Juvenile rat and pediatric trazodone studies: how to gain extra sensitivity to overcome bioanalytical challenges.

AIM: Trazodone (TZD) is used for the treatment of depression in adults and, off-label, as a sleep medication in adult and pediatric populations. The off-label use is well documented, however further clinical studies are needed to confirm its efficacy and safety for the treatment of sleep disorders. In this scenario, we developed a bioanalytical method to quantify low TZD concentrations in samples collected by capillary microsampling (CMS) to support dose finding, Good Laboratory Practice juvenile rat toxicokinetic and upcoming pediatric studies. METHODOLOGY: A method using only 8 µl of plasma was developed and successfully used for analyzing CMS samples from juvenile rats throughout toxicokinetic study. CONCLUSION: By harmoniously maximizing each analytical step, we achieved a sensitive method to quantify TZD in CMS samples.

Prevalence of glucose abnormalities and diagnostic value of fasting glucose and fasting insulin in urban Hispanics.

The prevalence of glucose abnormalities (GAs) and the diagnostic value of fasting plasma glucose and insulin in detecting impaired glucose tolerance (IGT) and diabetes mellitus (DM) were determined in urban Latin Americans. An oral glucose tolerance test was conducted in 592 subjects after administration of 75 g of glucose. Employing American Diabetes Association (ADA) and World Health Organization guidelines, GAs were found in 34% of the subjects, defined as impaired fasting glucose (IFG) (13.3%), IGT (6.9%), combined IFG + IGT (7.8%), and newly diagnosed type-2-DM (6.5%). All newly diagnosed diabetics had 2-hour glucose levels ≥200 mg/dL, but only 46.1% had fasting glucose ≥126 mg/dL. In addition, nearly half of the subjects with IGT (47%) had fasting glucose levels <100 mg/dL. The sensitivity, specificity, positive and negative predictive values of IFG in predicting IGT was 52.9%, 83%, 36.8%, and 90.4 %, respectively. Fasting insulin levels were not sensitive for differentiating glucose tolerant, from IFG and IGT; only the subjects with combined IFG + IGT had increased fasting insulin levels (22% above IFG) (P < 0.01). Two-hour insulin was increased by 30% in IGT and newly diagnosed diabetics, and by 46% in the subjects with combined IFG + IGT. In summary, the very high prevalence of undetected GA encountered in "healthy" subjects living in Caracas, Venezuela, requires immediate sanitary attention. With 50% of diabetic patients being unaware of their condition, half of IGT and DM not detected by ADA guidelines, and the poor sensitivity/specificity of fasting glucose in predicting 2-hour abnormalities, we recommend that 2-hour postload glucose be included when screening for GAs. Measurements of fasting and/or postload insulin are not cost effective for the diagnosis of GA, because they provide little additional clinical information in this context.

Increased low-grade inflammation is associated with lack of functional response to carvedilol in patients with systolic heart failure.

BACKGROUND: The aim of this study was to evaluate, according to functional response, the neuroendocrine and inflammatory status in patients with chronic heart failure before and after therapy with carvedilol. METHODS AND RESULTS: Serum tumor necrosis factor-α (TNF-α) soluble receptors (sTNF-R1 and sTNF-R2), interleukin (IL)-10 and IL-18, chromogranin A (CgA) and brain natriuretic peptide (pro-BNP) were measured in 37 New York Heart Association class II to IV heart failure patients, at baseline and after 6 months of therapy with carvedilol. Patients were divided in two groups according to whether, following carvedilol, left-ventricular ejection fraction (LVEF) had increased by at least 5% (17 patients) or not (20 patients). Baseline LVEF was higher in nonresponders (38 ± 5 vs. 31 ± 7%, P = 0.002). In responders, LVEF increased from 31 ± 7 to 51 ± 7% (P < 0.0001), whereas in nonresponders it decreased from 38 ± 5 to 33 ± 7%, (P = 0.02). sTNF-R1 (P = 0.019) and sTNF-R2 (P = 0.025) increased in nonresponders, whereas they did not change in responders. After carvedilol, IL-10 was significantly higher in responders (P = 0.03). Conversely, no significant IL-18 and CgA changes were observed in either group. CgA was not significantly different between groups at baseline and after carvedilol in either group, whereas pro-BNP significantly increased in nonresponders (from 438 ± 582 to 1324 ± 1664 pg/ml, P = 0.04) and decreased in responders (from 848 ± 1221 to 420 ± 530 pg/ml, P = 0.08). CONCLUSION: Increased inflammatory activation observed only in heart failure patients not improving left-ventricular function after carvedilol may indicate that inflammation, either as a direct cause or as a consequence, is associated with progressive ventricular dysfunction.

Identification of MK-5710 ((8aS)-8a-methyl-1,3-dioxo-2-[(1S,2R)-2-phenylcyclo- propyl]-N-(1-phenyl-1H-pyrazol-5-yl)hexahydro-imidazo[1,5-a]pyrazine-7(1H)-carboxamide), a potent smoothened antagonist for use in Hedgehog pathway dependent malignancies, part 2.

The Hedgehog (Hh-) signaling pathway is a key developmental pathway which gets reactivated in many human tumors, and smoothened (Smo) antagonists are emerging as novel agents for the treatment of malignancies dependent on the Hh-pathway, with the most advanced compounds demonstrating encouraging results in initial clinical trials. A novel series of potent bicyclic hydantoin Smo antagonists was reported in the preceding article, these have been resolved, and optimized to identify potent homochiral derivatives with clean off-target profiles and good pharmacokinetic properties in preclinical species. While showing in vivo efficacy in mouse allograft models, unsubstituted bicyclic tetrahydroimidazo[1,5-a]pyrazine-1,3(2H,5H)-diones were shown to epimerize in plasma. Alkylation of the C-8 position blocks this epimerization, resulting in the identification of MK-5710 (47) which was selected for further development.

Turbulent flow chromatography TFC-tandem mass spectrometry supporting in vitro/vivo studies of NCEs in high throughput fashion.

Turbulent Flow Chromatography (TFC) is a powerful approach for on-line extraction in bioanalytical studies. It improves sensitivity and reduces sample preparation time, two factors that are of primary importance in drug discovery. In this paper the application of the ARIA system to the analytical support of in vivo pharmacokinetics (PK) and in vitro drug metabolism studies is described, with an emphasis in high throughput optimization. For PK studies, a comparison between acetonitrile plasma protein precipitation (APPP) and TFC was carried out. Our optimized TFC methodology gave better S/N ratios and lower limit of quantification (LOQ) than conventional procedures. A robust and high throughput analytical method to support hepatocyte metabolic stability screening of new chemical entities was developed by hyphenation of TFC with mass spectrometry. An in-loop dilution injection procedure was implemented to overcome one of the main issues when using TFC, that is the early elution of hydrophilic compounds that renders low recoveries. A comparison between off-line solid phase extraction (SPE) and TFC was also carried out, and recovery, sensitivity (LOQ), matrix effect and robustness were evaluated. The use of two parallel columns in the configuration of the system provided a further increase of the throughput.

Dihydroxy-pyrimidine and N-methylpyrimidone HIV-integrase inhibitors: improving cell based activity by the quaternarization of a chiral center.

In the context of HIV-integrase, dihydroxypyrimidine and N-methyl pyrimidone inhibitors the cellular activity of this class of compounds has been optimized by the introduction of a simple methyl substituent in the alpha-position of the C-2 side chains. Enhanced passive membrane permeability has been identified as the key factor driving the observed cell-based activity improvement. The rat PK profile of the alpha-methyl derivative 26a was also improved over its des-methyl exact analog.

A novel strategy for reducing phospholipids-based matrix effect in LC-ESI-MS bioanalysis by means of HybridSPE.

A novel strategy to minimize phospholipids-based matrix effects in bioanalytical LC-MS/MS assays was evaluated. The phospholipids-based matrix effect was investigated with a commercially available electrospray ionization (ESI) source coupled with a triple quadrupole mass spectrometer. A systematic comparison approach of two sample preparation procedures was performed. In particular, the matrix effect on mass spectrometry response in rat and human plasma samples was studied by comparing sample extracts obtained by means of a conventional plasma protein precipitation with acetonitrile and the novel HybridSPE-Precipitation procedure. The HybridSPE dramatically reduced the levels of residual phospholipids in biological samples, leading to significant reduction in matrix effects. This new procedure which combines the simplicity of precipitation with the selectivity of SPE allows to obtain much cleaner extracts than with conventional procedures. The effective targeted removal of phospholipids and proteins in biological plasma samples achieved with the HybridSPE-Precipitation procedure provides significant improvement in bioanalysis and a practical and fast way to ensure the avoidance of phospholipids-based matrix effects.

[Changes in nitric oxide, prostaglandins and myeloperoxidase activity in acrolein-induced cystitis in rats]. / Cambios en el oxido nitrico, las prostaglandinas y en la actividad de la mieloperoxidasa, inducidos por la acroleína en vejiga urinaria de rata.

To investigate the role of substance P (sP), nitric oxide (ON) and prostaglandins (PGs) in acrolein (ACR)-induced cystitis, we studied the changes induced by ACR on bladder inducible nitric oxide synthase (iNOS) and mieloperoxidase (MPO) activities, along with PGs and NO metabolites levels. Sprague-Dawley male rats received i.p. ACR (5 mg/Kg) plus one of the following treatments: Group 1: saline 0.10 mL/100g i.p.; Group 2: Win-51.708 (WIN) 25 mg/Kg i.p.; Group 3: S-metilisothiourea (MITU) 35 mg/Kg i.p.; Group 4: Rofecoxib(ROF) 20 mg/Kg o.p.; Group 5: Meloxicam(MEL) 25 mg/Kg i.p.; Group 6: combination MITU+MEL. ACR-induced mortality was partially prevented by WIN (NK1 antagonist) and MITU (iNOS inhibitor). Animals that survived after 24h of ACR exposure, had histological inflammatory changes in bladder along with increased MPO activity. There was augmentation of nitrates+nitrites and of PGs. WIN didn't prevent any of these effects. ROF and MEL (COX-2 inhibitors) partially protected against bladder inflammation; MITU pre-treatment was able to prevent these changes and those of NO metabolites levels. The MITU+MEL combination produced the highest protection against ACR-induced damage. These results suggest that NO produced via iNOS and PGs produced by COX-1/COX-2, have an important role in the pathogenesis of cystitis induced by ACR. ACR could stimulate iNOS and COX-1/COX-2, producing lymphocyte migration and increases of NO and PGs.

Cambios en el óxido nítrico, las prostaglandinas y en la actividad de la mieloperoxidasa, inducidos por la acroleína en vejiga urinaria de rata / Changes in nitric oxide, prostaglandins and mieloperoxidase activity in acrolein-induced cystitis in rats

Se estudió el papel de la sustancia P, el óxido nítrico(ON) y las prostaglandinas (PGs) en la cistitis inducida por acroleína(ACR), para lo cual se estudiaron los cambios de las actividades de la óxido nítrico sintasa inducible(iNOS) y de la mieloperoxidasa(MPO) vesicales, así como en los niveles de PGs y de metabolitos del ON. Ratas macho Sprague-Dawley recibieron ACR (5mg/Kg, i.p), más uno de los siguientes tratamientos: Grupo 1: Salina, 0,10mL/100g i.p.; Grupo 2: Win-51.708(WIN), 25mg/Kg i.p.; Grupo 3: S-metilisotiourea(MITU), 35 mg/Kg i.p.; Grupo 4: Rofecoxib(ROF), 20mg/Kg v.o.; Grupo 5: Meloxicam(MEL), 25mg/Kg i.p.; Grupo 6: combinación MITU+ MEL. La mortalidad inducida por ACR fue parcialmente prevenida por WIN (antagonista NK1) y MITU (inhibidor iNOS). En los animales que sobrevivieron a 24 horas de exposición a ACR se encontraron cambios histológicos inflamatorios en vejiga, que se acompañaron de aumentos en la actividad MPO. También se observaron aumentos de nitratos+nitritos y PGs. El WIN no previno ninguno de estos cambios. El ROF y el MEL (inhibidores COX-2) protegieron parcialmente contra la inflamación vesical, mientras que el tratamiento con MITU fue capaz de prevenir estos cambios así como también el aumento de metabolitos del ON. La combinación MITU+MEL produjo una mayor protección contra los efectos inducidos por ACR. Estos resultados indican que el ON producido vía de la iNOS y las PGs producidas por la COX-1/COX-2, desempeñan un papel en la patogénesis de la cistitis por ACR. La ACR podría estimular a la iNOS y a las COX-1/COX-2, induciendo la migración linfocitaria, aumentos de nitratos+nitritos y de PGs.

To investigate the role of substance P(sP), nitric oxide (ON) and prostaglandins (PGs) in acrolein(ACR)-induced cystitis, we studied the changes induced by ACR on bladder inducible nitric oxide synthase(iNOS) and mieloperoxidase(MPO) activities, along with PGs and NO metabolites levels. Sprague-Dawley male rats received i.p. ACR (5mg/Kg) plus one of the following treatments: Group 1: saline 0.10 mL/100g i.p.; Group 2: Win-51.708 (WIN) 25mg/Kg i.p.; Group 3: S-metilisothiourea(MITU) 35mg/Kg i.p.; Group 4: Rofecoxib(ROF) 20mg/Kg o.p.; Group 5: Meloxicam(MEL) 25mg/Kg i.p.; Group 6: combination MITU+MEL. ACR-induced mortality was partially prevented by WIN (NK1 antagonist) and MITU (iNOS inhibitor). Animals that survived after 24h of ACR exposure, had histological inflammatory changes in bladder along with increased MPO activity. There was augmentation of nitrates+nitrites and of PGs. WIN didn’t prevent any of these effects. ROF and MEL (COX-2 inhibitors) partially protected against bladder inflammation; MITU pre-treatment was able to prevent these changes and those of NO metabolites levels. The MITU+MEL combination produced the highest protection against ACR-induced damage. These results suggest that NO produced via iNOS and PGs produced by COX-1/COX-2, have an important role in the pathogenesis of cystitis induced by ACR. ACR could stimulate iNOS and COX-1/COX-2, producing lymphocyte migration and increases of NO and PGs.

Óxido nítritico: sensibilidad a la sal y la obesidad / Nitric oxide: salt sensitivity and obesity

El óxido nítrico es una molécula neurotransmisora, mediadora de importantes funciones como la vasodilatación, estimulación de la síntesis de músculo liso vascular y antiagregación plaquetaria. El óxido nítrico es continuamente sintetizado y liberado desde el endotelio en condiciones fisiológicas. La inhibición crónica de la producción de óxido nítrico conduce a hipertensión arterial severa; por otra parte la hipertensión arterial se asocia con otros factores de riesgo, como lo es la sensibilidad a la sal. A pesar de que la hipertensión arterial inducida por sodio fue descrita hace medio siglo, aún se desconoce su etiología en humanos, y las implicaciones del óxido nítrico en su aparición. a) La ingesta elevada de sodio inhibe la producción del óxido nítrico; este efecto se revierte con la ingestión de sal. b) en sujetos obesos, la corrección de alteraciones metabólicas asociadas es capaz de corregir la sensibilidad a la sal y por lo tanto, de disminuir la hipertensión arterial en los individuos sal sensibles. También restablece la bioactividad normal del óxido nítrico. Estos hallazgos permiten afirmar que los factores de riesgo adquiridos juegan un papel importante en la patogénesis de la sensibilidad a la sal asociada a la obesidad, y que la corrección de la adiposidad abdominal mejora las anomalías metabólicas asociadas, reduce la reactividad de la presión arterial al sodio y mejora la producción de óxido nítrico.

Nitric oxide, as a neurotransmitter molecule, is a mediator of important functions including vasodilatation, synthesis of vascular smooth muscle and anti-aggregation of platelets. Under physiological conditions nitric oxide is synthesized and released from the endothelium. The chronic inhibition of nitric oxide production leads to severe hypertension; moreover hypertension is associated with other risk factors, such as salt sensitivity. Although hypertension induced by sodium was described half a century ago, its etiology remains unclear as well as the involvement of nitric oxide. A) high sodium intake inhibits the production of nitric oxide; this effect is reversed by reducing salt intake. B) in obese subjects, the reduction of central adiposity and the correction of related metabolic abnormalities can alter the salt sensitivy, and thereby decrease blood pressure in salt-sensitive individuals. It also restores the normal bioactivity of nitric oxide. These findings confirm the important role of risk factors in the pathogenesis of salt sensitivy associated with obesity, and that the correction of abdominal fat improves metabolic abnormalities, leading to the lowering of salt sensitivy and to an improvement in the production of nitric oxide.

Differential long-term effects of carvedilol on proinflammatory and antiinflammatory cytokines, asymmetric dimethylarginine, and left ventricular function in patients with heart failure.

Neuroendocrine/inflammatory and endothelial functions have been indicated as crucial for heart failure (HF) patients. We evaluated relation in HF patients among cytokines and asymmetric dimethylarginine (ADMA) and left ventricular ejection fraction (LVEF) at baseline and after long-term administration of carvedilol. Interleukin 10 (IL-10), interleukin 18 (IL-18), and ADMA were measured in 22 NYHA class II to IV HF patients at baseline and after 40 +/- 14 months of carvedilol treatment. Patients were divided into 2 groups according to whether, after treatment with carvedilol, LVEF had increased at least 5% (responders) or less than 5% (non-responders). In responders (11 of 22 patients), LVEF increased from 38 +/- 6% to 50 +/- 7%, (P < 0.001); in non-responders, it decreased from 36 +/- 9% to 31 +/- 6%, (P = 0.02); NYHA class significantly decreased in both groups. IL-18 decreased in responders (from 586.4 +/- 128 to 183.13 +/- 64.4 pg/mL; P < 0.001) and in non-responders (from 529.3 +/- 116.25 to 142.4 +/- 58.9 pg/mL; P < 0.001). IL-10 increased in responders (from 0.49 +/- 0.25 to 2.01 +/- 1.01 pg/mL; P < 0.001) and in non-responders (from 0.64 +/- 0.31 to 1.33 +/- 0.59 pg/mL; P < 0.001). Conversely, ADMA levels decreased only in responders (from 0.67 +/- 0.16 to 0.44 +/- 0.15 micromol/L; P < 0.001), and an inverse correlation was observed between basal ADMA levels and changes in LVEF after treatment. In HF patients, carvedilol appears to reduce symptoms and the expression of inflammation, regardless of the LV functional response. In those patients showing improvement of LVEF, the reduction of inflammation is paralleled by a reduction of ADMA. We surmise that carvedilol could be effective at various independent levels as a result of possible pleiotropic effects of this agent.

Salt-resistant and salt-sensitive phenotypes determine the sensitivity of blood pressure to weight loss in overweight/obese patients.

High blood pressure (BP) is extremely common in persons with obesity. However, not all obese individuals have high BP, nor does weight loss lower BP in all persons. In this study, the authors investigated whether the salt-sensitive (SS) or salt-resistant (SR) phenotype determines the degree of BP lowering induced by weight loss in a group of middle-aged individuals of whom 80% are of Hispanic descent. Overweight/obese participants classified as SS or SR (N=45; body mass index, 27-35 kg/m(2)) entered a 1-year program of dietary restriction, aerobic exercise, and metformin therapy. Comparable reductions in obesity (8%-10%), triglycerides (25%), and fasting insulin concentrations (40%) were observed in SR and SS individuals. In SS patients, the intervention lowered systolic BP/diastolic BP by 8.8/6.1 mm Hg, decreased albuminuria by 63%, and decreased the patient's salt sensitivity. Neither BP nor albuminuria was modified in SR persons by the intervention. In obese SS individuals, salt restriction induced comparable BP lowering as weight reduction. In summary, BP lowering induced by the lifestyle/metformin intervention appears to be determined by the SR/SS phenotype. Weight loss and correction of metabolic abnormalities lowers BP in obese SS persons but not in obese SR persons. Correcting adiposity in SS patients lowers BP by making the BP insensitive to dietary salt. The SR phenotype protects from obesity-induced increases in BP.

Lowering the threshold for defining microalbuminuria: effects of a lifestyle-metformin intervention in obese "normoalbuminuric" non-diabetic subjects.

BACKGROUND: We investigated whether levels of albuminuria (urinary albumin excretion (UAE)) below those conventionally accepted as microalbuminuria (<30 mg/day) are sensitive to correction of obesity and obesity-related risk factors. METHODS: The effects of a 12-month lifestyle modification-metformin program were evaluated in otherwise healthy overweight/obese "normoalbuminuric" subjects: group I with UAE of <10 mg/day (n = 23) and group II with UAE of 10-29 mg/day (n = 18). RESULTS: The subjects of group II were older and heavier, and had higher blood pressure (BP) and lower high-density lipoprotein (HDL) levels, than those of group I. Creatinine clearances were also higher in group II (148 +/- 14 ml/min) than in group I (108 +/- 9 ml/min). Although the intervention induced comparable reductions in obesity, BP, lipids and insulin levels in both groups, UAE was significantly reduced in group II (9.1 +/- 1.8 mg/24 h; 60% reduction; P < 0.001), and non-significantly in group I (0.75 +/- 0.5 mg/day; 12% reduction; P > 0.1). Additionally, greater reduction in creatinine clearance was observed in subjects with higher UAE rates. After the intervention, both groups achieved similar UAE rates (5.7 +/- 0.9 and 5.2 +/- 1.0 mg/day; P > 0.10). Basal UAE was related to the subjects' creatinine clearance (r = 0.38; P = 0.04). For both groups together, intervention-induced changes in UAE rates were not significantly related to BP, age, or body weight. However, for group II subjects, BP and UAE reduction were positively associated (r = 0.44; P = 0.03). CONCLUSIONS: UAE of 10-29 mg/day (hyperalbuminuria), below the conventionally used limit to define microalbuminuria, is already associated with a more adverse cardiovascular risk profile, and is exquisitely sensitive to interventions that reduce obesity, BP, and insulin resistance.

Discovery and synthesis of HIV integrase inhibitors: development of potent and orally bioavailable N-methyl pyrimidones.

The human immunodeficiency virus type-1 (HIV-1) encodes three enzymes essential for viral replication: a reverse transcriptase, a protease, and an integrase. The latter is responsible for the integration of the viral genome into the human genome and, therefore, represents an attractive target for chemotherapeutic intervention against AIDS. A drug based on this mechanism has not yet been approved. Benzyl-dihydroxypyrimidine-carboxamides were discovered in our laboratories as a novel and metabolically stable class of agents that exhibits potent inhibition of the HIV integrase strand transfer step. Further efforts led to very potent compounds based on the structurally related N-Me pyrimidone scaffold. One of the more interesting compounds in this series is the 2-N-Me-morpholino derivative 27a, which shows a CIC95 of 65 nM in the cell in the presence of serum. The compound has favorable pharmacokinetic properties in three preclinical species and shows no liabilities in several counterscreening assays.

Cyclophosphamide induced cystitis: role of nitric oxide synthase, cyclooxygenase-1 and 2, and NK(1) receptors.

PURPOSE: The role of substance P, inducible nitric oxide synthase, and cyclooxygenase-1 and 2 on the pathogenesis of cyclophosphamide induced cystitis was investigated in rats. MATERIALS AND METHODS: Sprague-Dawley male rats received 1 of certain treatments, including 1) 0.9 weight per volume saline (0.10 ml/100 gm intraperitoneally), 2) cyclophosphamide (75 mg/kg intraperitoneally), 3) cyclophosphamide plus the NK(1) receptor antagonist Win-51.708 (20 mg/kg intraperitoneally), 4) cyclophosphamide plus the inducible nitric oxide synthase inhibitor S-methylthiourea (20 mg/kg intraperitoneally), 5) cyclophosphamide plus the highly selective cyclooxygenase-2 inhibitor rofecoxib (15 mg/kg intraperitoneally), 6) cyclophosphamide plus the selective cyclooxygenase-2 inhibitor meloxicam (15 mg/kg intraperitoneally), 7) cyclophosphamide plus the nonselective cyclooxygenase inhibitor ketoprofen (20 mg/kg intraperitoneally) or 8) cyclophosphamide plus methylthiourea plus meloxicam. Parameters were evaluated 6 hours after cyclophosphamide administration, including plasma protein extravasation, histological changes, myeloperoxidase and inducible nitric oxide synthase activities in the bladder, plasmatic nitric oxide metabolites and urinary nitric oxide metabolites, and prostaglandin E(2) levels. RESULTS: Cyclophosphamide produced inflammatory and cytotoxic changes in the bladder, accompanied by increased nitric oxide metabolites, urinary prostaglandins, myeloperoxidase and inducible nitric oxide synthase activity. Pretreatment with Win-51.708 and with methylthiourea prevented all of these effects except myeloperoxidase activity, which was only prevented by Win-51.708. All inducible cyclooxygenases were able to prevent prostaglandin synthesis and increases in myeloperoxidase activity. Combined inhibition of inducible nitric oxide synthase and cyclooxygenase-2/cyclooxygenase-1 (methylthiourea plus meloxicam) did not provide any additional protection against bladder damage, increased inducible nitric oxide synthase activity or prostaglandin E(2) synthesis. Additionally, this combination was unable to prevent increased myeloperoxidase activity. CONCLUSIONS: The results of this study suggest that there is crosstalk between nitric oxide and the cyclooxygenase enzyme with cyclooxygenase-1/cyclooxygenase-2 isoforms having an important role in this relationship. Augmented myeloperoxidase activity seems to be associated with NK(1) receptor activation and low levels of nitric oxide with cyclooxygenase-1 having an important role.

Alpha-adducin polymorphism, salt sensitivity, nitric oxide excretion, and cardiovascular risk factors in normotensive Hispanics.

BACKGROUND: Genetic and environmental factors determine the blood pressure (BP) response to changes in salt intake. Mutations in the alpha-adducin gene may be associated with hypertension and salt-sensitive hypertension. We investigated whether one alpha-adducin polymorphism, the Gly460Trp (G/T) variant, was associated with salt sensitivity, nitric oxide (NO) production; and cardiovascular risk factors in healthy adult normotensive Venezuelans. METHODS AND RESULTS: Subjects (n = 126) were screened for salt sensitivity. The alpha-Adducin polymorphism was tested in salt-sensitive (SS) and salt-resistant (SR) subjects. The G/T and G/G (wild gene) groups had similar BP levels. The G/T subjects had higher LDL-cholesterol (P =.01) and postload glucose AUC (P =.03) than G/G individuals. Genotype frequencies were not associated with BP or salt sensitivity (G/G, 38.1% SS and 61.9% SR vs G/T, 40.7% SS and 59.3% SR). Shifting from high salt to low salt diet produced comparable reductions in systolic BP and diastolic BP in G/T and G/G groups. The G/G and G/T groups excreted similar amounts of sodium on high and low salt diets. The SR subjects carrying the wild or the mutated gene showed no changes in NO metabolite excretion at different levels of salt intake. In SS subjects, the level of NO metabolite excretion was highly dependent on salt intake. A combination of SS and 460Trp mutation enhanced the sodium-dependent modulation of NO production. CONCLUSIONS: In normotensive Venezuelans, the alpha-adducin G/T polymorphism was not associated with BP, salt sensitivity, or with sodium excretion during sodium loading or restriction. G/T was associated with increased LDL-cholesterol and postload glucose levels. In SS, G/T was associated with greater salt-dependent modulation of NO excretion. However, this larger increase in NO excretion was not associated with a larger decrease in BP.

Plasmatic nitric oxide, but not von Willebrand Factor, is an early marker of endothelial damage, in type 1 diabetes mellitus patients without microvascular complications.

Type 1 diabetes mellitus (IDDM) is associated with coronary artery disease and microvascular damage. Long-term glycemic control reduces but not fully prevents such complications. Recent evidence suggests that microvascular disease associated to IDDM begins with endothelial dysfunction. In this study, we evaluated changes in levels of nitric oxide (NO) and von Willebrand Factor (vWF) to detect early endothelial dysfunction in IDDM patients recently diagnosed. Subjects were included in one of the following groups: Group 1 (n=14): healthy subjects; Group 2 (n=14): IDDM patients recently diagnosed (<1 year), with no clinical evidence of microvascular disease; Group 3 (n=14): IDDM patients with microvascular disease (retinopathy and nephropathy). Urinary NO metabolites were similar in Group 1 (1.45+/-0.13) and Group 2 (1.6+/-0.2 micromol/mg creatinine) (P>.05), as well as vWF (99.6+/-5.7% and 84.3+/-5.1%, Groups 1 and 2, respectively, P>.05). Plasmatic NO metabolites were lower in Groups 2 and 3 (54.6+/-5.1 and 50.02+/-13.65 nmol/ml, respectively) compared with Group 1 (91.1+/-6.6 nmol/ml) (P=.0005). Also, in Group 3, urinary NO metabolites were lower (0.27+/-0.03 micromol/mg creatinine) and vWF was higher (184+/-25%) than Groups 1 and 2. There is evidence of early endothelial dysfunction even in IDDM patients recently diagnosed, with good glycemic control and without systemic hypertension, dyslipidemia or microvascular disease; this endothelial damage was detected as a decrease in plasmatic NO metabolite levels, before appearance of any clinical evidence of microvascular disease.