Possible involvement of oxidative stress in exercise-mediated platelet activation

In this study, we have attempted to verify whether a single bout of strenuous exercise performed by sedentary healthy individuals may interfere with the mechanisms controlling platelet sensitivity through exercise-related modifications of plasma oxidant/antioxidant equilibrium. Strenuous exercise resulted in an increased ADP- and collagen-evoked platelet aggregation associated with modified membrane fluidity and ion homeostasis. We also observed an enhanced plasma accumulation of secondary products of lipid peroxidation together with an increased susceptibility of low density lipoprotein (LDL) to in vitro oxidation and a decreased total plasma antioxidant potential. Notably, an acute elevation of nitrite/nitrate (NOx) amount was detected in plasma, whilst a decreased NOx content was measured in platelets. Findings of the current study suggest that oxidative stress induced by acute strenuous exertion may interfere with platelet responsiveness by promoting ox-LDL-mediated platelet activation and by decreasing platelet-derived nitric oxide bioactivity.     

Markers of activation of coagulation and fibrinolysis in patients with Cushing's syndrome

Patients with active Cushing's syndrome have an increased thrombotic tendency. We chose to reassess the mechanism underlying the thrombophilic state associated with this clinical condition using sensitive markers of coagulation and fibrinolysis activation in 17 patients with active disease. The results were compared with those obtained in 12 Cushing's patients successfully treated by surgery and in 20 normal individuals. The general pattern of results in patients with active disease was the finding of increased levels of von Willebrand factor (VWF: Ag), a marker of enhanced metabolic function of endothelial cells (VWF:Ag 181 +/- 42 vs 110 +/- 43, p<0.001 in normal subjects), accompanied by signs of heightened thrombin and plasmin generation, expressed by high levels of thrombin-antithrombin (TAT 5.59+/-3.6 vs 3.06+/-0.92 ng/ml in controls, p<0.01) and plasmin-antiplasmin complexes (PAP 407+/-176 vs 245+/-67 ng/ml in controls, p<0.01). VWF:Ag and TAT values were significantly higher in hypertensive than in normotensive patients with active disease (205+/-40 vs 155+/-26 U/dl, p<0.05 and 7.49+/-3.7 vs 3.45+/-1.8, p<0.01, respectively). Plasma levels of plasminogen activator inhibitor type 1 were higher, though not to a statistically significant extent, in patients with active disease compared to controls (12.8+/-12.3 vs 5.6+/-7.4 IU/ml, NS) and positively correlated with body mass index (r=0.66, p<0.01). After surgical control of Cushing's syndrome, there was a partial or complete reversal of the abnormalities to values similar to those found in normal individuals. Our data suggest that the thrombophilic state present in patients with active Cushing's syndrome is related to an enhanced metabolic function of endothelial cells; this in turn may be caused by an heightened production of thrombin with secondary hyperfibrinolysis. Primary prophylaxis with anticoagulants is recommended in these patients when they are exposed to a thrombophilic condition such as surgery.     

Hyperhomocysteinemia in patients with Cushing's syndrome

We evaluated serum homocysteine concentrations and the C677T polymorphism of the gene encoding for methylene tetrahydrofolate reductase, a key enzyme for homocysteine metabolism, in 57 patients with Cushing's syndrome, 41 with active disease, and 16 in remission after successful surgery and 105 blood donors. The patients with active Cushing's syndrome had significantly higher serum homocysteine levels and lower folate concentrations than either the patients in remission or controls. The presence of a statistically significant difference in homocysteine concentrations among the three groups was confirmed after adjustment for confounding variables. In a multiple regression model, homocysteine levels were significantly associated with midnight serum cortisol levels (beta = 0.33, P = 0.01), which is the most sensitive marker of endogenous hypercortisolism, and serum folate levels (beta = -0.32, P = 0.02). The distribution of methylene tetrahydrofolate reductase genotypes was not different between patients and controls. In conclusion, active hypercortisolism is associated with hyperhomocysteinemia and reduced serum folate concentrations, whereas the patients in remission have homocysteine concentrations comparable with healthy subjects. Low serum folate concentrations do not fully account for the increase in homocysteine levels that are positively correlated with cortisol levels. Hyperhomocysteinemia may be key to the prothrombotic state and increased cardiovascular risk of Cushing's syndrome.     

Effects of atorvastatin and rosuvastatin on thromboxane-dependent platelet activation and oxidative stress in hypercholesterolemia

ObjectivesWe examined the time-dependent effects of atorvastatin and rosuvastatin on in vivo oxidative stress and platelet activation, to assess whether these phenomena are related to any pleiotropic effect of any statin or to their LDL-lowering effect. We also asked whether the presence of specific allele frequencies in carriers of the 3′UTR/lectin-like oxidized LDL receptor-1 (LOX-1) polymorphism may influence the effect of either statin.MethodsWe included 60 hypercholesterolemic subjects, previously screened for LOX-1 3′UTR polymorphism, randomized, according to genetic profile (15 T and 15 C carriers for each arm), to atorvastatin 20 mg/day or rosuvastatin 10 mg/day.ResultsAfter 8 weeks, atorvastatin and rosuvastatin were associated with comparable, significant reductions in LDL cholesterol (40.8% and 43.6%, respectively), plasma hs-CRP (9.5% vs. 13.8%), urinary 11-dehydro-thromboxane (TX) B₂ (38.9% vs. 27.1%) and 8-iso-prostaglandin (PG) F_2α (39.4% vs. 19.4%). The impact of rosuvastatin or atorvastatin on CRP, 8-iso-PGF_2α, and 11-dehydro-TXB₂ did not differ according to the LOX-1 haplotype. On multiple regression analyses, only CRP and LDL were independent predictors of 11-dehydro-TXB₂, and only LDL was a significant predictor of 8-iso-PGF_2α.ConclusionsBoth atorvastatin and rosuvastatin cause comparable reductions of thromboxane-dependent platelet activation, lipid peroxidation and inflammation. The presence of 3′UTR/LOX-1 polymorphism does not affect the changes induced by either statin.     

Endothelial microparticles and platelet and leukocyte activation in patients with the metabolic syndrome

Accumulating evidence has shown a strong association between the metabolic syndrome (MS) and a chronic inflammatory state predisposing to atherosclerosis. We investigated leukocyte, platelet, and endothelial activation markers and cellular interactions in 33 patients with the MS and 25 healthy controls. Using flow cytometry, we measured: (1)P-selectin expression in platelets; (2) platelet microparticles identified by CD31 expression; (3) endothelial microparticles (EMPs) identified by expression of CD31 (EMP(31)), CD62E (EMP(62E)), and CD51 (EMP(51)); (4) conjugates of leukocytes with platelet microparticles/platelets and with EMPs identified by CD54 (EMP(54)); and (5) CD11b expression in leukocytes. Patients with the MS had markedly elevated EMP(31), although EMP(62E) levels were normal, suggesting that EMP(31) levels were increased because of endothelial cell apoptosis, rather than activation. EMP(51), EMP(54)-lymphocyte conjugates, platelet expression of P-selectin, CD11b expression in leukocytes, and platelet-lymphocyte conjugates were also increased in patients with the MS. Platelet-leukocyte conjugates correlated with leukocyte activation, suggesting that platelet binding to leukocytes regulates leukocyte activation in vivo. In conclusion, our data demonstrate endothelial cell microparticle release, platelet and leukocyte activation, and increased binding of EMPs and platelets to leukocytes in patients with the MS.     

Adipokine levels in Cushing's syndrome; elevated resistin levels in female patients with Cushing's syndrome

BACKGROUND: Cushing's syndrome (CS) is associated with central adiposity, insulin resistance and impaired glucose homeostasis. Adipose tissue is thought to regulates glucose homeostasis via circulating adipokines, such as resistin, leptin and adiponectin, although their role in the insulin resistance associated with CS has not been established. DESIGN: We examined the relationship between insulin resistance and adipokine levels in CS patients. We compared plasma levels of resistin, leptin and adiponectin in 10 women and four men patients with CS, with 14 health subjects matched for age, gender and body mass index. A subgroup of three women and four men with pituitary-dependent CS were re-examined at least 9 months after curative surgery. RESULTS: CS patients had significantly more truncal fat and less lean body mass as assessed by DEXA compared to control subjects. Total cholesterol, triglycerides and insulin resistance, as calculated using the homeostasis model assessment of insulin resistance (HOMA-R), was significantly increased in CS patients. Of the adipokines measured, only resistin was significantly different between female CS patients and female control subjects (5.05 +/- 0.56 vs. 2.91 +/- 0.39 micro g/l, P = 0.015). Curative surgery significantly reduced total body fat and truncal fat, leptin, total and low-density lipoprotein (LDL) cholesterol, glucose and HOMA-R. A reduction in both resistin and adiponectin was also observed but the differences between pre- and post-treatment levels did not achieve statistical significance. CONCLUSION: Here we report for the first time that resistin levels are significantly elevated in CS patients and may be important in the insulin resistance associated with glucocorticoid excess.     

Glucose fluctuations and activation of oxidative stress in patients with type 1 diabetes

AIMS/HYPOTHESIS: Glucose fluctuations may help predict diabetic complications. We evaluated the relation between glucose variability and oxidative stress in patients with type 1 diabetes. METHODS: Continuous glucose monitors were inserted subcutaneously in 25 patients. During the measurement, patients collected two 24 h urine samples, while 24 healthy controls collected one 24 h urine sample for determination of 15(S)-8-iso-prostaglandin [Formula: see text] using HPLC tandem mass spectrometry. Mean of the daily differences (MODD), mean amplitude of glycaemic excursions (MAGE) and continuous overlapping net glycaemic action calculated with n hour time-intervals (CONGA-n) were calculated as markers for glucose variability and correlation with [Formula: see text] excretion was calculated. RESULTS: Median [interquartile range (IQR)] urinary [Formula: see text] was higher in patients than healthy controls: 161 (140-217) pg/mg creatinine vs 118 (101-146) pg/mg creatinine (p = 0.001). Median (IQR) MODD was 3.7 (3.2-5.0) mmol/l, MAGE 7.6 (6.4-9.0) mmol/l and CONGA-1 2.3 (2.1-2.8) mmol/l. Univariate regression did not reveal an association for MODD (r (2) = 0.01), MAGE (0.08) or CONGA-1 (0.07) with [Formula: see text] excretion, nor was an association revealed when corrected for HbA(1c), age, sex and smoking. Spearman correlation coefficients (r) between [Formula: see text] excretion and MODD, MAGE and CONGA-1 were non-significant: -0.112, -0.381 and -0.177. CONCLUSIONS/INTERPRETATION: We report that there is no relationship between glucose variability and urinary [Formula: see text]. We also confirm that patients with type 1 diabetes have higher levels of urinary [Formula: see text] than healthy controls, suggesting that in addition to glucose variability, other factors favouring oxidative stress may exist. We did not see a relation between high glucose variability and elevated levels of oxidative stress in patients with type 1 diabetes.     

Enhanced prolactin responsiveness to galanin in patients with Cushing's disease

OBJECTIVE Galanin is believed to play a role in the control of prolactin (PRL) secretion in the rat. Such a role is uncertain in humans where the neuropeptide is expressed by the corticotrophs. However, in clinical conditions of enhanced ACTH secretion, increased PRL levels are often observed. Therefore, we evaluated the effect of galanin infusion on serum PRL levels in patients with Cushing's disease and in control subjects. For comparison, the PRL responses to TRH and metoclopramide were also investigated in the same patients. DESIGN Four tests were performed: (a) 40-minute infusion of 0–3 μg/kg/min of galanin; (b) infusion of normal saline only; (c) metoclopramide test (10 mg as i.v. bolus); (d) TRH test (200 mg as i.v. bolus). PATIENTS Twenty-four normal subjects and nine patients suffering from active Cushing's disease were investigated. MEASUREMENTS Serum concentrations of PRL were measured by radioimmunoassay on blood samples collected before and for 90 minutes after drug or saline administration. RESULTS Serum baseline PRL levels were superimposable in normal subjects and in patients with Cushing's disease. In normal subjects, infusion of galanin induced a distinct PRL increase compared to saline (mean ±SEM incremental areas 6514 ±2572 vs 540 ±571 μ/l/90 min, P= 005, respectively). In patients with Cushing's disease, galanin evoked a remarkable PRL rise with hormone levels which were significantly greater (P < 0.001) than those observed in the same patients after infusion of saline (21908 ± 4180 vs 534 ± 1556 mU/l/90 min) or after galanin administration in controls (P<0.01). The PRL response to TRH and, much more so, to metoclopramide was significantly lower in patients with Cushing's disease than in normal subjects (42125± 8000 vs 73181 ± 7246 mU/l/90 min, P<0.01 after TRH and 79095 ± 27265 vs 229049 ± 10602 mU/l/90 min, P < 001 after metoclopramide). CONCLUSIONS Galanin appears to be a specific, though weak, PRL secretagogue in normal subjects. The galanin-induced PRL release was significantly increased in patients with Cushing's disease. A number of hypothetical mechanisms may underlie the enhanced PRL reactivity to galanin in Cushing's disease. This finding together with the impaired PRL responsiveness to TRH and metoclopramide, also observed in this study, is a further example of a dysregulation of PRL secretion in patients with Cushing's disease.     

Thyroid axis in patients with Cushing's syndrome.

Thyroid hormone values and serum thyrotropin (thyroid-stimulating hormone [TSH]) responses to the intravenous administration of 400 micrograms of protirelin were determined in ten patients with Cushing's syndrome and in ten matched normal subjects. In patients with Cushing's syndrome, the serum thyroxine (T4) level was mildly depressed and free T4 level was normal. The mean (+/- SD) concentrations of serum triiodothyronine (T3) and free T3 were both reduced in patients compared with normal subjects (P less than .001). At 20 and 60 minutes after protirelin administration, serum TSH levels were, respectively, 3.3 +/- 2.7 microU/mL and 2.6 +/- 2.3 microU/mL in patients with Cushing's syndrome and 12.3 +/- 5.4 microU/mL and 10.7 +/- 5.4 microU/mL in normal subjects (P less than .001). The reduced serum T3 and free T3 levels are due to a glucocorticoid suppressive effect on the peripheral conversion of T4 to T3. The protirelin test is of limited value in assessing the thyroid status because the response of TSH is frequently blunted or absent due to glucocorticoid excess.     

Glucose metabolism in patients with subclinical Cushing's syndrome

This clinical review will summarize the available data regarding the effect of either physiological or increased glucocorticoid concentrations on glucose metabolism and insulin-sensitivity, in order to clarify the role, if any, of subclinical Cushing's syndrome (SCS), a status of altered hypothalamic-pituitary-adrenal axis secretion in the absence of the classical signs or symptoms of overt cortisol excess, in patients with adrenal incidentalomas (AI) and diabetes mellitus type 2. Focusing on patients with SCS associated to AI, while there is convincing evidence in the literature that even a mild hyper cortisolemia is associated with alterations of glucose metabolism, evidence is insufficient to conclude that the simple correction of chronic, even mild, hypercortisolism can completely revert metabolic, mainly glycemic alterations. At the same time, considering the variability of the prevalence of Cushing's syndrome in patients with diabetes mellitus type 2 reported in the literature, no agreement does exist whether screening for CS can be useful and recommended in those patients.     

Role for laparoscopic adrenalectomy in patients with Cushing's syndrome

Laparoscopic adrenalectomy is one of the most clinically important advances in the past 2 decades for the treatment of adrenal disorders. When compared to open adrenalectomy, laparoscopic adrenalectomy is equally safe, effective, and curative; it is more successful in shortening hospitalization and convalescence and has less long-term morbidity. The laparoscopic approach to the adrenal is the procedure of choice for the surgical management of cortisol-producing adenomas and for patients with corticotropin (ACTH) dependent Cushing's syndrome for whom surgery failed to remove the source of ACTH. The keys to successful laparoscopic adrenalectomy are appropriate patient selection, knowledge of anatomy, delicate tissue handling, meticulous hemostasis, and experience with the technique of laparoscopic adrenalectomy.     

production rates of testosterone in patients with Cushing's syndrome

Testosterone production rates were determined in 16 patients with Cushing's syndrome (4 men and 12 women) using the stable-isotope dilution technique and mass spectrometry. 1alpha,2alpha-D-Testosterone was infused for 10 hours at a dose of 20 microg/h (men) and 0.4 microg/h (women) and blood samples were obtained at 20-minute intervals during the last 4 hours of the observation period. Estimated production rates in men with Cushing's syndrome were 27, 73, 150, and 180 microg/h (mean, 106 +/- 70 microg/h; healthy men [n = 12], 210 +/- 70 microg/h). In the 12 women with Cushing's syndrome, testosterone production rates were 0.3 to 22.3 microg/h (healthy women [n = 5], 4.3 +/- 1.9 microg/h). There was no difference in testosterone production rates in female patients with central (n = 8) versus adrenal (n = 4) Cushing's syndrome. In summary, testosterone production rates are subnormal or low-normal in male patients with endogenous hypercortisolism, but not in female patients with the same disorder. We conclude that testosterone production in men, but not in women, is predominantly of gonadal origin and hence susceptible to a glucocorticoid-induced suppression of gonadotropin secretion.     

Reduced platelet activation and platelet aggregation in patients with alcoholic liver cirrhosis

Results from previous studies regarding platelet function in liver cirrhosis are discordant. The aim was to investigate platelet activation and platelet aggregation in patients with alcoholic liver cirrhosis. We included 27 patients with alcoholic liver cirrhosis and 22 healthy individuals. A recently established flow cytometric approach was used to measure platelet activation and platelet aggregation independent of sample platelet count. Platelet aggregation was further investigated using light transmission aggregometry (LTA) (for platelet count >100 × 10⁹/L). Platelet agonists were adenosine diphosphate, thrombin receptor-activating peptide, arachidonic acid, collagen, and collagen-related peptide. Patients had lower median platelet count than healthy individuals, 125 × 10⁹/L (interquartile range [IQR] 90?185) versus 240 × 10⁹ (IQR 204?285), p < 0.001. Platelet activation levels in stimulated samples were lower in patients versus healthy individuals, e.g., after collagen-related peptide stimulation, the median percentage of platelets positive for activated glycoprotein IIb/IIIa was 85% (IQR 70–94) in patients versus 97% (IQR 94–99) in healthy individuals, p < 0.001; lower platelet activation capacity being associated with low platelet count and Child–Pugh class B/C cirrhosis. Flow cytometric platelet aggregation was reduced in patients for collagen-related peptide and for adenosine diphosphate, e.g., platelet aggregation (mean ± standard deviation) was 57% ± 4 in patients versus 70% ± 1 in healthy individuals for collagen-related peptide, p = 0.01. Light LTA showed reduced collagen-induced platelet aggregation in some patients compared with healthy individuals. In conclusion, platelet function was reduced in some patients with alcoholic liver cirrhosis and the severity was associated with platelet count and severity of liver cirrhosis.