A Novel Multiplexed Enzyme-Linked Immunosorbent Assay for the Detection of IgG Seroreactivity to Cytomegalovirus (CMV) UL144.

Infection with human cytomegalovirus (CMV) is common and may have grave consequences in transplant recipients and congenitally infected children. Diagnosis of CMV infection is based on detection of specific antibodies and molecular assays. The incorporation of CMV serological assays into diagnostic algorithms requires careful evaluation and interpretation. Very few serological assays measure CMV infection by a specific strain. We developed an enzyme-linked immunosorbent assay (ELISA) using CMV-encoded UL144 as the antigen. UL144 encodes three major genotypes, A, B, and C, and recombinants. The ELISA was developed with the three UL144 proteins and optimized as a multiplex assay. Sera from 55 positive and 59 negative CMV IgG, determined by the clinical microbiology laboratory, were used for evaluation and optimization. A cutoff optical density (OD) that distinguishes UL144 antibody-positive from antibody-negative sera was established. UL144 A, B, C, and combinations of these antigens were detected in sera. An assay threshold of 0.1 was established and, from a total of 303 sera, the overall sensitivity, specificity, and positive and negative predictive values of the multiplex ELISA were 86.72% (95% confidence interval [CI] 79.59% to 92.07%), 96.57% (92.69% to 98.73%), 94.40% (88.45% to 97.38%), and 91.60% (87.50% to 94.44%), respectively. The inter- and intraassay median coefficients of variation were 0.06 (interquartile range [IQR] 0.56, 0.2) and 0.171 (IQR 0.038, 0.302), respectively. No cross-reactivity was observed with HSV-positive CMV-negative sera. This ELISA gives simple and reproducible results for detection of anti-CMV UL144 IgG. It may assist in differentiating natural infection from CMV vaccines that lack UL144, and may provide an important tool for epidemiological studies of CMV strains.

Asymmetric dimethylarginine and symmetric dimethylarginine prospectively relates to carotid wall thickening in black men: the SABPA study.

The relationship of both asymmetric (ADMA) and symmetric (SDMA) dimethylarginine with carotid wall thickness is inconclusive especially among black populations. We aimed to compare carotid intima media thickness (cIMT) and dimethylarginine levels in 75 black and 91 white men at baseline and after a 3-year follow-up, and to investigate associations of percentage change in cIMT with percentage change in dimethylarginine levels (ADMA and SDMA). Plasma levels of ADMA and SDMA were determined with a liquid chromatography mass spectrometry method and B-mode ultrasonography was used to determine the cIMT at baseline and follow-up. In black men, mean cIMT (p = 0.79) and ADMA levels (p = 0.67) remained the same, but SDMA levels were lower (p < 0.001) when comparing baseline and follow-up. In white men, cIMT increased (p < 0.001), but both mean ADMA and SDMA levels decreased (p < 0.001) over time. In black men, percentage change in cIMT was positively associated with percentage change in ADMA (R 2 = 0.49; ß = 0.46; p < 0.001) and percentage change in SDMA (R 2 = 0.46; ß = 0.41; p < 0.001). These associations were absent in the white men. Despite lower mean SDMA and similar ADMA and cIMT in black men, percentage change in cIMT was independently associated with percentage change in ADMA and percentage change in SDMA. These results suggest an important role for ADMA and SDMA lowering strategies to delay carotid wall thickening, especially in black populations prone to the development of cardiovascular disease.

New cell-signaling pathways for controlling cytomegalovirus replication.

Cytomegalovirus (CMV) is increasingly recognized as an accomplished modulator of cell-signaling pathways, both directly via interaction between viral and cellular proteins, and indirectly by activating metabolic/energy states of infected cells. Viral genes, as well as captured cellular genes, enable CMV to modify these pathways upon binding to cellular receptors, up until generation of virus progeny. Deregulation of cell-signaling pathways appears to be a well-developed tightly balanced virus strategy to achieve the desired consequences in each infected cell type. Importantly and perhaps surprisingly, identification of new signaling pathways in cancer cells positioned CMV as a sophisticated user and abuser of many such pathways, creating opportunities to develop novel therapeutic strategies for inhibiting CMV replication (in addition to standard of care CMV DNA polymerase inhibitors). Advances in genomics and proteomics allow the identification of CMV products interacting with the cellular machinery. Ultimately, clinical implementation of candidate drugs capable of disrupting the delicate balance between CMV and cell-signaling will depend on the specificity and selectivity index of newly identified targets.

Asymmetric dimethylarginine as an independent risk marker for mortality in ambulatory patients with peripheral arterial disease.

BACKGROUND: Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide (NO) synthesis causing endothelial dysfunction, an early sign of atherogenesis. Symmetric dimethylarginine (SDMA) does not inhibit NO synthases. Peripheral arterial disease (PAD) is a systemic indication of atherosclerosis. METHODS: We assessed the associations between both ADMA and SDMA blood levels and major cardiovascular and cerebrovascular events or death from any cause within a 5-year follow-up in the multicentre getABI trial. From a cohort of 6821 primary care patients, aged ≥65 years, all 1260 patients with prevalent PAD were compared with a random sample of 1187 non-PAD controls. A total of 11,544 patient-years were documented. Multivariate risks were calculated by Cox proportional hazard models, adjusting for PAD, renal dysfunction and other important cardiovascular risk factors. RESULTS: We documented 390 deaths, 296 cardiovascular events and 98 cerebrovascular events. Increased ADMA levels in the 4th quartile were significantly associated with total mortality [hazard ratio (HR) 1.41; 95% CI 1.14-1.74] and with cardiovascular events (HR 1.32; 95% CI 1.03-1.69), but there was a nonsignificant association with cerebrovascular events (HR 1.50; 95% CI 0.98-2.29). Increased SDMA was only just significantly associated with mortality (HR 1.27; 95% CI 1.01-1.59). In PAD patients compared with non-PAD controls, only mean SDMA concentration was considerably increased (0.52 µmol L(-1) vs. 0.48 µmol L(-1); P < 0.001) mainly because of a highly significant association with impaired renal function. CONCLUSION: These data suggest that ADMA but not SDMA is an independent risk marker for death from any cause or from cardiovascular events. The association between SDMA and mortality is in part explained by a close link between SDMA and renal function.

A survey on clinical study participation and study infrastructure at non-university hospitals in Germany.

OBJECTIVE: In order to participate in multicenter clinical trials a fair amount of infrastructure and human resources has to be provided by hospitals. Therefore clinical trials are carried out predominantly in university hospitals. Data concerning participation in clinical trials and the infrastructure of study centers in non-university hospitals in Germany do not exist. A survey was thus conducted to evaluate the current status of clinical study performance in non-university hospitals. MATERIALS AND METHODS: A questionnaire comprising 10 questions covering hospital infrastructure, local study history, and the individual interest in performing studies was sent to 790 non-university hospitals in Germany. RESULTS: 58.7% of questionnaires were returned for evaluation. 74.1% of nonuniversity hospitals participate in clinical studies. Hospital size is a significant predictor of study participation. 25.5% of hospitals have established a multidisciplinary study center. 86.2% have a certified study nurse and in 34.5% this nurse is the only person running the study center. Only 25.5% of hospitals were not interested in participating in clinical studies at all, even if an individual tailored concept were to be offered. CONCLUSIONS: The demand for more hospitals to participate in clinical trials is urgent since high quality studies are a fundamental part of clinical research. Even though 75% of non-university hospitals in Germany already participate in clinical trials, it may be possible to increase this number. In addition by establishing and developing study centers in hospitals the quality of studies will presumably rise, and due to the concentration of study resources, the number of clinical trials may increase.

Interaction of angiotensin receptor type 1 blockers with ATP-binding cassette transporters.

ATP-binding cassette (ABC)-transporters, such as P-glycoprotein (P-gp/ABCB1), multidrug resistance-associated proteins (MRPs/ABCCs) and breast cancer resistance protein (BCRP/ABCG2) transport numerous drugs thus regulating their absorption, distribution and excretion. Angiotensin receptor type 1 blockers (ARBs), used to treat hypertension and heart failure, are commonly administered in combination therapy. However, their interaction potential is not well studied and their effect on ABC-transporters remains elusive. The study therefore aimed to elucidate the effect of various ARBs (telmisartan, candesartan, candesartan-cilexetil, irbesartan, losartan, olmesartan, olmesartan-medoxomil, eprosartan) on ABC-transporter activity in vitro. P-gp inhibition was assessed by calcein assay, BCRP inhibition by pheophorbide A efflux assay, and MRP2 inhibition by a MRP2 PREDIVEZ Kit. Induction of P-gp, BCRP and MRP2 was assessed by real time reverse transcriptase polymerase chain reaction and for P-gp also in a functional assay. Telmisartan was identified as one of the most potent inhibitors of P-gp currently known (IC(50)=0.38+/-0.2 microM for murine P-gp) and it also inhibited human BCRP (IC(50)=16.9+/-8.1 microM) and human MRP2 (IC(50)=25.4+/-0.6 microM). Moreover, the prodrug candesartan-cilexetil, but not candesartan itself, significantly inhibited P-gp and BCRP activity. None of the compounds tested induced mRNA transcription of P-gp or BCRP but eprosartan and olmesartan induced MRP2 mRNA expression. In conclusion, telmisartan substantially differed from other ARBs with respect to its potential to inhibit ABC-transporters relevant for drug pharmacokinetics and tissue defense. These findings may explain the known interaction of telmisartan with digoxin and suggest that it may modulate the bioavailability of drugs whose absorption is restricted by P-gp and possibly also by BCRP or MRP2.

Nitric oxide may mediate the hemodynamic effects of recombinant growth hormone in patients with acquired growth hormone deficiency. A double-blind, placebo-controlled study.

We studied the effects of recombinant growth hormone on systemic nitric oxide (NO) formation and hemodynamics in a double-blind, placebo-controlled trial in adult patients with acquired growth hormone deficiency. 30 patients were randomly allocated to either recombinant human growth hormone (r-hGH; 2.0 IU/d) or placebo for 12 mo. In the subsequent 12 mo, the study was continued with both groups of patients receiving r-hGH. In months 1, 3, 6, 9, and 12 of each year, urine and plasma samples were collected for the determination of urinary nitrate and cyclic GMP as indices of systemic NO production, and of plasma IGF-1 levels. Cardiac output was measured in months 1, 12, and 24 by echocardiography. r-hGH induced a fourfold increase in plasma IGF-1 concentrations within the first month of treatment. Urinary nitrate and cyclic GMP excretion rates were low at baseline in growth hormone-deficient patients (nitrate, 96.8+/-7.4 micromol/mmol creatinine; cyclic GMP, 63.6+/-7.1 nmol/mmol creatinine) as compared with healthy controls (nitrate, 167.3+/-7.5 micromol/mmol creatinine; cyclic GMP, 155.2+/-6.9 nmol/mmol creatinine). These indices of NO production were significantly increased by r-hGH, within the first 12 mo in the GH group, and within the second 12 mo in the placebo group. While systolic and diastolic blood pressure were not significantly altered by r-hGH, cardiac output significantly increased by 30-40%, and total peripheral resistance decreased by approximately 30% in both groups when they were assigned to r-hGH treatment. In the second study year, when both groups were given r-hGH, there were no significant differences in plasma IGF-1, urinary nitrate, or cyclic GMP excretion, or hemodynamic parameters between both groups. In conclusion, systemic NO formation is decreased in untreated growth hormone-deficient patients. Treatment with recombinant human growth hormone normalizes urinary nitrate and cyclic GMP excretion, possibly via IGF-1 stimulation of endothelial NO formation, and concomitantly decreases peripheral arterial resistance. Increased NO formation may be one reason for improved cardiovascular performance of patients with acquired hypopituitarism during growth hormone therapy.

Asymmetric dimethylarginine is associated with parameters of glucose metabolism in Caucasian but not in African women from South Africa.

AIM: Ethnic differences in obesity and obesity related disorders prompted us to search for possible contributors. The impact of the novel cardiovascular risk factor asymmetric dimethylarginine (ADMA) has been never determined in the African population. The present observational study aimed to compare ADMA levels between healthy African (102) and Caucasian women (115) from South Africa, and its impact on glucose metabolism. METHODS: All participants underwent an oral glucose tolerance test with measurements of glucose, insulin, C-peptide, proinsulin and free fatty acids before and after 30, 60, 90, 120 minutes. Fasting serum ADMA was measured by ELISA assay and obesity was determined by anthropometry. RESULTS: Serum ADMA did not differ between the ethnic groups. After stratification according to ADMA quartiles Caucasian women in the upper quartile had significantly higher body mass index and waist circumference as well as elevated insulin resistance, insulin, C-peptide and proinsulin levels with no differences in serum glucose compared to women in the lowest quartile. There was a significant stronger postchallenge insulin response in Caucasian women of the upper quartile. No differences were found in African women. CONCLUSIONS: Despite similar ADMA levels in both ethnic groups ADMA was positively correlated with parameters of glucose metabolism in the Caucasian but not in the African women from South Africa.

Increased urinary excretion of 8-iso-prostaglandin F2alpha in patients with HFE-related hemochromatosis: a case-control study.

The hypothesis according to which iron overload could be harmful has been extensively and controversially discussed in the literature. One underlying pathological mechanism may be elevated oxidative stress. Thus, we studied the correlation between hemochromatosis and an established marker of oxidative stress, 8-iso-prostaglandin F2alpha (8-iso-PGF2alpha, iPF2alpha-III, 15-F2t-IsoP). We enrolled 21 patients with hemochromatosis, positive for the homozygous C282Y mutation in the HFE gene, and 21 healthy controls frequency-matched by age and gender in a case-control study design. The objective was to show that iron overload in HFE-related hemochromatosis is associated with increased oxidative stress assessed through 8-iso-PGF(2alpha) urinary excretion, and that oxidative stress is impacted by iron-removal treatment (phlebotomy). Study parameters were transferrin saturation, 8-iso-PGF(2alpha) urine excretion, transferrin, ferritin, serum iron, and vitamins A and E for all participants. Iron concentration in the liver and non-transferrin-bound iron were measured in patients only. We found a significant difference in 8-iso-PGF2alpha in patients (245 [interquartile range 157-348] pg/mg creatinine) compared with controls (128 [106-191] pg/mg creatinine, P = 0.002). Vitamin A was significantly reduced in cases (0.34 [0.25-1.83] microg/ml compared to 3.00 [2.11-3.39] microg/ml, P < 0.001), while vitamin E did not show a significant difference in cases (14.7 [11.5-18.1] microg/ml) compared with controls (14.9 [13.1-19.2] microg/ml, P = 0.52). After phlebotomy treatment and normalization of the iron parameters in the hemochromatosis group, serum vitamin A levels were significantly increased (1.36 [1.08-1.97] microg/ml, P = 0.035 vs. baseline, P < 0.001 vs. controls) and 8-iso-PGF2alpha urinary excretion was lowered to control levels (146 [117-198] pg/mg creatinine, P = 0.38 vs. controls). In our study, HFE-related hemochromatosis was associated with increased oxidative stress and hypovitaminemia A in C282Y homozygotes. The increased oxidative stress was reversible by normalization of the iron load by phlebotomy. Thus, phlebotomy is an effective and adequate means for reducing oxidative stress in these patients.

Restoring vascular nitric oxide formation by L-arginine improves the symptoms of intermittent claudication in patients with peripheral arterial occlusive disease.

BACKGROUND: Administration of L-arginine improves nitric oxide (NO) formation and endothelium-dependent vasodilation in atherosclerotic patients. OBJECTIVES: We investigated in this double-blind, controlled study whether prolonged intermittent infusion therapy with L-arginine improves the clinical symptoms of patients with intermittent claudication, as compared with the endothelium-independent vasodilator prostaglandin E1, and control patients. METHODS: Thirty-nine patients with intermittent claudication were randomly assigned to receive 2 x 8 g L-arginine/day, or 2 x 40 microg prostaglandin E1 (PGE1)/day or no hemodynamically active treatment, for 3 weeks. The pain-free and absolute walking distances were assessed on a walking treadmill at 3 km/h, 12% slope, and NO-mediated, flow-induced vasodilation of the femoral artery was assessed by ultrasonography at baseline, at 1, 2 and 3 weeks of therapy and 6 weeks after the end of treatment. Urinary nitrate and cyclic guanosine-3', 5'-monophosphate (GMP) were assessed as indices of endogenous NO production. RESULTS: L-Arginine improved the pain-free walking distance by 230+/-63% and the absolute walking distance by 155+/-48% (each p < 0.05). Prostaglandin E1 improved both parameters by 209+/-63% and 144+/-28%, respectively (each p < 0.05), whereas control patients experienced no significant change. L-Arginine therapy also improved endothelium-dependent vasodilation in the femoral artery, whereas PGE1 had no such effect. There was a significant linear correlation between the L-arginine/asymmetric dimethylarginine (ADMA) ratio and the pain-free walking distance at baseline (r=0.359, p < 0.03). L-Arginine treatment elevated the plasma L-arginine/ADMA ratio and increased urinary nitrate and cyclic GMP excretion rates, indicating normalized endogenous NO formation. Prostaglandin E1 therapy had no significant effect on any of these parameters. Symptom scores assessed on a visual analog scale increased from 3.51+/-0.18 to 83+/-0.4 (L-arginine) and 7.0+/-0.5 (PGE1; each p < 0.05), but did not significantly change in the control group (4.3+/-0.4). CONCLUSIONS: Restoring NO formation and endothelium-dependent vasodilation by L-arginine improves the clinical symptoms of intermittent claudication in patients with peripheral arterial occlusive disease.

An endogenous inhibitor of nitric oxide synthase regulates endothelial adhesiveness for monocytes.

OBJECTIVES: We sought to determine whether asymmetric dimethylarginine (ADMA) inhibits nitric oxide (NO) elaboration in cultured human endothelial cells and whether this is associated with the activation of oxidant-sensitive signaling mediating endothelial adhesiveness for monocytes. BACKGROUND: Endothelial NO elaboration is impaired in hypercholesterolemia and atherosclerosis, which may be due to elevated concentrations of ADMA, an endogenous inhibitor of NO synthase. METHODS: Human umbilical vein endothelial cells (ECV 304) and human monocytoid cells (THP-1) were studied in a functional binding assay. Nitric oxide and superoxide anion (O2-) were measured by chemiluminescence; ADMA by high pressure liquid chromatography; monocyte chemotactic protein-1 (MCP-1) by ELISA and NF-KB by electromobility gel shift assay. RESULTS: Incubation of endothelial cells with ADMA (0.1 microM to 100 microM) inhibited NO formation, which was reversed by coincubation with L-arginine (1 mM). The biologically inactive stereoisomer symmetric dimethylarginine did not inhibit NO release. Asymmetric dimethylarginine (10 microM) or native low-density lipoprotein cholesterol (100 mg/dL) increased endothelial O2- to the same degree. Asymmetric dimethylarginine also stimulated MCP-1 formation by endothelial cells. This effect was paralleled by activation of the redox-sensitive transcription factor NF-KB. Preincubation of endothelial cells with ADMA increased the adhesiveness of endothelial cells for THP-1 cells in a concentration-dependent manner. Asymmetric dimethylarginine-induced monocyte binding was diminished by L-arginine or by a neutralizing anti-MCP-1 antibody. CONCLUSIONS: We concluded that the endogenous NO synthase inhibitor ADMA is synthesized in human endothelial cells. Asymmetric dimethylarginine increases endothelial oxidative stress and potentiates monocyte binding. Asymmetric dimethylarginine may be an endogenous proatherogenic molecule.

Endothelium-dependent vasodilation is independent of the plasma L-arginine/ADMA ratio in men with stable angina: lack of effect of oral L-arginine on endothelial function, oxidative stress and exercise performance.

OBJECTIVES: This study was designed to determine the effect of two weeks' treatment with L-arginine on the ratio of plasma L-arginine to asymmetric dimethylarginine (ADMA), oxidative stress, endothelium-dependent vasodilatation to acetylcholine, exercise performance and heart rate variability in men with stable angina. BACKGROUND: The ratio of plasma L-arginine:ADMA has been proposed as a determinant of endothelium-dependent dilation; dietary supplementation with L-arginine has been shown to improve endothelium-dependent vasodilation and symptoms in some conditions. METHODS: Men (n = 40) with stable angina, at least one epicardial coronary artery with a stenosis >50% and a positive exercise test were randomized to receive L-arginine (15 g daily) or placebo for two weeks according to a double-blind parallel-group design. Plasma L-arginine, ADMA, 8-epi-prostaglandin F2alpha (a marker of oxidative stress) and forearm vasodilator responses to brachial artery infusion of nitroprusside and acetylcholine (+/-L-arginine) were measured. A standard Bruce protocol exercise test was performed before and at the end of the treatment period. RESULTS: Plasma L-arginine increased after oral L-arginine, whereas ADMA remained unchanged, leading to an increase in the L-arginine/ADMA ratio of 62 +/- 11% (mean +/- SE, p < 0.01). Despite a significant enhancement in acetylcholine response by intra-arterial L-arginine at baseline, this response remained unchanged after oral L-arginine. Measures of oxidative stress and exercise performance after L-arginine/placebo were similar in placebo and active groups. CONCLUSIONS: In men with stable angina, an increase in plasma L-arginine/ADMA ratio after two weeks' oral supplementation with L-arginine is not associated with an improvement in endothelium-dependent vasodilatation, oxidative stress or exercise performance.

Urinary tract infections with low and high colony counts in young women. Spontaneous remission and single-dose vs multiple-day treatment.

BACKGROUND: The aim of this study was to test whether the spontaneous cure rate is higher in dysuric women with low urinary colony counts (10(2) to 10(4) colony-forming units [cfu] per milliliter) than in women with high colony counts (> or = 10(5) cfu/mL) and whether the response of patients with low colony counts to single-dose treatment is better than those with high colony counts. METHODS: Dysuric women underwent a clinical interview, examination, urine culture, and microscopic examination of urine, and were asked to postpone treatment for 2 days. At that time, the urine tests were repeated. Women with bacterial urinary tract infection were randomized to a group given a single dose of 1200 mg of norfloxacin and to a group treated with 400 mg of norfloxacin twice daily for 7 days. Cure rates were tested at 1 and 5 weeks after treatment. RESULTS: Of 146 women with urinary tract complaints, 113 (25 patients with sterile urine cultures, 21 with low colony counts, and 67 with high colony counts) agreed to postpone antibiotic treatment. Two days after the initial urine culture, only one patient with a low initial colony count had a sterile urine culture (5%, 95% confidence interval, 0% to 14%); and 10 patients (48%, 95% confidence interval, 27% to 69%) had a colony count of 10(5) cfu/mL or more. The rate of spontaneous cure in women with high initial colony count was 7%, 95% confidence interval, 1% to 13%. One week after treatment, the cure rates were 48 (84%) of 57 patients given single-dose treatment vs 49 (98%) of 50 patients treated for 7 days. Five weeks after treatment the rates were 63% and 83%, respectively. The efficacy of single-dose treatment in patients with low urinary counts were similar to those with high counts, and less than that achieved by 7 days of treatment. CONCLUSIONS: The spontaneous cure rate for a 2-day period is minimal in patients with both a low and a high colony count, but half of the patients with low urinary counts will have high colony counts after this interval. The response to single-dose treatment is similar in patients with low and high colony counts, and lower than with multiday treatment.

Long-term administration of L-arginine, L-NAME, and the exogenous NO donor molsidomine modulates urinary nitrate and cGMP excretion in rats.

OBJECTIVE: The effects of long term oral administration of L-arginine, NG-nitro-L-arginine methyl-ester (L-NAME), or molsidomine v placebo on blood pressure and the urinary excretion rates of NO3- and cyclic GMP were studied in Munich Wistar Frömter (MWF) rats. METHODS: L-arginine (2 g.kg-1 body weight, n = 8), NG-nitro-L-arginine methylester (L-NAME; 5 mg.kg-1, n = 8), or molsidomine (3 mg.kg-1, n = 8) were given in drinking water and compared with placebo (n = 8) over a period of five months. Urinary excretion rates of NO3- (by gas chromatography) and cyclic GMP (by radioimmunoassay) were assessed in monthly intervals, as well as systolic blood pressure (tail plethysmography). RESULTS: Mean basal blood pressure was 143.5(SEM 2.2) mm Hg. It was unaffected by L-arginine or molsidomine, but continuously and significantly increased during L-NAME treatment to 199.3(6.4) mm Hg (p < 0.05). Urinary excretion of NO3- increased by 20-41% v controls in L-arginine and molsidomine treated rats (p < 0.05), and decreased by 5-15% in L-NAME treated rats (p < 0.05). Urinary excretion of cyclic GMP increased by 9-38% v controls in the L-arginine and molsidomine treated groups and decreased by 5-20% in the L-NAME treated animals. Consistent with their higher blood pressure, L-NAME treated animals displayed cardiac hypertrophy. CONCLUSIONS: Determination of urinary NO3- excretion by gas chromatography is a sensitive and specific method to assess NO formation in vivo. Long term oral administration of L-arginine in MWF rats increases NO production (as assessed by the urinary excretion rates of NO3- and cyclic GMP), but does not significantly influence systolic blood pressure, whereas L-NAME induces sustained hypertension and cardiac hypertrophy due to inhibition of NO formation.

Chronic dietary supplementation with L-arginine inhibits platelet aggregation and thromboxane A2 synthesis in hypercholesterolaemic rabbits in vivo.

OBJECTIVES: L-arginine exerts anti-atherosclerotic effects in hypercholesterolaemic rabbits via modulating endogenous NO production. We investigated whether L-arginine inhibits thromboxane formation in vivo and platelet aggregation ex vivo in this animal model. METHODS: The urinary excretion rates of 2,3-dinor-6-keto-PGF1 alpha (major urinary metabolite of PGI2) and 2,3-dinor-TXB2 (major urinary metabolite of thromboxane A2) were used as indicators of platelet-endothelial cell interactions in vivo. Rabbits were fed 1% cholesterol (Cholesterol group, N = 8), 1% cholesterol plus 2.25% L-arginine (Cholesterol + L-arginine, N = 8), or normal rabbit chow (Control, N = 4) for 12 weeks. Urine samples were collected in weekly intervals. At the end of the study period platelet aggregation ex vivo and endothelium-dependent and -independent vascular function of isolated aortic rings in vitro was assessed. RESULTS: Urinary 2,3-dinor-TXB2 excretion significantly increased in the cholesterol group (p < 0.05), and endogenous NO formation (measured as urinary nitrate excretion) decreased (p < 0.05). Both parameters were significantly correlated with each other (R = 0.48, p < 0.01). L-arginine partly restored urinary nitrate excretion and significantly reduced TXA2 production to values even below those in the control group (p < 0.001). Urinary 2,3-dinor-6-keto-PGF1 alpha excretion increased in early hypercholesterolaemia and returned to control values in the second half of the study period. The early increase in urinary 2,3-dinor-6-keto-PGF1 alpha excretion was attenuated by L-arginine. Platelet aggregation was significantly enhanced in cholesterol-fed rabbits and attenuated by dietary L-arginine. L-arginine also improved the impaired endothelium-dependent relaxations to ADP, and normalized the vasoconstrictor effects of 5-HT in isolated aortic rings. CONCLUSIONS: Cholesterol-feeding enhances platelet aggregation and TXA2 formation, and stimulates platelet-endothelial cell interaction in rabbits. These effects are probably due to impaired NO elaboration, as indicated by decreased urinary nitrate excretion. Chronic dietary supplementation with L-arginine elevates systemic NO elaboration and significantly increases the PGI2/TXA2 ratio. It thus beneficially influences the homeostasis between vasodilator and vasoconstrictor prostanoids in vivo.

Role of nitric oxide in the vasodilator effect of recombinant human growth hormone in patients with dilated cardiomyopathy.

OBJECTIVE: Dilated cardiomyopathy is characterized by elevated arterial vascular resistance and impaired nitric oxide (NO)-dependent vasodilation. Insulin-like growth factor-I (IGF-I) has been shown to stimulate endothelial NO-synthase resulting in endothelium-dependent vasodilation. Growth hormone (GH) substitution therapy leads in GH-deficient patients to significant increases of IGF-I which may alter systemic vascular resistance by stimulating NO production. This study was designed to evaluate the effects of treatment with recombinant human growth hormone (GH) on NO production and NO-dependent vascular effects in patients with dilated cardiomyopathy. METHODS: 50 patients with dilated cardiomyopathy were randomly assigned to double-blind treatment with 2 I.U. of GH or placebo for 3 months. Central hemodynamics were determined by Swan-Ganz catheter and cardiac output was obtained by the thermodilution method. Serum GH and IGF-I levels were measured and systemic NO production was determined from urinary nitrate and cyclic GMP excretion rates in 42 patients. RESULTS: GH treatment caused in comparison to the placebo group a significant increase of IGF-I by 91 ng/ml (P = 0.0001). Urinary excretion rates of nitrate and cyclic GMP increased also significantly by 38 mumol/mmol creatinine (P = 0.027) and 65 nmol/mmol creatinine (P = 0.003), respectively. The parallel increase of both marker molecules indicates increased systemic NO production during GH treatment. CONCLUSION: GH treatment induces a significant, but moderate increase of systemic NO production in patients with dilated cardiomyopathy. This effect may be mediated by IGF-I stimulating endothelial NO synthase.

Prolonged duration of blood pressure response to enalkiren, the novel dipeptide renin inhibitor, in essential hypertension.

The effects of sustained renin inhibition by repeated administration of enalkiren (A-64662), the novel dipeptide renin inhibitor, were evaluated in a randomized, double-blind, placebo-controlled, parallel-group study of 32 inpatients (eight per group) with essential hypertension who were maintained on a diet containing 60 meq/day sodium. Three different dosage regimens of enalkiren were studied: 1) 1.2 mg/kg quotid., 2) 0.3 mg/kg q.i.d., and 3) 0.1 mg/kg q.i.d. Each patient received an intravenous infusion every 6 hours for 1 week. Placebo infusions were used to mimic the 4 times/day dosing schedule. Blood pressure was measured periodically via 24-hour automated monitoring equipment. Mean plasma renin activity in the patient groups ranged from 1.58 to 2.68 ng angiotensin I/ml/hr. Plasma renin activity was promptly suppressed in all groups receiving enalkiren. Prolonged duration of plasma renin activity suppression (greater than or equal to 24 hours) was demonstrated after the administration of 1.2 mg/kg enalkiren. The 0.3 mg/kg q.i.d. and 1.2 mg/kg quotid. regimens produced statistically significant reductions (p less than or equal to 0.05) in systolic and diastolic blood pressures with clear evidence of persistent antihypertensive activity for 12 hours or more when compared with the placebo group. Despite relatively large reductions in mean systolic and diastolic blood pressure, mean pulse rates were essentially unchanged. The prolonged reduction in blood pressure with enalkiren without evidence of tachyphylaxis after 1 week of treatment suggests that renin inhibitors may emerge as useful therapeutic agents for the treatment of hypertension.

Hemodynamic and humoral effects of the new renin inhibitor enalkiren in normal humans.

The effect of the renin inhibitor enalkiren (Abbott-64662) was evaluated in eight normal volunteer subjects on a standardized sodium diet (100 mmol/day) by measurement of various components of the renin-angiotensin system and drug levels in plasma. On day 1, vehicle and doses of 0.001, 0.003, and 0.01 mg/kg i.v. were administered within 2 minutes at 90-minute intervals. On day 2, vehicle and doses of 0.01, 0.03, and 0.1 mg/kg i.v. were given. With the higher doses, blood pressure tended to decrease slightly with no change in heart rate. Plasma renin activity and plasma angiotensin-(1-8)octapeptide (angiotensin II) fell markedly in a dose-dependent manner. Inhibition of plasma renin activity was maximal 5 minutes after administration of the drug and persisted 90 minutes after the doses of 0.03 and 0.1 mg/kg. Not surprisingly, there was a close correlation between plasma renin activity and plasma angiotensin II levels (r = 0.81, n = 28, p less than 0.001). In contrast, active and total renin measured directly by monoclonal antibodies rose in dose-related fashion in response to renin inhibition. Pharmacokinetic parameters were calculated using the plasma drug concentrations obtained up to 6 hours after the 0.1 mg/kg dose. By means of a two-compartment model, plasma mean half-life of the drug was estimated at 1.60 +/- 0.43 hours.

Longitudinal assessment of everolimus in de novo renal transplant recipients over the first post-transplant year: pharmacokinetics, exposure-response relationships, and influence on cyclosporine.

OBJECTIVE: Our objective was to characterize the steady-state pharmacokinetics of everolimus and cyclosporine (INN, ciclosporin) when coadministered in de novo kidney allograft recipients during the first year after transplantation. METHOD: This study was a multicenter randomized double-blind study of 101 patients who were randomly assigned 1:1:1 to receive everolimus tablets at doses of 0.5 mg, 1 mg, or 2 mg twice daily with cyclosporine and prednisone. Blood sampling for the pharmacokinetics of everolimus and cyclosporine was performed on day 1, on weeks 1, 2, 3, and 4, and on months 2, 3, 6, 9, and 12. Everolimus dose-proportionality and stability over time were assessed in the context of linear regression and ANOVA models. Everolimus exposure-response relationships between area under the blood concentration-time curve (AUC) and changes in platelets, leukocytes, and lipids were explored with the median-effect model. Potential differences in cyclosporine dosing and pharmacokinetics at different levels of everolimus exposure were assessed in the context of ANOVA. RESULTS: Everolimus steady state was reached on or before day 7, with a median 3-fold accumulation of drug exposure compared with that after the first postoperative dose. Both steady-state maximum concentration and AUC were dose proportional over the full dose range when assessed on day 1, as well as for the full duration of the study at steady state. There was evidence for longitudinal stability in AUC of everolimus during the course of the study. The interindividual pharmacokinetic variability for AUC was 85.4% and intraindividual, interoccasion variability was 40.8%. Age (range, 17-69 years), weight (range, 49-106 kg), and sex (65 men and 36 women) were not significant contributors to variability. There was an increasing incidence of transient thrombocytopenia (< or =100 x 10(9)/L) with increasing everolimus AUC (P = .03). Cyclosporine doses, trough concentrations, and AUC exhibited similar temporal patterns during the course of the study regardless of the co-administered everolimus dose level (P = .13, .82, and .76, respectively). CONCLUSIONS: Everolimus exhibited dose-proportional, stable exposure during the first post-transplant year. For a 4-fold range of everolimus doses there were no differential effects on cyclosporine dosing or pharmacokinetics.

Endogenous nitric oxide synthase inhibitors are responsible for the L-arginine paradox.

L-Arginine, the substrate of nitric oxide (NO) synthases (NOSs), is found in the mammalian organism at concentrations by far exceeding K(M) values of these enzymes. Therefore, additional L-arginine should not enhance NO formation. In vivo, however, increasing L-arginine concentration in plasma has been shown repeatedly to increase NO production. This phenomenon has been named the L-arginine paradox; it has found no satisfactory explanation so far. In the present work, evidence for the hypothesis that the endogenous NOS inhibitors methylarginines, asymmetric dimethylarginine being the most powerful (IC(50) 1.5 microM), are responsible for the L-arginine paradox is presented.