Improving the Accuracy of Predictive 2D-LC Optimization Strategies: Incorporation of Simulated Elution Profiles to Account for Injection Band Broadening in Online Comprehensive Two-Dimensional Liquid Chromatography.

Method development in online comprehensive two-dimensional liquid chromatography (LC × LC) requires the selection of a large number of experimental parameters. The complexity of this process has led to several computer-based LC × LC optimization algorithms being developed to facilitate LC × LC method development. One particularly relevant challenge for predictive optimization software is to accurately model the effect of second dimension (2D) injection band broadening under sample solvent mismatch and/or sample volume overload conditions. We report a novel methodology that combines a chromatographic numerical simulation model capable of predicting elution profiles of analytes under conditions where peak distortion occurs with a predictive multiparameter Pareto optimization approach for online LC × LC. Preliminary method optimization is performed using a theoretical model to predict 2D injection profiles, and optimal experimental configurations obtained from the Pareto fronts are then subjected to further optimization using the simulation model. This approach drastically reduces the number of simulations and therefore the computational demand. We show that the optimal experimental conditions obtained in this manner are similar to those obtained using a complete optimization using only the simulation model. Online HILIC × RP-LC separation of phenolic compounds was used to compare experimental data to simulated two- and three-dimensional contour plots. The main advantage of the proposed approach is the ability to predict the formation of split or deformed peaks in the 2D, a significant benefit in online LC × LC method optimization, especially for separation combinations with mismatched mobile phases. A further benefit is that simulated elution profiles can be used for the visualization of predicted two-dimensional chromatograms for method selection.

Transcriptional responses of neonatal mouse lung to hyperoxia by Nrf2 status.

UNLABELLED: Hyperoxia exposure can inhibit alveolar growth in the neonatal lung through induction of p21/p53 pathways and is a risk factor for the development of bronchopulmonary dysplasia (BPD) in preterm infants. We previously found that activation of nuclear factor erythroid 2 p45-related factor (Nrf2) improved survival in neonatal mice exposed to hyperoxia likely due to increased expression of anti-oxidant response genes. It is not known however, whether hyperoxic induced Nrf2 activation attenuates the growth impairment caused by hyperoxia in neonatal lung. To determine if Nrf2 activation modulates cell cycle regulatory pathway genes associated with growth arrest we examined the gene expression in the lungs of Nrf2(-/-) and Nrf2(+/+) neonatal mice at one and 3days of hyperoxia exposure. METHODS: Microarray analysis was performed in neonatal Nrf2(+/+) and Nrf2(-/-) lungs exposed to one and 3days of hyperoxia. Sulforaphane, an inducer of Nrf2 was given to timed pregnant mice to determine if in utero exposure attenuated p21 and IL-6 gene expression in wildtype neonatal mice exposed to hyperoxia. RESULTS: Cell cycle regulatory genes were induced in Nrf2(-/-) lung at 1day of hyperoxia. At 3days of hyperoxia, induction of cell cycle regulatory genes was similar in Nrf2(+/+) and Nrf2(-/-) lungs, despite higher inflammatory gene expression in Nrf2(-/-) lung. CONCLUSION: p21/p53 pathways gene expression was not attenuated by Nrf2 activation in neonatal lung. In utero SUL did not attenuate p21 expression in wildtype neonatal lung exposed to hyperoxia. These findings suggest that although Nrf2 activation induces expression of anti-oxidant genes, it does not attenuate alveolar growth arrest caused by exposure to hyperoxia.

Characterization of a high throughput approach for large scale retention measurement in liquid chromatography.

Many contemporary challenges in liquid chromatography-such as the need for "smarter" method development tools, and deeper understanding of chromatographic phenomena-could be addressed more efficiently and effectively with larger volumes of experimental retention data than are available. The paucity of publicly accessible, high-quality measurements needed for the development of retention models and simulation tools has largely been due to the high cost in time and resources associated with traditional retention measurement approaches. Recently we described an approach to improve the throughput of such measurements by using very short columns (typically 5 mm), while maintaining measurement accuracy. In this paper we present a perspective on the characteristics of a dataset containing about 13,000 retention measurements obtained using this approach, and describe a different sample introduction method that is better suited to this application than the approach we used in prior work. The dataset comprises results for 35 different small molecules, nine different stationary phases, and several mobile phase compositions for each analyte/phase combination. During the acquisition of these data, we have interspersed repeated measurements of a small number of compounds for quality control purposes. The data from these measurements not only enable detection of outliers but also assessment of the repeatability and reproducibility of retention measurements over time. For retention factors greater than 1, the mean relative standard deviation (RSD) of replicate (typically n=5) measurements is 0.4%, and the standard deviation of RSDs is 0.4%. Most differences between selectivity values measured six months apart for 15 non-ionogenic compounds were in the range of +/- 1%, indicating good reproducibility. A critically important observation from these analyses is that selectivity defined as retention of a given analyte relative to the retention of a reference compound (kx/kref) is a much more consistent measure of retention over a time span of months compared to the retention factor alone. While this work and dataset also highlight the importance of stationary phase stability over time for achieving reliable retention measurements, we are nevertheless optimistic that this approach will enable the compilation of large databases (>> 10,000 measurements) of retention values over long time periods (years), which can in turn be leveraged to address some of the most important contemporary challenges in liquid chromatography. All the data discussed in the manuscript are provided as Supplemental Information.

Neonatal hyperoxia contributes additively to cigarette smoke-induced chronic obstructive pulmonary disease changes in adult mice.

The extent by which early postnatal lung injury contributes to the development of chronic obstructive pulmonary disease (COPD) in the adult is unclear. We hypothesized that exposure to hyperoxia during early postnatal life can augment lung changes caused by adult chronic cigarette smoke (CS) exposure. C57BL/6J mice (1 d old) were exposed to hyperoxia (O(2)) for 5 days. At 1 month of age, half of the O(2)-exposed mice and half of the control mice were placed in a CS chamber for 6 months. After exposure to CS, mice underwent quasi-static pressure-volume curve and mean chord length measurements; quantification of pro-Sp-c expression; and measurement of lung IL-8/ KC, CXCR2/IL8Rα, TNF-α, and IL-6 mRNA by real-time PCR. Adult mice exposed to O(2)+CS had significantly larger chord length measurements (P < 0.02) and lung volumes at 35 cm H(2)O (P < 0.05) compared with all other groups. They also had significantly less pro-Sp-c protein and surfactant protein C mRNA expression (P < 0.003). Mice exposed to O(2)+CS and CS-only mice had significantly higher lung resistance and longer mean time constants (P < 0.01), significantly more inflammatory cells in the bronchoalveolar lavage fluid (P < 0.03), and significantly higher levels of lung CXCR2/IL8Rα mRNA compared with mice not exposed to smoke (P < 0.02). We conclude that exposure to early postnatal hyperoxia contributed additively to CS-induced COPD changes in adult mice. These results may be relevant to a growing population of preterm children who sustained lung injury in the newborn period and may be exposed to CS in later life.

Predicting neurally mediated syncope based on pulse arrival time: algorithm development and preliminary results.

BACKGROUND: Neurally mediated syncope (NMS) is a common disorder that is triggered by orthostatic stress. The circulatory adjustments to orthostatic stress occur just prior to a sudden loss of consciousness. NMS prediction would protect patients from falls or accidents. METHODS AND RESULTS: Based on simultaneously recorded heart rate (HR) and pulse wave during 70° head-up tilt (HUT) table testing we investigated a syncope warning system. In 14 patients with a history of suspected NMS we tested 2 algorithms based on HR and/or pulse arrival time (PAT). When the cumulative risk exceeded the threshold, which was calculated during the first 2 minutes following the posture change to upright position, a syncope prediction alarm was triggered. All syncopes (n = 7) were detected more than 16 seconds before the onset of dizziness or unconsciousness by using a prediction alarm based on HR and PAT (syncope prediction algorithm 2). No false alarm was generated in patients with negative HUT (n = 7). Syncope prediction was improved by detecting the slope of HR changes as compared with monitoring PAT changes alone (syncope prediction algorithm 1). The duration between the prediction alarm and the occurrence of syncope was 99 ± 108 seconds. CONCLUSION: Predicting NMS is feasible by monitoring HR and the onset of the pulse wave at the periphery. This approach might improve NMS management.

Exposure to neonatal cigarette smoke causes durable lung changes but does not potentiate cigarette smoke-induced chronic obstructive pulmonary disease in adult mice.

The impact of early childhood cigarette smoke (CS) exposure on CS-induced chronic obstructive pulmonary disease (COPD) is unknown. This study was performed to evaluate the individual and combined effects of neonatal and adult CS exposure on lung structure, function, and gene expression in adult mice. To model a childhood CS exposure, neonatal C57/B6 mice were exposed to 14 days of CS (Neo CS). At 10 weeks of age, Neo CS and control mice were exposed to 4 months of CS. Pulmonary function tests, bronchoalveolar lavage, and lung morphometry were measured and gene expression profiling was performed on lung tissue. Mean chord lengths and lung volumes were increased in neonatal and/or adult CS-exposed mice. Differences in immune, cornified envelope protein, muscle, and erythrocyte genes were found in CS-exposed lung. Neonatal CS exposure caused durable structural and functional changes in the adult lung but did not potentiate CS-induced COPD changes. Cornified envelope protein gene expression was decreased in all CS-exposed mice, whereas myosin and erythrocyte gene expression was increased in mice exposed to both neonatal and adult CS, suggesting an adaptive response. Additional studies may be warranted to determine the utility of these genes as biomarkers of respiratory outcomes.

Modelling of analyte profiles and band broadening generated by interface loops used in multi-dimensional liquid chromatography.

Currently, the shape and variance of the analyte band entering the second dimension column when injected from an open loop interface in two-dimensional liquid chromatography is not fully understood. This is however important as it is connected to several other variables encountered when developing 2D-LC methods, including the first dimension flow rate, the sampling (modulation) time and the loop volume. Both numerical simulation methods and experimental measurements were used to understand and quantify the dispersion occurring in open tubular interface loops. Variables included are the analyte diffusion coefficient (Dmol), loop filling and emptying rates (Ffill & Fempty), loop inner diameter or radius (Rloop) and loop volume (Vloop). For a straight loop capillary, we find that the concentration profile (as measured at the loop outlet) depends only on a single dimensionless parameter tempty*=VloopFempty·DmolRloop2 and the ratio of the filling and emptying flow rates Fempty/Ffill. A model depending only on these two parameters was developed to predict of the peak variance resulting from the filling and emptying of a straight capillary operated in the first-in-last-out (FILO) modulation mode. Comparison of the concentration profiles and the corresponding variances obtained by either numerical simulation or experiments with straight capillaries shows the results generally agree very well. When the straight capillary is replaced by a tightly coiled loop, significantly smaller (20-40%) peak variances are observed compared to straight capillaries. The magnitude of these decreases is not predicted as well by simulations, however the simulation results are still useful in this case, because they represent an upper boundary (i.e., worst-case scenario) on the predicted variance.

Implications of dispersion in connecting capillaries for separation systems involving post-column flow splitting.

It is common practice in liquid chromatography to split the flow of the effluent exiting the analytical column into two or more parts, either to enable parallel detection (e.g., coupling the separation to two destructive detectors such as light scattering and mass spectrometry (MS)), or to accommodate flow rate limitations of a detector (e.g., electrospray ionization mass spectrometry). In these instances the user must make choices about split ratio and dimensions of connecting tubing that is used between the split point and the detector, however these details are frequently not mentioned in the literature, and rarely justified. In our own work we often split the effluent following the second dimension (2D) column in two-dimensional liquid chromatography systems coupled to MS detection, and we have frequently observed post 2D column peak broadening that is larger than we would expect to result from dispersion in the MS ionization source itself. For the present paper we describe a series of experiments aimed at understanding the impact of the split ratio and post-split connecting tubing dimensions on dispersion of peaks exiting an analytical column. We start with the simple idea - based on the principle of conservation of mass - that analyte peaks entering the split point are split into two parts such that the analyte mass (and thus peak volume) entering and exiting the split point is conserved, and directly related to the ratio of flow rates entering and exiting the split point. Measurements of peak width and variance after the split point show that this simple view of the splitting process - along with estimates of additional dispersion in the post-split tubing - is sufficient to predict peak variances at the detector with accuracy that is sufficient to guide experimental work (median error of about 10% over a wide range of conditions). We feel it is most impactful to recognize that flow splitting impacts apparent post-column dispersion not because anything unexpected happens in the splitting process, but because the split dramatically reduces the volume of the analyte peak, which then is more susceptible to dispersion in connecting tubing that would not cause significant dispersion under conditions where splitting is not implemented. These results will provide practitioners with a solid basis on which rational decisions about split ratios and dimensions of post-split tubing can be made.

Characterization of the non-invasive assessment of the cutaneous microcirculation by laser Doppler perfusion scanner.

OBJECTIVE: Microcirculatory dysfunction contributes to morbidity and mortality in vascular diseases. Here, we aimed at establishing a sensitive and valid method to measure microvascular reactivity during post-occlusive reactive hyperemia (PORH) using scanning laser Doppler perfusion imaging (LDPI) of the forearm. METHODS: In a first series, LDPI was methodologically evaluated on the volar forearm of healthy volunteers (n = 10) before and after one to five minutes of upper arm occlusion. In a second series, readings were performed in 20 healthy subjects and 20 patients with coronary artery disease (CAD). RESULTS: Three minutes of forearm occlusion were sufficient to induce maximal vasodilation during PORH as indicated by maximal increase in perfusion unit (PU) amplitude that did not further increase after five-minute occlusion. Five-minute occlusion led to a significant prolongation of PORH with greater area under curve (AUC) suggesting longer lasting vasodilation of microvessels. The five-minute occlusion was associated with lower variability as compared with three minutes (intraindividual variability: 9-17% vs. 12-21%; interindividual variability: 13-24% vs. 14-26%). CAD patients exhibited significantly reduced amplitude (105 +/- 49 vs. 164 +/- 35 PU; p < 0.001), ratio (4.7 +/- 1.8 vs. 7.1 +/- 1.8; p < 0.001), and AUC (1656 +/- 1070 vs. 2723 +/- 864 PU x minutes; p = 0.001). CONCLUSION: Scanning LDPI is a feasible and reproducible method for non-invasive assessment of the cutaneous microcirculatory response during PORH.

z-Tree unleashed: A novel client-integrating architecture for conducting z-Tree experiments over the Internet.

We present z-Tree unleashed, a novel approach and set of scripts to aid the implementation of computerized behavioral experiments outside the laboratory. z-Tree unleashed enables subjects to join the experiment using a web portal that requires no software apart from a web browser. Experimenters are likewise enabled to administer their experiments from anywhere in the world. Except for z-Tree itself, z-Tree unleashed is entirely based on free and open-source software. In this paper we give a high-level overview of z-Tree unleashed's features and benefits and its design. We also show how to set up the server and demonstrate the steps required for conducting an entire experiment. We subsequently explain how to leverage the security and routing features of a virtual private network with z-Tree unleashed, enabling servers to securely run behind routers.

Numerical and experimental investigation of analyte breakthrough from sampling loops used for multi-dimensional liquid chromatography.

Two-dimensional liquid chromatography is increasingly being used to address challenging separations in fields ranging from pharmaceutical analysis to the food industry. A significant impediment to development of more methods is the lack of a complete theoretical foundation upon which sound development decisions can be made. One parameter that is currently not fully understood is the extent of filling of sampling loops in the case where effluent from the first dimension separation is transferred to the second dimension separation through this type of open loop interface. This is a highly important parameter because it is connected to several other variables in a 2D-LC system, including the first dimension flow rate, the sampling (modulation) time, and the loop volume. In this study we have used both numerical simulation methods and experimental measurements to understand the extent to which sampling loops can be filled before a significant fraction of the analyte is lost from the end of the loop. Variables included in the study are the analyte diffusion coefficient (Dmol), loop filling rate (Ffill), loop inner diameter or radius (Rloop) and loop volume (Vloop). For a straight loop capillary we find that analyte breakthrough curves (as measured at the loop outlet) depend only on a single the dimensionless parameter t*=VloopFfill·DmolRloop2 . As a function of this parameter, the fraction of analyte lost from the loop outlet for different extents of loop filling could be calculated, allowing to develop guidelines for the maximum permissible extent of filling before a specified level of analyte loss is reached. Breakthrough measurements using a coiled loop capillary show that less breakthrough is observed compared to the straight capillary at high filling flow rates, presumably due to secondary flows that increase radial dispersion. These measurements enabled the calculation of apparent radial diffusion coefficients for use with coiled capillaries such that the same relation for t* can be used to predict analyte loss due to breakthrough. These results should be very useful to practitioners of 2D-LC, enabling them to make rational decisions about the extent of loop filling on the basis of experimental conditions and analyte type.

Serial measurements of whole blood nitrite in an intensive care setting.

Nitrite plays an eminent role in cardiovascular physiology and pathology, mediating hypoxic vasodilation, reducing ischemia-reperfusion injury, and regulating cardiac energetics and function. The role of circulating nitrite in critically ill patients has not been examined so far. To investigate whether whole blood nitrite can be determined reproducibly in an intensive care setting, 30 patients from a cardiology intensive care unit were enrolled in this study, no matter what the underlying disease. Blood was drawn from an arterial catheter and whole blood nitrite was determined, using a tri-iodide/ozone-based chemiluminescence assay after incubation with a ferricyanide-containing stabilization solution. Whole blood nitrite levels ranged from 35 to 1193 nmol/L (mean+/-SEM: 220+/-20 nmol/L). Myocardial infarction was associated with lower whole blood nitrite levels (200+/-53 nmol/L for elevated serum CK MB levels vs 432+/-95 nmol/L in the normal CK MB range, p=0.039). Neither impaired kidney function nor an inflammatory state was associated with higher or lower whole blood nitrite levels. In conclusion, whole blood nitrite can be measured easily and reproducibly in critically ill patients, regardless of renal function and inflammation. The origin of decreased nitrite levels in myocardial infarction is currently unclear and needs to be further elucidated.

The SDF-1/CXCR4 ligand/receptor pair is an important contributor to several types of ocular neovascularization.

Hypoxia causes increased expression of several proteins that have the potential to promote neovascularization. Vascular endothelial growth factor (VEGF) is up-regulated by hypoxia in the retina and plays a central role in the development of several types of ocular neovascularization, but the effects of other hypoxia-regulated proteins are less clear. Stromal-derived factor-1 (SDF-1) and its receptor, CXCR4, have hypoxia response elements in the promoter regions of their genes and are increased in hypoxic liver and heart. In this study, we found that SDF-1 and CXCR4 are increased in hypoxic retina, with SDF-1 localized in glial cells primarily near the surface of the retina and CXCR4 localized in bone marrow-derived cells. Glial cells also expressed CXCR4, which suggested the possibility of autocrine stimulation, but influx of bone marrow-derived cells is the major source of increased levels of CXCR4. High levels of VEGF in the retina in the absence of hypoxia also increased levels of Cxcr4 and Sdf1 mRNA. CXCR4 antagonists reduced influx of bone marrow-derived cells into ischemic retina and strongly suppressed retinal neovascularization, VEGF-induced subretinal neovascularization, and choroidal neovascularization. These data suggest that SDF-1 and CXCR4 contribute to the involvement of bone marrow-derived cells and collaborate with VEGF in the development of several types of ocular neovascularization. They provide new targets for therapeutic intervention that may help to bolster and supplement effects obtained with VEGF antagonists.

Resting microvascular resistance and conduit artery tone: relevance to endothelium-dependent flow-mediated dilation.

BACKGROUND: Conduit arteries respond to increases in flow by dilating, which is mediated by endothelium-derived nitric oxide. The significance of the interaction between microcirculation and macrocirculation in the flow-mediated dilation (FMD) is poorly understood. HYPOTHESIS: We hypothesize that baseline conduit artery vasomotor tone (CAVT) and resting microvascular resistance (MVR) predict FMD. METHODS: We investigated resting diameter and FMD of the brachial artery using high-resolution ultrasound and forearm blood flow with plethysmography in 60 healthy individuals. CAVT was calculated as change of the arterial diameter from baseline to maximal dilation expressed as percentage of the maximum dilation. Resting MVR was determined as quotient of mean arterial blood pressure and forearm blood flow. RESULTS: Mean FMD was 10.7+/-2.0%, indicating normal endothelial function. The extent of brachial artery FMD was not only related to baseline CAVT (r=0.70, P<0.01), but inversely to resting MVR (r=-0.69, P<0.01). Moreover, in a simple regression analysis, baseline CAVT was inversely related to resting MVR (r=-0.82, P<0.01). In a multivariate linear regression analysis, baseline CAVT and MVR were identified as independent predictors of brachial artery FMD. CONCLUSION: Our data imply a close interaction of resting microvascular resistance and baseline CAVT modulating flow-mediated conduit artery dilation.

Evidence for in vivo transport of bioactive nitric oxide in human plasma.

Although hitherto considered as a strictly locally acting vasodilator, results from recent clinical studies with inhaled nitric oxide (NO) indicate that NO can exert effects beyond the pulmonary circulation. We therefore sought to investigate potential remote vascular effects of intra-arterially applied aqueous NO solution and to identify the mechanisms involved. On bolus application of NO into the brachial artery of 32 healthy volunteers, both diameter of the downstream radial artery and forearm blood flow increased in a dose-dependent manner. Maximum dilator responses were comparable to those after stimulation of endogenous NO formation with acetylcholine and bradykinin. Response kinetics and pattern of NO decomposition suggested that despite the presence of hemoglobin-containing erythrocytes, a significant portion of NO was transported in its unbound form. Infusion of NO (36 micromol/min) into the brachial artery increased levels of plasma nitroso species, nitrite, and nitrate in the draining antecubital vein (by < 2-fold, 30-fold, and 4-fold, respectively), indicative of oxidative and nitrosative chemistry. Infused N-oxides were inactive as vasodilators whereas S-nitrosoglutathione dilated conduit and resistance arteries. Our results suggest that NO can be transported in bioactive form for significant distances along the vascular bed. Both free NO and plasma nitroso species contribute to the dilation of the downstream vasculature.

Recent methodological advances in the analysis of nitrite in the human circulation: nitrite as a biochemical parameter of the L-arginine/NO pathway.

Nitric oxide (NO) plays a pivotal role in the modulation of multiple physiological processes. It acts as a messenger molecule within the cardiovascular system. NO is a highly unstable free radical in circulating blood and is oxidized rapidly to nitrite and nitrate. Recent studies suggest that nitrite has the potential to function as a surrogate of NO production under physiological and pathophysiological conditions and could therefore be of high relevance as a biochemical parameter in experimental and clinical studies. Under hypoxic conditions nitrite is reduced to bioactive NO by deoxyhemoglobin. This mechanism may represent a dynamic cycle of NO generation to adapt the demand and supply for the vascular system. Because of these potential biological functions the concentration of nitrite in blood is thought to be of particular importance. The determination of nitrite in biological matrices represents a considerable analytical challenge. Methodological problems often arise from pre-analytical sample preparation, sample contamination due to the ubiquity of nitrite, and from lack of selectivity and sensitivity. These analytical difficulties may be a plausible explanation for reported highly diverging concentrations of nitrite in the human circulation. The aim of this article is to review the methods of quantitative analysis of nitrite in the human circulation, notably in plasma and blood, and to discuss pre-analytical and analytical factors potentially affecting accurate quantification of nitrite in these human fluids.

Nitric oxide synthase-derived plasma nitrite predicts exercise capacity.

BACKGROUND: Nitrite is the main oxidation product of nitric oxide (NO) in plasma. It sensitively reflects changes in endothelial NO synthase (eNOS) activity under fasting conditions and serves as an endocrine NO donor, contributing to the regulation of blood flow through reaction with haemoglobin. As NO is necessary to maintain an adequate vascular response to the increased demands of blood flow, it is believed to be important for vasodilation induced by exercise. OBJECTIVE: To investigate whether the capacity of the vasculature to produce nitrite is associated with exercise performance. DESIGN: With the use of chemiluminescence detection, nitrite concentrations in 55 healthy subjects (mean (SEM) age 40 (2) years; 22 men) were studied before and after an exercise test, and endothelial function was determined by measuring flow-mediated dilation of the brachial artery using high-resolution ultrasound. In a subset of subjects, the NOS inhibitor, N(G)-monomethyl-L-arginine, was applied to elucidate the effect of eNOS on changes in nitrite. RESULTS: Exercise significantly (p<0.001) increased plasma nitrite from 97 (6) to 125 (8) nM. The relative increase in plasma nitrite was related to flow-mediated dilation (6.1 (0.3)%; r = 0.36; p = 0.01). N(G)-Monomethyl-L-arginine blocked increases in nitrite. Post-exercise nitrite concentration correlated with exercise performance, as determined by maximally reached stress power (r = 0.37; p<0.007), and inversely with age. Multivariate analysis showed that both age and post-exercise nitrite concentration were independent predictors of stress endurance and power. CONCLUSION: The results suggest a role for plasma nitrite in the adaptation of haemodynamics during exercise. An impaired increase in plasma nitrite may limit exercise capacity.

Plasma nitrite reserve and endothelial function in the human forearm circulation.

Attenuation of endothelium-derived nitric oxide (NO) synthesis is a hallmark of endothelial dysfunction. Early detection of this disorder may have therapeutic and prognostic implications. Plasma nitrite mirrors acute and chronic changes in endothelial NO-synthase activity. We hypothesized that local plasma nitrite concentration increases during reactive hyperemia of the forearm, reflecting endothelial function. In healthy subjects (n = 11) plasma nitrite and nitrate were determined at baseline and during reactive hyperemia of the forearm using reductive gas-phase chemiluminescence and flow-injection analysis, respectively. Endothelium-dependent dilation of the brachial artery was measured as flow-mediated dilation (FMD) using high-resolution ultrasound. Results were compared to patients with endothelial dysfunction as defined by reduced FMD (n = 11). Reactive hyperemia of the forearm increased local plasma nitrite concentration from 68 +/- 5 to 126 +/- 13 nmol/L (p < 0.01), whereas in endothelial dysfunction nitrite remained unaffected (116 +/- 12 to 104 +/- 10 nmol/L; n.s.), corresponding to nitrite reserves of 94 +/- 21 and -8 +/- 4%. This was accompanied by a significantly greater increase in brachial artery diameter (FMD: 8.5 +/- 0.4% vs 2.9 +/- 0.5%, for healthy subjects and endothelial dysfunction, respectively; p < 0.001). This observation suggests that nitrite changes reflect endothelial function. Assessment of local plasma nitrite during reactive hyperemia may open new avenues in the diagnosis of vascular function.

Plasma nitrite concentrations reflect the degree of endothelial dysfunction in humans.

A reduced nitric oxide availability is a hallmark of endothelial dysfunction occurring early in atherosclerosis. Recently, we have shown that plasma nitrite mirrors acute changes in endothelial nitric oxide synthase activity in various mammals, including humans. Here, we examined the hypothesis that plasma nitrite levels are reduced in humans with endothelial dysfunction and the decrease is correlated with increasing numbers of cardiovascular risk factors (RF). Plasma nitrite concentrations were quantified by flow-injection analysis. The coefficient of variation for repeated measurements of plasma nitrite was <8%, and heart rate and blood pressure at the time of blood sampling had no significant effect on nitrite values measured (n=10). Baseline levels of plasma nitrite followed a normal distribution in each group studied and decreased progressively with increasing numbers of cardiovascular risk factors (n=351, p<0.001): 351+/-13 (0 RF), 261+/-10 (1 RF), 253+/-11 (2 RF), 222+/-18 (3 RF), and 171+/-29 nmol/L (4 RF). Intima media thickness (IMT) and flow-mediated dilation (FMD) were determined via ultrasound. Plasma nitrite and FMD levels were lower, whereas IMT was greater in individuals with endothelial dysfunction (n=12) compared to healthy volunteers (n=12). Nitrite correlated significantly with FMD (r=0.56, p<0.001) and inversely with IMT (r= -0.49, p<0.01). Plasma nitrite levels are reliably measurable in humans, indicate endothelial dysfunction, and correlate with cardiovascular risk factors. Future studies are necessary to identify the prognostic relevance of plasma nitrite determination in patients suffering from cardiovascular disease.

Plasma nitrosothiols contribute to the systemic vasodilator effects of intravenously applied NO: experimental and clinical Study on the fate of NO in human blood.

Higher doses of inhaled NO exert effects beyond the pulmonary circulation. How such extrapulmonary effects can be reconciled with the presumed short half-life of NO in the blood is unclear. Whereas erythrocytes have been suggested to participate in NO transport, the exact role of plasma in NO delivery in humans is not clear. Therefore, we investigated potential routes of NO decomposition and transport in human plasma. NO consumption in plasma was accompanied by a concentration-dependent increase in nitrite and S-nitrosothiols (RSNOs), with no apparent saturation limit up to 200 micro mol/L. The presence of red blood cells reduced the formation of plasma RSNOs. Intravenous infusion of 30 micro mol/min NO in healthy volunteers increased plasma levels of RSNOs and induced systemic hemodynamic effects at the level of both conduit and resistance vessels, as reflected by dilator responses in the brachial artery and forearm microvasculature. Intravenous application of S-nitrosoglutathione, a potential carrier of bioactive NO, mimicked the vascular effects of NO, whereas nitrite and nitrate were inactive. Changes in plasma nitrosothiols were correlated with vasodilator effects after intravenous application of S-nitrosoglutathione and NO. These findings demonstrate that in humans the pharmacological delivery of NO solutions results in the transport and delivery of NO as RSNOs along the vascular tree.