrane fluidity, and Na(+)/K(+)-adenosine triphosphatase (ATPase) activity were determined in erythrocyte membrane from 10 nonpregnant women (HNPW), 16 pregnant women affected by gestational diabetes mellitus (GDM), and 25 healthy pregnant women (HPW). In GDM patients the membrane erythrocyte SA content was significantly increased compared with HNPW and membrane fluidity was significantly increased in comparison with HPW. Erythrocyte membrane Na(+)/K(+)-ATPase activity was significantly reduced in GDM patients compared both to HNPW and to HPW subjects. A significant inverse correlation was found between 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) anisotropy and erythrocyte membrane SA content in HNPW and in HPW, while this significant correlation was not observed in GDM. The present results indicate that in comparison with normal pregnancy GDM is characterized by deep alterations of the erythrocyte plasma membrane physicochemical properties (increased fluidity) and functional activities (reduced Na(+)/K(+)-ATPase activity). These modifications might be at the basis of the altered blood viscosity and placental perfusion observed under such conditions. Moreover, these results show that in physiological pregnancy and in the nonpregnant state, the erythrocyte surface membrane fluidity is inversely correlated with SA content, while in GDM there is an unbalance of this relation, which might be associated with the microcirculatory abnormality present in this disease.

Reduced susceptibility to peroxidation of erythrocyte plasma membranes from centenarians.

The plasma membrane composition affects intracellular processes and the cellular susceptibility to free radical attack, which has been associated with the impairment of cellular functions occurring during senescence. The study of the modifications of the plasma membrane in centenarians might elucidate the biological mechanisms at the basis of longevity and successful aging. The work was performed in 190 subjects, divided into five groups according to the age range: (1) 21-40 years (n=25); (2) 41-60 years (n=30); (3) 61-80 years (n=30); (4) 81-99 years (n=50); and (5) centenarians (> or = 100 years) (n=55). The following determinations were performed on erythrocyte membranes: (i) the lipid peroxide level (Lp) evaluated as malondialdehyde content; (ii) susceptibility to in vitro oxidation evaluated as difference in the content of thiobarbituric acid-reactive substances before and after phenylhydrazine addition; (iii) unsaturated/saturated fatty acid ratio and individual polyunsaturated fatty acid composition measured by gas chromatography; and (iv) fluidity studied by means of the anisotropy of the probe 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH). Erythrocyte membranes from centenarians showed: (i) decreased basal lipid peroxide levels and reduced susceptibility to peroxidation in comparison with elderly subjects; (ii) increased unsaturated/saturated fatty acid ratio in comparison with every other age group; (iii) higher levels of eicosapentaenoic and docosahexaenoic acid and reduced content of linoleic and arachidonic acid in comparison with elderly subjects; and (iv) decreased anisotropy of TMA-DPH, i.e. higher fluidity compared with all the other age groups. In conclusion, the present work demonstrates that erythrocyte membranes from centenarians show some distinct features in comparison with elderly subjects that might act in a protective way against injuries.

Homocysteine-induced inhibition of nitric oxide production in platelets: a study on healthy and diabetic subjects.

AIMS/HYPOTHESIS: The molecular mechanisms involved in the platelet activation observed in hyperhomocysteinemia are not known. We aimed to discover if homocysteine concentrations are associated with abnormal platelet nitric oxide production in healthy and diabetic subjects. METHODS: The study cohort included 28 patients with Type I (insulin-dependent) diabetes mellitus, 30 patients with Type II (non-insulin-dependent) diabetes mellitus, and 34 healthy subjects. Homocysteine plasma concentrations were measured by high-performance liquid chromatography. Platelet nitric oxide production was measured using a nitric oxide meter before and after a 3-h incubation with 100 micromol/l homocysteine. Stimulation experiments were done in vitro by the addition of alpha-thrombin (0.2 U/ml). RESULTS: Basal platelet nitric oxide production was lower in diabetic patients than in healthy subjects. Nitric oxide release was reduced by in vitro homocysteine incubation, being lower in platelets from diabetic patients than in platelets from control subjects. Thrombin increased nitric oxide synthesis in platelets from healthy subjects both in the presence and absence of homocysteine. In diabetic subjects thrombin increased nitric oxide release in the absence of homocysteine. But in the presence of homocysteine the response was reduced. An inverse relation was found between plasma homocysteine levels and basal platelet nitric oxide release in diabetic and healthy subjects. CONCLUSION/INTERPRETATION: Homocysteine could exert its atherogenic action in healthy and diabetic subjects partly by inhibiting platelet nitric oxide production with the subsequent increased platelet activation and aggregation.

Increased susceptibility to peroxidation of VLDL from non-insulin-dependent diabetic patients: a possible correlation with fatty acid composition.

Recent studies suggested that both oxidized very low density lipoproteins (VLDL) and oxidized high density lipoproteins (HDL) might play a role in the pathogenesis of atherosclerosis. The aim of the present work was to analyse the susceptibility to in vitro peroxidation of VLDL and HDL from apparently normolipidemic subjects affected by insulin-dependent diabetes mellitus (IDDM) and non-insulin-dependent diabetes mellitus (NIDDM) in good metabolic control and to examine the possible relations between oxidisability and lipoprotein fatty acid composition. VLDL and HDL were isolated from 13 IDDM patients, 12 NIDDM patients and 18 healthy subjects. The degree of lipoprotein oxidation was determined by the measurement of hydroperoxide levels and thiobarbituric acid-reactive substances (TBARS) before and after in vitro peroxidative stress with CuSO4. Fatty acid analysis was performed by gas chromatography. VLDL and HDL from NIDDM patients showed a decrease in the saturated fatty acid content with a concomitant increase in unsaturated fatty acids and higher basal peroxide levels compared with healthy subjects. Oxidisability of VLDL from NIDDM subjects was higher than in controls and was significantly related with the unsaturated fatty acid content. The present work suggests that alterations in the composition and functions of both VLDL and HDL able to produce more atherogenic lipoproteins are present in NIDDM.

Tyrosine phosphorylation in type-1 diabetes by immunogold detection: an in vitro human aortic endothelial cell (HAEC) study in the presence of diabetic low density lipoproteins (LDL).

An immunomorphometric study of tyrosine phosphorylation was performed by the immunogold technique on cultured human aortic endothelial cells (HAEC) with a view to demonstrating their impaired signal transduction status, induced in vitro by incubation with low-density lipoproteins from the plasma of Type-1 diabetic patients. The results seem to sustain the hypothesis that extranuclear bioenergetic derangement induced by low-density lipoproteins from Type-1 diabetic patients may be associated with an up-regulation of the nuclear energetic machinery aimed at maintaining intracellular metabolic equilibrium. Our data demonstrate that phosphorylated tyrosine is a useful marker to monitor this metabolic condition.

Influence of low density lipoprotein from insulin-dependent diabetic patients on platelet functions.

In the present work we studied in vitro the action of low density lipoproteins (LDL) isolated from normolipemic insulin-dependent diabetic (IDDM) patients on transmembrane cation transport, nitric oxide synthase (NOS) activity, and aggregating response to stimuli of platelets from healthy subjects to elucidate whether the modified interaction between circulating lipoproteins and cells might be one of the pathogenetic mechanisms of the increased platelet activation in IDDM. LDL were obtained by discontinuous gradient ultracentrifugation from 15 IDDM out-patients and 15 sex- and age-matched healthy subjects and used for incubation experiments with control platelets. Lipid composition and hydroperoxide concentrations were studied in LDL. Platelet aggregation responses to ADP, NOS activity, cytosolic Ca2+ concentrations, and platelet membrane Na+/K+-adenosine triphosphatase (Na+/K+-ATPase) and Ca2+-ATPase activities were measured after incubation. IDDM LDL showed an increased lysophosphatidylcholine content compared with that of control LDL. IDDM LDL significantly increased the platelet aggregating response to ADP, cytosolic Ca2+ concentrations, and plasma membrane Ca2+-ATPase activity and significantly reduced NOS activity and platelet membrane Na+/K+-ATPase activity compared with those of platelets incubated in buffer or cells incubated with control LDL. The effects exerted by IDDM LDL on platelet suspensions from healthy subjects mimic the alterations observed in platelets from diabetic subjects in basal conditions. Both the decreased activity of NOS and the higher cytoplasmic concentrations of Ca2+ might cause increased platelet activation, as observed in IDDM. In conclusion, the present study suggests a new mechanism with a potential role in the early development of atherosclerosis in diabetic patients, i.e. an altered interaction between circulating lipoproteins and platelets.

Modifications induced by LDL from type 1 diabetic patients on endothelial cells obtained from human umbilical vein.

The aim of the present work was to analyze the effect of LDL obtained from type 1 diabetic patients in good metabolic control on human umbilical vein endothelial cells (HUVECs) after a short incubation period to detect possible atherogenic modifications of endothelial properties. Cultured HUVECs were incubated for 3 h with culture medium alone (control HUVEC), with native LDL from 12 healthy men (control LDL), or with native LDL from 12 type 1 diabetic men (type 1 LDL) (100 pg/ml). After the incubation, the following parameters were evaluated: nitric oxide synthase (NOS) activity, cytoplasmic Ca2+ levels, Na+-K+-ATPase activity, plasma membrane fluidity determined by means of 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), and plasma membrane conjugated diene (CD) content. The same experiments were repeated after bradykinin stimulation or in the presence of the antioxidant butylated hydroxytoluene (BHT), and nitric oxide (NO) production in intact HUVECs was also evaluated. HUVECs incubated with control LDL in comparison with control HUVECs showed a decreased fluidity of the membrane surface evaluated by TMA-DPH and a higher CD content. These alterations were prevented by the presence of BHT. HUVECs incubated with type 1 LDL in comparison with both control HUVECs and cells incubated with control LDL showed 1) increased NOS and Na+-K+-ATPase activity, cytoplasmic Ca2+ levels, and CD content, and 2) decreased fluidity of the membrane surface evaluated by TMA-DPH. These modifications were blunted--but not abolished--by the presence of BHT. After bradykinin stimulation either in the absence or in the presence of BHT, both cytoplasmic Ca2+ levels and NO production were increased in control HUVECs and in HUVECs incubated with control LDL, while a reduced response was observed in HUVECs incubated with type 1 LDL. The alterations observed in the endothelial function after the cell-LDL interaction might play a central role in the atherogenic process in diabetes.

Evidence for iNOS-dependent peroxynitrite production in diabetic platelets.

AIMS/HYPOTHESIS: The aim of the present study was twofold. Firstly, to determine whether diabetic platelets produce more peroxynitrite than normal platelets and secondly to correlate the peroxynitrite production with the intraplatelet induction of the inducible isoform of nitric oxide-synthase. METHODS: Intraplatelet peroxynitrite production was monitored with dichlorofluorescin acetate with a combination of confocal microscopy and steady-state fluorescence. The platelets were probed for the induction of the inducible-nitric oxide-synthase by western immunoblotting. RESULTS: In the presence of extracellular L-arginine (100 micromol/l), platelets from subjects with Type I (insulin-dependent) diabetes displayed about 5 times higher fluorescence than those from control subjects. To determine whether inducible-nitric oxide-synthase was the source of peroxynitrite, dichlorofluorescein production was quantified as a function of L-arginine as well as nitric oxide-synthase inhibitors, in platelets from control subjects, subjects with Type I diabetes and subjects with Type II (non-insulin-dependent) diabetes mellitus. Platelets from subjects with Type I yielded about sevenfold and those from Type II about threefold larger amounts of L-arginine/nitric oxide-synthase-dependent dichlorofluorescein fluorescence than those from control subjects. The platelets were then immunologically probed for inducible-nitric oxide-synthase, which has previously been implicated in peroxynitrite production and detected in megakaryocytes of subjects with coronary heart disease. Western immunoblots of intraplatelet proteins indicated that the inducible-nitric oxide-synthase was absent in control subjects. Platelets from both Type I and Type II diabetic subjects, however, contained inducible-nitric oxide-synthase. CONCLUSION/INTERPRETATION: Inducible-nitric oxide-synthase-derived peroxynitrite is a source of platelet damage in diabetes.