Stefano Angelo Santini

Defective Plasma Antioxidant Defenses and Enhanced Susceptibility to Lipid Peroxidation in Uncomplicated IDDM

Oxidative stress is postulated to be increased in patients with IDDM. Accumulating evidence suggests that oxidative cell injury caused by free radicals contributes to the development of IDDM complications. On the other side, a decreased efficiency of antioxidant defenses (both enzymatic and nonenzymatic) seems to correlate with the severity of pathological tissue changes in IDDM. Thus, we determined plasma antioxidant defenses, measuring the total radical-trapping antioxidant capacity (TRAP) and the two markers of oxidative stress, lipid hydroperoxides (ROOHs) and conjugated dienes, in 72 patients with well-controlled IDDM and without evident complications, compared with 45 nondiabetic subjects. Compared with control subjects, IDDM patients showed significantly reduced plasma TRAP (669 ±131 vs. 955 ± 104 μmol/1, P < 0.001) and significantly increased levels of ROOHs (7.13 ± 2.11 vs. 2.10 ± 0.71 μmol/1, P < 0.001) and conjugated dienes (0.0368 ± 0.0027 vs. 0.0328 ± 0.0023 arbitrary units [AU], P < 0.01), especially in the trans-trans conformation (0.0340 ± 0.0028 vs. 0.0259 ± 0.0022 AU, P < 0.001), with a concurrent reduction of conjugated dienes in the cis-trans conformation (0.0028 ± 0.0011 vs. 0.0069 ± 0.0012 AU, P < 0.001). The oxidative parameters studied did not appear to be correlated with metabolic control (HbA1c levels) and lipid profile (cholesterol or triglyceride levels). The reduced TRAP and the increased ROOH and conjugated diene plasma levels, together with the decreased ratio of cis-trans/trans-trans conjugated dienes, which reflects an altered redox status of plasma, indicate that in IDDM patients, oxidative stress is enhanced and antioxidant defenses are defective, regardless of diabetes duration, metabolic control, or presence of complications.

Subclinical Hypercortisolism among Outpatients Referred for Osteoporosis

Context The Cushing syndrome is a well-recognized secondary cause of osteoporosis. Contributions The researchers looked for hypercortisolism in asymptomatic patients referred for osteoporosis testing. They identified 7 patients with the condition. Six had functioning adrenal masses and 1 had an adrenocorticotropic hormonesecreting pituitary adenoma. The prevalence of subclinical hypercortisolism among patients with T-scores of 2.5 or less and vertebral fractures was 10.8%. Caution The findings come from a referral setting and might not apply to patients in the community. Implication Subclinical hypercortisolism may be more common than is generally recognized in patients with osteoporosis. The Editors Hypercortisolism is a frequent cause of secondary osteoporosis (1). Overt endogenous hypercortisolism (Cushing syndrome) is a well-recognized cause of osteoporosis (2), but because its prevalence in the general population is low (1 per 500000 persons) (2), its contribution to osteoporosis in general populations is trivial. The terms subclinical Cushing syndrome and subclinical hypercortisolism describe altered adrenocorticotropic hormone (ACTH)cortisol homeostasis without the classic signs or symptoms of the Cushing syndrome (3). Subclinical hypercortisolism is more common than overt hypercortisolism, with an estimated prevalence of about 0.8 per 1000 individuals in the general population (3); however, this prevalence is probably underreported because of the lack of symptoms or signs in these patients (37). Several cross-sectional and longitudinal studies have suggested that these patients are at high risk for complications of hypercortisolism, such as diabetes and osteoporosis (816). Recent studies have indicated that subclinical hypercortisolism is more prevalent than previously thought in patients with type 2 diabetes (1719). However, studies on the prevalence of subclinical hypercortisolism in patients with osteoporosis are lacking. Some evidence suggests that osteoporotic fractures may be the presenting manifestations of otherwise-asymptomatic hypercortisolism (20). Moreover, a recent paper showed a difference in cortisol secretion between healthy participants and patients with established osteoporosis, possibly due to mild autonomous cortisol hypersecretion in some individuals (21). Thus, the prevalence of subclinical hypercortisolism in patients with osteoporosis may be underestimated. We therefore designed a study to assess the prevalence of subclinical hypercortisolism in patients referred to our outpatient clinics for evaluation of osteoporosis. Methods Setting and Participants The study was done at the Casa Sollievo della Sofferenza Scientific Institute, San Giovanni Rotondo, Foggia, Italy, and the San Giuseppe-Fatebenefratelli Hospital, Fatebenefratelli Research Association, Milan, Italy, from January 2005 to December 2005. We recruited 219 consecutive patients (200 women and 19 men) referred to our outpatient clinics for prevention or diagnosis and treatment of osteoporosis and who met the following inclusion criteria: 1) absence of any known secondary causes of osteoporosis (that is, past or current thyrotoxicosis, bowel disease, precocious or surgical menopause, chronic renal failure, chronic hepatic disease, eating disorders, or rheumatologic or hematologic disease); 2) absence of depression and alcoholism, which may enhance cortisol secretion; 3) no administration of drugs influencing bone, cortisol, and dexamethasone metabolism or cortisol secretion; and 4) no signs or symptoms of cortisol excess, including moon facies, striae rubrae, skin atrophy, or buffalo hump. All participants signed consent forms, and local ethical committees approved the study in accordance with the second Declaration of Helsinki. Testing Sequence The Figure shows the study flow diagram. All patients had spinal and femoral dual-energy x-ray absorptiometry and spinal radiography. They had outpatient testing for secondary causes of osteoporosis (general chemistry profile, calcium homeostasis measurements [serum calcium, phosphorus, alkaline phosphatase total activity, 24-hour urinary calcium], thyroid-stimulating hormone, antigliadin antibodies, and serum testosterone in men) and blood for cortisol measurement drawn at 8:00 a.m. after a 1-mg overnight dexamethasone suppression test. Participants with altered thyroid-stimulating hormone levels were tested for free thyroxine, antithyroglobulin, and antithyroperoxidase antibodies; those with high serum calcium levels were tested for serum parathyroid hormone. In patients with normal antigliadin antibodies but clinical suspicion of celiac disease, antiendomysial antibodies were also measured. Figure. Study flow diagram. All patients were subdivided on the basis of bone mineral density (BMD) (T-score of 2.5 or less [low BMD] or greater than 2.5 [normal BMD]) and vertebral fractures. ACTH = adrenocorticotropic hormone; Fx+ = presence of vertebral fractures; Fx = absence of vertebral fractures. Participants with serum cortisol levels greater than 50.0 nmol/L after the 1-mg overnight dexamethasone suppression test were hospitalized for further diagnostic investigations (case participants). Those with cortisol levels less than 50.0 nmol/L had no further evaluation, but antiosteoporotic therapy was started in those with osteoporosis. Among hospitalized patients, catheters were inserted in the forearm vein on the day of admission, and blood testing began the day after to avoid stress-related hypopituitaryadrenal axis activation due to venipuncture. Because inpatient status can in theory increase cortisol secretion (19), a control group of inpatients was recruited to estimate the prevalence of subclinical hypercortisolism in hospitalized participants (control participants). This group comprised 56 age- and sex-matched inpatients without diabetes, osteoporosis, or vertebral fractures who were consecutively hospitalized from January 2005 to December 2005. All hospitalized participants had serum cortisol levels measured at 9:00 a.m. after 2 days of low-dose (0.5 mg every 6 hours) dexamethasone suppression and at midnight, 2 measurements of 24-hour urinary free cortisol, and measurement of ACTH at 8:00 a.m. Subclinical hypercortisolism was diagnosed if participants had incomplete suppression of cortisol (>50.0 nmol/L) after the low-dose dexamethasone suppression test and a 24-hour urinary free cortisol level greater than 165.6 nmol/d (normal range, 22.2 to 165.6 nmol/d) and/or midnight cortisol level greater than 207 nmol/L (normal range, 0.0 to 138.5 nmol/L) (3, 7, 8, 2123). The cutoff value of 165.6 nmol/d for urinary free cortisol corresponds to the 97th percentile value of 70 healthy control participants (20 men and 50 women; age, 35 to 65 years; body mass index, 20 to 40 kg/m2) who were recruited in our center as a reference population for urinary free cortisol. The cutoff value of 207.0 nmol/L for midnight cortisol is the standard for diagnosing hypercortisolism when overt Cushing syndrome is clinically suspected (2). Terzolo and colleagues (24) proposed a cutoff value of 148.8 nmol/L for diagnosing subclinical hypercortisolism, but we used the greater value because we lack reference midnight cortisol values in our center and wanted to increase specificity. Participants with subclinical hypercortisolism and an ACTH level of 2.2 pmol/L or less (normal range, 1.1 to 11.0 pmol/L) had abdominal computed tomography. Patients with subclinical hypercortisolism and ACTH levels greater than 2.2 pmol/L had abdominal computed tomography, nuclear magnetic resonance of the pituitary region, and additional biochemical tests (serum cortisol measurement after 8-mg overnight dexamethasone suppression and serum ACTH and cortisol measurement after stimulation with corticotropin-releasing hormone). Whole-body computed tomography was done when an ectopic source of ACTH hypersecretion was suspected (25). Subclinical hypercortisolism in patients with type 2 diabetes can be attributed mainly to adrenal masses (19). Because incidentally discovered adrenal lesions (adrenal incidentalomas) are frequently found in otherwise-healthy persons (4), we performed abdominal computed tomography in a subset of patients who tested positive after the 1-mg overnight dexamethasone suppression test but were classified as having no subclinical hypercortisolism. Testing Procedures In all patients, bone mineral density (BMD) was measured by dual-energy x-ray absorptiometry (Hologic Discovery, Bedford, Massachusetts) at the spine (in vivo precision at L1 to L4, 1.0%) and total and femoral neck (in vivo precision, 1.8% and 2.3%, respectively). Individual BMD values were expressed as SD units (T-scores) relative to the reference population of our center, which included 382 healthy female participants (26). Conventional spinal radiographs in lateral (T4 to L4) and anteroposterior (L1 to L4) projections were obtained in all participants by using a standard technique. Two trained radiologists who were blinded to BMD and hormonal data independently reviewed the radiographs. Vertebral fractures were diagnosed on visual inspection by using the semiquantitative method described by Genant and colleagues (27), in which fractures assessed on lateral thoracolumbar spine radiographs were defined as a reduction of more than approximately 20% in anterior, middle, or posterior vertebral height. Fractures were graded by severity and were graded as I, II, or III on the basis of the height reduction (20% to 25%, 25% to 40%, or >40%, respectively). The radiologists discussed questionable cases for consensus on a diagnosis; the interrater reliability between the 2 radiologists was good (= 0.85). The 2-day, low-dose dexamethasone suppression test was done after ACTH, 24-hour urinary free cortisol, and midnight cortisol levels were measured. Every 6 hours, 0.5 mg of dexamethasone was administrated orally, and serum cortisol was measured at 9:00 a.m., 4

Chronic taurine supplementation ameliorates oxidative stress and Na+K+ATPase impairment in the retina of diabetic rats

Summary. This study evaluates the effect of 4 months supplementation with 2% and 5% taurine (w/w) on the retina of diabetic rats. In non-diabetic rats, taurine does not modify glycemia, body weight, retinal conjugated dienes (CD), lipid hydroperoxide (LP), and Na+K+ATPase activity. In diabetic rat, at 2, 4, 8, 16 weeks following the onset of diabetes, retinal CD and LP are significantly and progressively increased, while pump activity is gradually and significantly reduced. In taurine supplemented diabetic rats, glycemia is not affected but lipid peroxidation is significantly decreased. Finally, taurine preserves ATPase activity being 5% more effective than 2% taurine. We conclude that taurine supplementation ameliorates biochemical retinal abnormalities caused by diabetes, thereby suggesting that taurine may have a role in the prevention of retinal changes in diabetes.

Paradoxical inhibition of cardiac lipid peroxidation in cancer patients treated with doxorubicin. Pharmacologic and molecular reappraisal of anthracycline cardiotoxicity.

Anticancer therapy with doxorubicin (DOX) and other quinone anthracyclines is limited by severe cardiotoxicity, reportedly because semiquinone metabolites delocalize Fe(II) from ferritin and generate hydrogen peroxide, thereby promoting hydroxyl radical formation and lipid peroxidation. Cardioprotective interventions with antioxidants or chelators have nevertheless produced conflicting results. To investigate the role and mechanism(s) of cardiac lipid peroxidation in a clinical setting, we measured lipid conjugated dienes (CD) and hydroperoxides in blood plasma samples from the coronary sinus and femoral artery of nine cancer patients undergoing intravenous treatments with DOX. Before treatment, CD were unexpectedly higher in coronary sinus than in femoral artery (342 +/- 131 vs 112 +/- 44 nmol/ml, mean +/- SD; P < 0.01), showing that cardiac tissues were spontaneously involved in lipid peroxidation. This was not observed in ten patients undergoing cardiac catheterization for the diagnosis of arrhythmias or valvular dysfunctions, indicating that myocardial lipid peroxidation was specifically increased by the presence of cancer. The infusion of a standard dose of 60 mg DOX/m(2) rapidly ( approximately 5 min) abolished the difference in CD levels between coronary sinus and femoral artery (134 +/- 95 vs 112 +/- 37 nmol/ml); moreover, dose fractionation studies showed that cardiac release of CD and hydroperoxides decreased by approximately 80% in response to the infusion of as little as 13 mg DOX/m(2). Thus, DOX appeared to inhibit cardiac lipid peroxidation in a rather potent manner. Corollary in vitro experiments were performed using myocardial biopsies from patients undergoing aortocoronary bypass grafting. These experiments suggested that the spontaneous exacerbation of lipid peroxidation probably involved preexisting Fe(II) complexes, which could not be sequestered adequately by cardiac isoferritins and became redox inactive when hydrogen peroxide was included to simulate DOX metabolism and hydroxyl radical formation. Collectively, these in vitro and in vivo studies provide novel evidence for a possible inhibition of cardiac lipid peroxidation in DOX-treated patients. Other processes might therefore contribute to the cardiotoxicity of DOX.

Large, sustained cardiac lipid peroxidation and reduced antioxidant capacity in the coronary circulation after brief episodes of myocardial ischemia.

OBJECTIVES We sought to investigate whether a brief episode of myocardial ischemia produces a detectable cardiac oxidative stress in patients undergoing elective coronary angioplasty (PTCA). BACKGROUND Although cardiac oxidative stress has been clearly demonstrated in experimental models of ischemia-reperfusion, its presence in patients after transient myocardial ischemia is still unclear. METHODS In order to evaluate oxidative stress in ischemic cardiac regions, plasma conjugated dienes (CD), lipid hydroperoxides (ROOHs) and total antioxidant capacity (TRAP), independent indexes of oxidative stress, were measured in the aorta and great cardiac vein (GCV) before (t0), 1, (t1), 5 (t5) and 15 min (t15) after first balloon inflation in 15 patients undergoing PTCA on left anterior descending coronary artery (Group 1); six patients with right coronary artery stenosis (Group 2), which is not drained by the GCV, were studied as controls. RESULTS In Group 1 at baseline, CD and ROOHs levels were higher in GCV than in aorta (p < 0.01 for both), and TRAP levels were lower (p < 0.01). Aortic levels of CD, ROOHs and TRAP did not change at any time after to; venous levels of CD and ROOHs levels markedly increased at t1, at t5 and remained elevated at t15 (p < 0.01 for all comparisons vs. to); venous levels of TRAP decreased at t1 and t5 (p < 0.01 vs. t0) and returned to normal at t15. In Group 2, CD, ROOHs and TRAP levels were similar in the aorta and GCV and did not change throughout the study. CONCLUSIONS Short episodes of myocardial ischemia during PTCA induce a sustained oxidative stress, which is detectable in the venous effluent of reperfused myocardium.

Potential Therapeutic Effect of Antioxidants in Experimental Diabetic Retina: A Comparison between Chronic Taurine and Vitamin E Plus Selenium Supplementations

Although good glycaemic control can delay the development and progression of diabetic retinopathy, new therapies are needed to obtain a better control of this diabetic complication. Oxidative stress seems to be a contributing factor in diabetic retinal alterations, therefore, it has been suggested that antioxidants may be beneficial in reducing diabetic retinal changes. However, many questions are still open. In fact, it remains to be ascertained which antioxidants are the most active when they are chronically administered in vivo and their effective dosages. Therefore, we compared the effect of chronic taurine supplementations versus a mixture of vitamin E+selenium on biochemical retinal changes induced by diabetes at different stages of the disease. Briefly, streptozotocin (STZ) diabetic rats ware administered for 4 months following the dietary supplements: (a) 2% (w/w) taurine; (b) 5% (w/w) taurine; (c) 200 IU vitamin E+8 mg selenium/kg diet (d) 500 IU vitamin E+8 mg selenium/kg diet. In STZ diabetic rat in poor metabolic control (i.e. serum glucose >16.5 mmol/l), at 2, 4, 8, 16 weeks following the onset of diabetes, retinal conjugated dienes (CD) and lipid hydroperoxides (LP) were significantly and progressively increased, while sodium pump activity was gradually and significantly reduced. In taurine and vitamin E+selenium supplemented diabetic rats, glycaemia and body weight were not significantly different from those of non-supplemented diabetic animals. In diabetic rats, 2 and 5% taurine significantly decreased CD. This reduction is long lasting. Regarding CD, both vitamin E+selenium supplementations reduced CD only during the first 4 weeks of diabetes. Two percent taurine supplementation significantly lowered LP for the first 8 weeks of the disease while 5% taurine-induced-reduction lasted for the whole experimental time. A 200 IU vitamin E+8 mg selenium supplementation did not significantly modify LP, while 500 IU vitamin E+8 mg selenium significantly lowered them for the whole studied period. Finally, taurine preserved ATPase activity being more effective at 5% than 2%. Two hundred IU vitamin E+8 mg selenium did not generally modify pump activity, while 500 IU vitamin E+8 mg selenium partially prevented the decrease in pump activity. We conclude that taurine and vitamin E+selenium supplementations ameliorate biochemical retinal abnormalities caused by diabetes. These effects are dose- and time-dependent. Moreover, the effect of taurine on CD is longer lasting than that of vitamin E+selenium. In addition, taurine seems to better preserve ATPase activity in comparison with vitamin E+selenium. Finally, in diabetic animals a negative correlation is found between CD and LP on one side and Na + K + ATPase activity on the other; thus, lipid peroxidation and pump activity seem to be associated. The same inverse correlations are present in vitamin E+selenium supplemented diabetic rats, but are lost in taurine supplemented animals. Therefore, taurine effects may not be simply mediated by its antioxidant activity. Thus, chronical (4 months) taurine and vitamin E+selenium supplementations reduce biochemical retinal alterations in diabetic rat in poor metabolic control.

Role of cytochrome P4502D6 functional polymorphisms in the efficacy of donepezil in patients with Alzheimer's disease.

Objective Cytochrome P450 (CYP) 2D6 enzyme is the major responsible for the metabolism of donepezil, an inhibitor of acetyl cholinesterase currently used for the symptomatic treatment of mild-to-moderate Alzheimers disease (AD). Functional polymorphisms in the CYP2D6 gene may affect enzyme activity and thus, the metabolism of donepezil. The aim of this study was to evaluate the effect of 16 functional polymorphisms in the CYP2D6 gene on the clinical response to donepezil treatment in patients with mild-to-moderate AD. Methods In this multicenter prospective cohort study we evaluated 57 unrelated Caucasians clinically diagnosed as AD according to the National Institute of Neurological and Communicative Disorders and Stroke-Alzheimers Disease and Related Disorders Association Work Group criteria. Patients were treated with donepezil (5–10 mg/daily) for 6 months. The response to donepezil treatment was evaluated at 6-month follow-up according to the National Institute for Health and Clinical Excellence requirements. The identification of 16 clinically relevant CYP2D6 gene variants was performed by a high-throughput genetic analysis. Results Thirty-eight of 57 patients (67%) were responders and 19 patients (33%) were nonresponders to donepezil treatment. A significantly higher frequency of gene variants conferring decreased or absent enzyme activity was observed in responder than in nonresponder patients (73.68% vs. 36.84%; P=0.005). The presence of gene variants conferring decreased or absent activity of the CYP2D6 enzyme was significantly associated with a clinical response to donepezil treatment (odds ratio=6.286; 95% confidence interval=1.828–21.667). Conclusions Functional polymorphisms in the CYP2D6 gene can influence the clinical efficacy of donepezil. The analysis of CYP2D6 genotypes may be useful in identifying subgroups of AD patients with different clinical response to donepezil treatment.

Long-term taurine supplementation reduces mortality rate in streptozotocin-induced diabetic rats

Summary.Oxidative stress is implicated in the pathogenesis of diabetes mellitus. Taurine and vitamin E+selenium supplementation has some benefits in experimental models of diabetes mellitus. This study evaluates whether taurine and vitamin E+selenium supplementations reduce a hard end-point such as mortality due to diabetes. Streptozotocin-induced diabetic rats were fed with standard diet or taurine (5%, w/w) or vitamin E (500 UI/Kg)+selenium (8 mg/Kg) enriched diets. Taurine significantly decreased mortality rate (p < 0.04), while vitamin E failed to increase survival. In the late phase of the disease, taurine significantly decreased glycaemia, being vitamin E ineffective. No correlation between glycaemia and survival was found. None of supplementations modified body weight. Thus, only taurine decreases the mortality rate and glycaemia. These results encourage new research in the field, since classical hypoglycaemic agents are unable to decrease mortality in diabetic patients.

Role of asymmetric-dimethyl-L-arginine (ADMA) and nitrite/nitrate (NOx) in the pathogenesis of oxidative stress in female subjects with uncomplicated type 1 diabetes mellitus.

AIMS To explore the role of asymmetric-dimethyl-L-arginine (ADMA), an endogenous nitric oxide synthetases (NOS) inhibitor, and nitrite/nitrate (NOx) in the pathogenesis of oxidative stress in early stages of type 1 diabetes mellitus. METHODS We measured in 99 female subjects with uncomplicated type 1 diabetes (duration disease <10 years) and in 44 sex-matched controls (comparable for age, smoking habit, diet and physical activity) plasma levels of NOx, glycosylated haemoglobin (HbA1c), glucose, uric acid, total cholesterol, high density lipoprotein (HDL) cholesterol, triglycerides and serum ADMA. RESULTS Type 1 diabetic subjects have higher levels of glycemia, HbA1c, LDL cholesterol and NOx, but lower ADMA and serum uric acid (UA), compared with the control group; no further differences were found. A significant linear and inverse correlation was found between NOx and ADMA levels (R(2)=0.237, p<0.001). CONCLUSIONS This study suggests a reduced ADMA inhibition of NOS as possible mechanism involved in the pathogenesis of oxidative stress in female subjects with a short duration and uncomplicated type 1 diabetes.

Regulation of PTH secretion by 25-hydroxyvitamin D and ionized calcium depends on vitamin D status: a study in a large cohort of healthy subjects

INTRODUCTION Previous papers investigating vitamin D status have often outlined the significant relationships between serum parathyroid hormone (PTH) and 25-hydroxyvitamin D (25OHD), but the influence of ionized calcium levels has not been concomitantly considered. DESIGN Cross-sectional. MATERIALS AND METHODS In 1050 healthy men (547) and women (503), serum ionized calcium (iCa), creatinine (Cr), albumin, 25OHD, and PTH were measured. After conventional analysis, a regression tree was fitted on the data set. RESULTS 25OHD and PTH values showed significant opposite seasonal changes. 25OHD levels negatively correlated with PTH, which in turn negatively correlated with iCa. A regression tree was fitted to the whole data set using PTH as the response variable and 25OHD and iCa as covariates. PTH concentration depended on that of iCa only in subjects with 25OHD levels>16.35 ng/mL, while for 25OHD<16.35 ng/mL it depended on 25OHD values. CONCLUSIONS Our results indicated that PTH levels were highly conditioned by those of 25OHD in subjects with 25OHD values lower than 16.35 ng/mL and by those of iCa only for higher 25OHD concentration.