H2S prevents the disruption of the blood-brain barrier in rats with prenatal hyperhomocysteinemia.

Elevation of the homocysteine concentration in the plasma called hyperhomocysteinemia (hHCY) during pregnancy causes a number of pre- and postnatal developmental disorders. The aim of our study was to analyze the effects of H2S donors -NaHS and N-acetylcysteine (NAC) on blood-brain barrier (BBB) permeability in rats with prenatal hHCY. In rats with mild hHCY BBB permeability assessed by Evans Blue extravasation in brain increased markedly throughout life. Administration of NaHS or NAC during pregnancy attenuated hHCY-associated damage and increased endogenous concentrations of sulfides in brain tissues. Acute application of dl-homocysteine thiolactone induced BBB leakage, which was prevented by the NMDA receptor antagonist MK-801 or H2S donors. Rats with hHCY demonstrated high levels of NO metabolite - nitrites and proinflammatory cytokines (IL-1ß, TNF-α, IL-6) in brain. Lactate dehydrogenase (LDH) activity in the serum was higher in rats with hHCY. Mitochondrial complex-I activity was lower in brain of hHCY rats. NaHS treatment during pregnancy restored levels of proinflammatory cytokines, nitrites and activity of the respiratory chain complex in brain as well as the LDH activity in serum. Our data suggest that H2S has neuroprotective effects against prenatal hHCY-associated BBB disturbance providing a potential strategy for the prevention of developmental impairments in newborns.

Relationships between nitric oxide biomarkers and physiological outcomes following dietary nitrate supplementation.

Dietary nitrate (NO3-) supplementation can increase nitric oxide (NO) bioavailability, reduce blood pressure (BP) and improve muscle contractile function in humans. Plasma nitrite concentration (plasma [NO2-]) is the most oft-used biomarker of NO bioavailability. However, it is unclear which of several NO biomarkers (NO3-, NO2-, S-nitrosothiols (RSNOs)) in plasma, whole blood (WB), red blood cells (RBC) and skeletal muscle correlate with the physiological effects of acute and chronic dietary NO3- supplementation. Using a randomized, double-blind, crossover design, 12 participants (9 males) consumed NO3--rich beetroot juice (BR) (∼12.8 mmol NO3-) and NO3--depleted placebo beetroot juice (PL) acutely and then chronically (for two weeks). Biological samples were collected, resting BP was assessed, and 10 maximal voluntary isometric contractions of the knee extensors were performed at 2.5-3.5 h following supplement ingestion on day 1 and day 14. Diastolic BP was significantly lower in BR (-2 ± 3 mmHg, P = 0.03) compared to PL following acute supplementation, while the absolute rate of torque development (RTD) was significantly greater in BR at 0-30 ms (39 ± 57 N m s-1, P = 0.03) and 0-50 ms (79 ± 99 N m s-1, P = 0.02) compared to PL following two weeks supplementation. Greater WB [RSNOs] rather than plasma [NO2-] was correlated with lower diastolic BP (r = -0.68, P = 0.02) in BR compared to PL following acute supplementation, while greater skeletal muscle [NO3-] was correlated with greater RTD at 0-30 ms (r = 0.64, P=0.03) in BR compared to PL following chronic supplementation. We conclude that [RSNOs] in blood, and [NO3-] in skeletal muscle, are relevant biomarkers of NO bioavailability which are related to the reduction of BP and the enhanced muscle contractile function following dietary NO3- ingestion in humans.

Nitrate ingestion blunts the increase in blood pressure during cool air exposure: a double-blind, placebo-controlled, randomized, crossover trial.

Cold exposure increases blood pressure (BP) and salivary flow rate (SFR). Increased cold-induced SFR would be hypothesized to enhance oral nitrate delivery for reduction to nitrite by oral anaerobes and to subsequently elevate plasma [nitrite] and nitric oxide bioavailability. We tested the hypothesis that dietary nitrate supplementation would increase plasma [nitrite] and lower BP to a greater extent in cool compared with normothermic conditions. Twelve males attended the laboratory on four occasions. Baseline measurements were completed at 28°C. Subsequently, participants ingested 140 mL of concentrated nitrate-rich (BR; ∼13 mmol nitrate) or nitrate-depleted (PL) beetroot juice. Measurements were repeated over 3 h at either 28°C (Norm) or 20°C (Cool). Mean skin temperature was lowered compared with baseline in PL-Cool and BR-Cool. SFR was greater in BR-Norm, PL-Cool, and BR-Cool than PL-Norm. Plasma [nitrite] at 3 h was higher in BR-Cool (592 ± 239 nM) versus BR-Norm (410 ± 195 nM). Systolic BP (SBP) at 3 h was not different between PL-Norm (117 ± 6 mmHg) and BR-Norm (113 ± 9 mmHg). SBP increased above baseline at 1, 2, and 3 h in PL-Cool but not BR-Cool. These results suggest that BR consumption is more effective at increasing plasma [nitrite] in cool compared with normothermic conditions and blunts the rise in BP following acute cool air exposure, which might have implications for attenuating the increased cardiovascular strain in the cold.NEW & NOTEWORTHY Compared with normothermic conditions, acute nitrate ingestion increased plasma [nitrite], a substrate for oxygen-independent nitric oxide generation, to a greater extent during cool air exposure. Systolic blood pressure was increased during cool air exposure in the placebo condition with this cool-induced blood pressure increase attenuated after acute nitrate ingestion. These findings improve our understanding of environmental factors that influence nitrate metabolism and the efficacy of nitrate supplementation to lower blood pressure.

Migraine Attacks Triggered by Ingestion of Watermelon.

INTRODUCTION: Ingesting some foods can trigger headache attacks in migraine patients. Diet-sourced citrulline activates the l-arginine-nitric oxide pathway, acting on the pathophysiology of migraine. METHODS: The study was a clinical trial, interventional, controlled, and with group comparison. The sample was non-random, composed of 38 volunteers with migraine and 38 without headache (control). Both groups ingested a portion of watermelon to determine the onset of headache attacks. Before and after ingesting watermelon, they underwent blood collections to determine serum nitrite levels. RESULTS: There were 38 volunteers diagnosed with migraine without aura and 38 controls, whose mean age was, respectively, 22.4 ± 1.5 and 22.9 ± 3.1 years (p = 0.791). Headache was triggered by watermelon ingestion after 124.3 ± 20.5 min of ingestion in 23.7% (9/38) of the migraine volunteers and in none of the controls (p = 0.002). There was an increase in serum nitrite levels, both in migraine volunteers (23.4%) and in the control group (24.3%), after watermelon ingestion. This difference was significant (p < 0.001). DISCUSSION: Watermelon ingestion triggered headache attacks in migraine patients and increased serum nitrite levels, attesting to a possible activation of the l-arginine-nitric oxide pathway.

Characterization of the L-Arginine/Nitric Oxide Pathway and Oxidative Stress in Pediatric Patients with Atopic Diseases.

INTRODUCTION: L-Arginine (Arg) is a semi-essential amino acid. Constitutive and inducible nitric oxide synthase (NOS) isoforms convert Arg to nitric oxide (NO), a potent vaso- and bronchodilator with multiple biological functions. Atopic dermatitis (AD) and bronchial asthma (BA) are atopic diseases affecting many children globally. Several studies analyzed NO in airways, yet the systemic synthesis of NO in AD and BA in children with BA, AD or both is elusive. METHODS: In a multicenter study, blood and urine were obtained from 130 of 302 participating children for the measurement of metabolites of the Arg/NO pathway (BA 31.5%; AD 5.4%; AD + BA 36.1%; attention deficit hyperactivity disorder (ADHD) 12.3%). In plasma and urine amino acids Arg and homoarginine (hArg), both substrates of NOS, asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA), both inhibitors of NOS, dimethylamine (DMA), and nitrite and nitrate, were measured by gas chromatography-mass spectrometry. Malondialdehyde (MDA) was measured in plasma and urine samples to evaluate possible effects of oxidative stress. RESULTS: There were no differences in the Arg/NO pathway between the groups of children with different atopic diseases. In comparison to children with ADHD, children with AD, BA or AD and BA had higher plasma nitrite (p < 0.001) and nitrate (p < 0.001) concentrations, suggesting higher systemic NO synthesis in AD and BA. Urinary excretion of DMA was also higher (p = 0.028) in AD and BA compared to patients with ADHD, suggesting elevated ADMA metabolization. DISCUSSION/CONCLUSION: The Arg/NO pathway is activated in atopic diseases independent of severity. Systemic NO synthesis is increased in children with an atopic disease. Plasma and urinary MDA levels did not differ between the groups, suggesting no effect of oxidative stress on the Arg/NO pathway in atopic diseases.

Tadalafil ameliorates memory deficits, oxidative stress, endothelial dysfunction and neuropathological changes in rat model of hyperhomocysteinemia induced vascular dementia.

AIM: The present study investigates the potential of Tadalafil, a phosphodiesterase-5 inhibitor, in a rat model of hyperhomocysteinemia induced vascular dementia. METHODS: Hyperhomocysteinemia induced vascular dementia in Wistar rats was produced by administering l-Methionine (1.7 g/kg/day; p.o.×8 weeks). Learning and memory was assessed by employing Morris water maze (MWM) test. Endothelial dysfunction was assessed through acetylcholine-induced endothelial-dependent vasorelaxation and serum nitrite levels. Various other biochemical and histopathological estimations were also performed. RESULTS: l-Methionine produced significant impairment in acetylcholine-induced endothelium-dependent vasorelaxation and a decrease in serum nitrite levels indicating endothelial dysfunction. Further, these animals performed poorly on Morris water maze, depicting impairment of learning and memory. There was a significant rise in brain oxidative stress level (indicated by an increase in brain thiobarbituric acid reactive species and a decrease in reduced glutathione levels). Increase in brain acetylcholinesterase activity; brain myeloperoxidase activity and brain neutrophil infiltration (a marker of inflammation) were also observed. Tadalafil (5 and 10 mg/kg, p.o.)/Donepezil (0.5 mg/kg, i.p., serving as standard) treatment ameliorated l-Methionine induced endothelial dysfunction; memory deficits; biochemical and histopathological changes in a significant manner. CONCLUSIONS: It may be concluded that tadalafil has shown efficacy in the rat model of l-Methionine induced vascular dementia and that phosphodiesterase-5 can be considered as an important therapeutic target for the treatment of vascular dementia.

Physiological and performance effects of dietary nitrate and N-acetylcysteine supplementation during prolonged heavy-intensity cycling.

The purpose of this study was to investigate effects of concurrent and independent administration of dietary nitrate (NO3-), administered as NO3--rich beetroot juice (BR; ~12.4 mmol of NO3-), and N-acetylcysteine (NAC; 70 mg·kg-1) on physiological responses during prolonged exercise and subsequent high-intensity exercise tolerance. Sixteen recreationally active males supplemented with NO3--depleted beetroot juice (PL) or BR for 6 days and ingested an acute dose of NAC or maltodextrin (MAL) 1 h prior to performing 1 h of heavy-intensity cycling exercise immediately followed by a severe-intensity time-to-exhaustion (TTE) test in four conditions: 1) PL+MAL, 2) PL+NAC, 3) BR+MAL and 4) BR+NAC. Pre-exercise plasma [NO3-] and nitrite ([NO2-]) were elevated following BR+NAC  and BR+MAL (both P < 0.01) compared with PL+NAC and PL+MAL; plasma [cysteine] was increased in PL+NAC  and BR+NAC (both P < 0.01) compared to PL+MAL. Muscle excitability declined over time during the prolonged cycling bout in all conditions  but was better preserved in PL+NAC  compared to BR+NAC (P < 0.01) and PL+MAL (P < 0.05). There was no effect of supplementation on subsequent TTE . These findings indicate that co-ingestion of BR and NAC does not appreciably alter physiological responses during prolonged heavy-intensity cycling or enhance subsequent exercise tolerance.

Placental Ischemia Says "NO" to Proper NOS-Mediated Control of Vascular Tone and Blood Pressure in Preeclampsia.

In this review, we first provide a brief overview of the nitric oxide synthase (NOS) isoforms and biochemistry. This is followed by describing what is known about NOS-mediated blood pressure control during normal pregnancy. Circulating nitric oxide (NO) bioavailability has been assessed by measuring its metabolites, nitrite (NO2) and/or nitrate (NO3), and shown to rise throughout normal pregnancy in humans and rats and decline postpartum. In contrast, placental malperfusion/ischemia leads to systemic reductions in NO bioavailability leading to maternal endothelial and vascular dysfunction with subsequent development of hypertension in PE. We end this article by describing emergent risk factors for placental malperfusion and ischemic disease and discussing strategies to target the NOS system therapeutically to increase NO bioavailability in preeclamptic patients. Throughout this discussion, we highlight the critical importance that experimental animal studies have played in our current understanding of NOS biology in normal pregnancy and their use in finding novel ways to preserve this signaling pathway to prevent the development, treat symptoms, or reduce the severity of PE.

Supplemental nitrite increases choroidal neovascularization in mice.

Low doses of nitrite, close to physiological levels, increase blood flow in normal and ischemic tissues through a nitric oxide (NO) dependent mechanism. Given that nitrite therapy and dietary supplementation with vegetables high in nitrate (e.g. beets) are gaining popularity we decided to determine if low doses of nitrite impact the development of choroidal neovascularization (CNV), a key feature of wet age related macular degeneration (AMD). Sodium nitrite (at 50 mg/L, 150 mg/L, and 300 mg/L), nitrate (1 g/L) or water alone were provided in the drinking water of C57BL/6 J mice aged 2 or 12 months. Mice were allowed to drink ad libitum for 1 week at which time laser-induced choroidal neovascularization (L-CNV) was induced. The mice continued to drink the supplemented water ad libitum for a further 14 days at which point optical coherence tomography (OCT) was performed to determine the volume of the CNV lesion. Blood was drawn to determine nitrite and nitrate levels and eyes taken for histology. CNV volume was 2.86 × 107 µm3 (±0.4 × 107) in young mice on water alone but CNV volume more than doubled to >6.9 × 107 µm3 (±0.8 × 107) in mice receiving 300 mg/L nitrite or 7.34 × 107 µm3 (±1.4 × 107) in 1 g/L nitrate (p < 0.01). A similar trend was observed in older mice. CNV volume was 5.3 × 107 µm3 (±0.5 × 107) in older mice on water alone but CNV volume almost doubled to approximately 9.3 × 107 µm3 (±1.1 × 107) in mice receiving 300 mg/L nitrite or 8.7 × 107 µm3 (±0.9 × 107) 1 g/L nitrate (p < 0.01). Plasma nitrite levels were highest in young mice receiving 150 mg/L in the drinking water with no changes in plasma nitrate observed. In older mice, drinking water nitrite did not significantly change plasma nitrite, but plasma nitrate was increased. Plasma nitrate was elevated in both young and old mice provided with nitrate supplemented drinking water. Our data demonstrate that the CNV lesion is larger in older mice compared to young and that therapeutic levels of oral nitrite increase the volume of CNV lesions in both young and older mice. Therapeutic nitrite or nitrate supplementation should be used with caution in the elderly population prone to CNV.

Antioxidant tempol modulates the increases in tissue nitric oxide metabolites concentrations after oral nitrite administration.

Nitric oxide (NO) metabolites have physiological and pharmacological importance and increasing their tissue concentrations may result in beneficial effects. Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) has antioxidant properties that may improve NO bioavailability. Moreover, tempol increases oral nitrite-derived gastric formation of S-nitrosothiols (RSNO). We hypothesized that pretreatment with tempol may further increase tissue concentrations of NO-related species after oral nitrite administration and therefore we carried out a time-dependent analysis of how tempol affects the concentrations of NO metabolites in different tissues after oral nitrite administration to rats. NO metabolites (nitrate, nitrite and RSNO) were assessed by ozone-based reductive chemiluminescence assays in plasma, stomach, aorta, heart and liver samples obtained from anesthetized rats at baseline conditions and 15 min, 30 min, 2 h or 24 h after oral nitrite (15 mg/kg) was administered to rats pretreated with tempol (18 mg/kg) or vehicle 15 min prior to nitrite administration. Aortic protein nitrosation was assessed by resin-assited capture (SNO-RAC) method. We found that pretreatment with tempol transiently enhanced nitrite-induced increases in nitrite, RSNO and nitrate concentrations in the stomach and in the plasma (all P < 0.05), particularly for 15-30 min, without affecting aortic protein nitrosation. Pretreatment with tempol enhanced nitrite-induced increases in nitrite (but not RSNO or nitrate) concentrations in the heart (P < 0.05). In contrast, tempol attenuated nitrite-induced increases in nitrite, RSNO or nitrate concentrations in the liver. These findings show that pretreatment with tempol affects oral nitrite-induced changes in tissue concentrations of NO metabolites depending on tissue type and does not increase nitrite-induced vascular nitrosation. These results may indicate that oral nitrite therapy aiming at achieving increased nitrosation of cardiovascular targets requires appropriate doses of nitrite and is not optimized by tempol.

Nitrite in paraffin-stimulated saliva correlates with blood nitrite.

Nitrite anion (NO2-) is a circulating nitric oxide (NO) metabolite considered an endothelial function marker. Nitrite can be produced from nitrate (NO3-) secreted from plasma into saliva. The nitrate reductase of oral bacteria converts salivary nitrate to nitrite, which is swallowed and absorbed into circulation. In this study, we aimed to examine the relevance between these species' salivary and blood levels. We collected three whole saliva samples (unstimulated, paraffin-stimulated, and post-chlorhexidine mouthwash stimulated saliva) and blood from 75 healthy volunteers. We measured the nitrite and nitrate by the chemiluminescence method. The nitrite levels in stimulated saliva and post-mouthwash stimulated saliva exhibited weak correlations with blood nitrite. There was no correlation between nitrite in unstimulated saliva with blood nitrite. The baseline platelet activity, determined as P-selectin expression, negatively correlated with nitrite in plasma and post-mouthwash stimulated saliva. The salivary nitrate in all saliva samples showed correlations with its plasma levels. We conclude that nitrite in stimulated saliva correlates with blood nitrite.

High-Throughput Griess Assay of Nitrite and Nitrate in Plasma and Red Blood Cells for Human Physiology Studies under Extreme Conditions.

The metabolism of nitric oxide plays an increasingly interesting role in the physiological response of the human body to extreme environmental conditions, such as underwater, in an extremely cold climate, and at low oxygen concentrations. Field studies need the development of analytical methods to measure nitrite and nitrate in plasma and red blood cells with high requirements of accuracy, precision, and sensitivity. An optimized spectrophotometric Griess method for nitrite-nitrate affords sensitivity in the low millimolar range and precision within ±2 µM for both nitrite and nitrate, requiring 100 µL of scarcely available plasma sample or less than 50 µL of red blood cells. A scheduled time-efficient procedure affords measurement of as many as 80 blood samples, with combined nitrite and nitrate measurement in plasma and red blood cells. Performance and usefulness were tested in pilot studies that use blood fractions deriving from subjects who dwelt in an Antarctica scientific station and on breath-holding and scuba divers who performed training at sea and in a land-based deep pool facility. The method demonstrated adequate to measure low basal concentrations of nitrite and high production of nitrate as a consequence of water column pressure-triggered vasodilatation in deep-water divers.

Direct comparison of inorganic nitrite and nitrate on vascular dysfunction and oxidative damage in experimental arterial hypertension.

Arterial hypertension is one of the major health risk factors leading to coronary artery disease, stroke or peripheral artery disease. Dietary uptake of inorganic nitrite (NO2-) and nitrate (NO3-) via vegetables leads to enhanced vascular NO bioavailability and provides antihypertensive effects. The present study aims to understand the underlying vasoprotective effects of nutritional NO2- and NO3- co-therapy in mice with angiotensin-II (AT-II)-induced arterial hypertension. High-dose AT-II (1 mg/kg/d, 1w, s. c.) was used to induce arterial hypertension in male C57BL/6 mice. Additional inorganic nitrite (7.5 mg/kg/d, p. o.) or nitrate (150 mg/kg/d, p. o.) were administered via the drinking water. Blood pressure (tail-cuff method) and endothelial function (isometric tension) were determined. Oxidative stress and inflammation markers were quantified in aorta, heart, kidney and blood. Co-treatment with inorganic nitrite, but not with nitrate, normalized vascular function, oxidative stress markers and inflammatory pathways in AT-II treated mice. Of note, the highly beneficial effects of nitrite on all parameters and the less pronounced protection by nitrate, as seen by improvement of some parameters, were observed despite no significant increase in plasma nitrite levels by both therapies. Methemoglobin levels tended to be higher upon nitrite/nitrate treatment. Nutritional nitric oxide precursors represent a non-pharmacological treatment option for hypertension that could be applied to the general population (e.g. by eating certain vegetables). The more beneficial effects of inorganic nitrite may rely on superior NO bioactivation and stronger blood pressure lowering effects. Future large-scale clinical studies should investigate whether hypertension and cardiovascular outcome in general can be influenced by dietary inorganic nitrite therapy.

DNA damage in leukocytes and serum nitrite concentration are negatively associated in type 1 diabetes.

Chronic hyperglycaemia leads to DNA damage in diabetes and might be associated with nitrosative stress. In this study, we aimed at assessing the level of DNA strand breaks in leukocytes, serum nitrite and nitrate in patients with type 1 diabetes and healthy controls and associations of these parameters with diabetes-related outcomes in a prospective study. The level of DNA damage was determined in 71 patients with type 1 diabetes and 57 healthy controls by comet assay and scored with arbitrary units (AU). The chemiluminescence method was used to measure nitrite and nitrate. Clinical information and data on consumption of alcohol, physical activity and smoking were collected. Progression of complications in patients with diabetes was assessed after a follow-up time of 4-5 years. We observed a higher level of DNA damage in leukocytes of patients with type 1 diabetes compared with healthy subjects [type 1 diabetes AU 50 (36-74.5); control AU 30 (24.1-43), P < 0.001]. According to regression, type 1 diabetes leads to a 2-fold increase in DNA damage. In the group of type 1 diabetes, DNA damage correlated positively with total cholesterol (R = 0.262, P = 0.028) and negatively with serum glucose level (R = -0.284; P = 0.018) and serum nitrite (R = -0.335; P = 0.008). DNA damage was not significantly associated with HbA1c, diabetes duration, complications and lifestyle factors. However, DNA damage > 57 AU was associated with statistically significantly lower serum nitrite and 1.52 higher risk of progression of complications of diabetes over the follow-up period. The latter result was not statistically significant due to insufficient study power [relative risk 1.52 (95% confidence interval = 0.68, 3.42, P = 0.31)]. Our results confirm that type 1 diabetes is associated with a higher level of DNA strand breaks in leukocytes when compared with the reference group and demonstrate the negative association between DNA damage and serum nitrite concentration.

Marching to the Beet: The effect of dietary nitrate supplementation on high altitude exercise performance and adaptation during a military trekking expedition.

PURPOSE: The aim was to investigate the effect of dietary nitrate supplementation (in the form of beetroot juice, BRJ) for 20 days on salivary nitrite (a potential precursor of bioactive nitric oxide), exercise performance and high altitude (HA) acclimatisation in field conditions (hypobaric hypoxia). METHODS: This was a single-blinded randomised control study of 22 healthy adult participants (12 men, 10 women, mean age 28 ± 12 years) across a HA military expedition. Participants were randomised pre-ascent to receive two 70 ml dose per day of either BRJ (~12.5 mmol nitrate per day; n = 11) or non-nitrate calorie matched control (n = 11). Participants ingested supplement doses daily, beginning 3 days prior to departure and continued until the highest sleeping altitude (4800 m) reached on day 17 of the expedition. Data were collected at baseline (44 m altitude), at 2350 m (day 9), 3400 m (day 12) and 4800 m (day 17). RESULTS: BRJ enhanced the salivary levels of nitrite (p = 0.007). There was a significant decrease in peripheral oxygen saturation and there were increases in heart rate, diastolic blood pressure, and rating of perceived exertion with increasing altitude (p=<0.001). Harvard Step Test fitness scores significantly declined at 4800 m in the control group (p = 0.003) compared with baseline. In contrast, there was no decline in fitness scores at 4800 m compared with baseline (p = 0.26) in the BRJ group. Heart rate recovery speed following exercise at 4800 m was significantly prolonged in the control group (p=<0.01) but was unchanged in the BRJ group (p = 0.61). BRJ did not affect the burden of HA illness (p = 1.00). CONCLUSIONS: BRJ increases salivary nitrite levels and ameliorates the decline in fitness at altitude but does not affect the occurrence of HA illness.

Predialysis and Dialysis Therapies Differently Affect Nitric Oxide Synthetic Pathway in Red Blood Cells from Uremic Patients: Focus on Peritoneal Dialysis.

Red blood cells (RBCs) have been found to synthesize and release both nitric oxide (NO) and cyclic guanosine monophosphate (cGMP), contributing to systemic NO bioavailability. These RBC functions resulted impaired in chronic kidney disease (CKD). This study aimed to evaluate whether predialysis (conservative therapy, CT) and dialysis (peritoneal dialysis, PD; hemodialysis, HD) therapies used during CKD progression may differently affect NO-synthetic pathway in RBCs. Our data demonstrated that compared to PD, although endothelial-NO-synthase activation was similarly increased, HD and CT were associated to cGMP RBCs accumulation, caused by reduced activity of cGMP membrane transporter (MRP4). In parallel, plasma cGMP levels were increased by both CT and HD and they significantly decreased after hemodialysis, suggesting that this might be caused by reduced cGMP renal clearance. As conceivable, compared to healthy subjects, plasma nitrite levels were significantly reduced by HD and CT but not in patients on PD. Additionally, the increased carotid intima-media thickness (IMT) values did not reach the significance exclusively in patients on PD. Therefore, our results show that PD might better preserve the synthetic NO-pathway in CKD-erythrocytes. Whether this translates into a reduced development of uremic vascular complications requires further investigation.

Control of rat muscle nitrate levels after perturbation of steady state dietary nitrate intake.

The roles of nitrate and nitrite ions as nitric oxide (NO) sources in mammals, complementing NOS enzymes, have recently been the focus of much research. We previously reported that rat skeletal muscle serves as a nitrate reservoir, with the amount of stored nitrate being highly dependent on dietary nitrate availability, as well as its synthesis by NOS1 enzymes and its subsequent utilization. We showed that at conditions of increased NO need, this nitrate reservoir is used in situ to generate nitrite and NO, at least in part via the nitrate reductase activity of xanthine oxidoreductase (XOR). We now further investigate the dynamics of nitrate/nitrite fluxes in rat skeletal muscle after first increasing nitrate levels in drinking water and then returning to the original intake level. Nitrate/nitrite levels were analyzed in liver, blood and several skeletal muscle samples, and expression of proteins involved in nitrate metabolism and transport were also measured. Increased nitrate supply elevated nitrate and nitrite levels in all measured tissues. Surprisingly, after high nitrate diet termination, levels of both ions in liver and all muscle samples first declined to lower levels than the original baseline. During the course of the overall experiment there was a gradual increase of XOR expression in muscle tissue, which likely led to enhanced nitrate to nitrite reduction. We also noted differences in basal levels of nitrate in the different types of muscles. These findings suggest complex control of muscle nitrate levels, perhaps with multiple processes to preserve its intracellular levels.

Effects of arginase genetic polymorphisms on nitric oxide formation in healthy pregnancy and in preeclampsia.

BACKGROUND AND AIMS: Preeclampsia is associated with reduced nitric oxide (NO) bioavailability. Arginase is related to NO synthesis, but relatively unexplored in preeclampsia. However, no previous study has examined whether variations in ARG1 and ARG2 genes affect NO bioavailability and the risk of preeclampsia. Here, we compared the alleles and genotypes of single nucleotide polymorphisms (SNPs) in ARG1 (rs2781659; rs2781667; rs2246012; rs17599586) and ARG2 (rs3742879; rs10483801) in healthy pregnant women and preeclampsia, and examined whether these SNPs affect plasma nitrite concentrations (a marker of NO formation) in these groups. METHODS: Genotypes for the ARG1 and ARG2 SNPs were determined by Taqman probe and plasma nitrite by an ozone-based chemiluminescence assay. RESULTS: Regarding ARG1 SNPs, the GG genotype and G allele frequencies for rs2781659, and the C allele frequencies for rs2246012 were higher in preeclampsia compared to healthy pregnant women. Moreover, the GG genotype for rs2781659 and the TT genotype for rs2781667 were associated with higher plasma nitrite in healthy pregnant. We found no association of ARG2 polymorphisms with preeclampsia or nitrite levels in the study groups. CONCLUSIONS: Our results suggest that SNPs of ARG1 increase the risk of preeclampsia and modulate plasma nitrite levels in healthy pregnant women.