Urinary Nitrate Levels Research Articles

Urinary Nitrite Excretion after Prophylactic Intravenous Immunoglobulin in Premature Infants

Aims: To investigate the correlation between the prophylactic administration of intravenous immunoglobulin (IVIG) to preterm infants and urinary nitrite levels, which can be utilized as an index of endogenous nitric oxide (NO) formation, and to determine if NO formation plays a role in both therapeutic and adverse effects of IVIG. Methods: 28 healthy preterm infants were included in this prospective study. They had a mean gestational age of 29.4 ± 2.2 weeks and weight of 1,387 ± 371 g. Prophylactic IVIG infusion at a dose of 0.5 g/kg/day was administered when they were 3–10 days old. Urine samples of the neonates were obtained for analysis on days 1, 2 and 3 after IVIG administration as well as 1 day before. Urinary nitrite levels obtained in the subjects were normalized for urinary creatinine concentrations. Results: The mean urinary nitrite levels were: 2.77 ± 1.66 μmol/mmol creatinine before IVIG administration; 4.33 ± 3.88 μmol/mmol creatinine on the 1st day of IVIG; 3.77 ± 2.73 μmol/mmol creatinine on the 2nd day, and 3.64 ± 3.28 μmol/mmol creatinine on the 3rd day. There was a significant increase in urinary nitrite levels between before and after IVIG administration. There was no statistical difference in urinary nitrate levels between days 1, 2 and 3 after IVIG administration. Conclusion: We demonstrated that urinary nitrite excretion is significantly elevated in preterm infants after prophylactic IVIG administration and this result suggests that endogenous NO formation may play an important role in both the therapeutic and adverse effects of IVIG.

Diminished Nitric Oxide Production Following Administration of Transforming Growth Factor-β1 Using a Noninflammatory Wound Repair Model

The effects of transforming growth factor-β1 (TGF-β1) on systemic nitric oxide (NO) production and wound repair were evaluated using a noninflammatory mouse perforated mesentery connective tissue repair model. Urinary nitrates were monitored as a measure of NO production. TGF-β1 [bovine serum albumin (BSA) carrier and TGF-β1 BSA carrier free] were administered on days 0 and 1 and were evaluated in mice over a 10-day period. A significant decrease in the average postwounding urinary nitrate levels compared to prewounding levels was observed within the TGF-β1 treatment group animals (P ≤ 0.001) with an insignificant change for the phosphate-buffered saline control animals (P ≤ 0.10). Additionally, TGF-β1-treated animals showed significant connective tissue repair compared to controls without a concurrent increase in postwounding urinary nitrate levels, supporting the noninflammatory nature of the perforated mesentery model. Our findings suggest that an unforseen consequence of TGF-β1 administration is diminished basal nitric oxide production. When using TGF-β1 in a wound repair or other therapeutic model, the administration of an exogenous NO donor compound may be necessary in order to ensure homeostasis, thereby avoiding undesired physiological consequences due to diminished basal NO production.

Increased urinary nitric oxide oxidation products in children with active coeliac disease

Background: Nitric oxide (NO) production catalyzed by iNOS (inducible NO synthase) is thought to take place mainly in macrophages after activation by inflammatory mediators. NO is subsequently oxidized to nitrite and nitrate, which are excreted in urine. The concentration of inflammatory mediators in small bowel biopsy specimens from patients with coeliac disease is increased. The latter could induce increased NO production by stimulation of intestinal macrophage iNOS, resulting in high levels of urinary NO oxidation products, nitrite and nitrate (NOx). Aim: In the present study we evaluated the urinary NOx/creatinine ratios in children with active coeliac disease (n = 22), coeliac disease patients on a gluten‐free diet (n = 9), healthy (n=11) and sick control children (n = 18). Methods: The Griess reagent method was used for measuring urinary NOx. Results: Median NOx/creatinine ratios of active coeliac disease patients, coeliac disease patients on a gluten‐free diet, healthy and sick control patients were 1.21, 0.19, 0.10 and 0.13 mmol/mmol, respectively. All active coeliac disease patients showed increased NOx/ creatinine ratios. Urinary NOx/creatinine ratios of the active coeliac disease patients were significantly higher than those of healthy controls (p < 0.0001), sick controls (p < 0.0001) and coeliac disease patients on a gluten‐free diet (p < 0.0001). Conclusion: The urinary NOx/creatinine ratio is increased in patients with active coeliac disease and reverts to normal on a gluten‐free diet. □Coeliac disease, nitric oxide, urinary nitrate

The effects of human burn injury on urinary nitrate excretion

Different studies have demonstrated both an increase and a decrease in the biosynthesis of nitric oxide (NO) during the first 2 days following experimental and human burn trauma. This study investigated changes in urinary nitrate excretion in humans following thermal injury in order to determine the temporal relationship between NO release and the initial injury. Urinary nitrate was measured in daily 24-h urine collections taken on days 1–7 following burn injury from 15 patients. The control group consisted of 11 healthy, age- and sex-matched patients who kept a nitrate-restricted diet for five days prior to collection of a single 24-h urine sample. The burns group had a mean age of 41.9±19.4 (mean±S.D.) years and a mean total burn surface area (TBSA) of 30.2±24.9% (mean±S.D.). In the burn injured patients, urinary nitrate levels peaked at day 4 and a 2-fold increase relative to day 1 was observed. Urinary nitrate levels were significantly higher in the burns group than the control group on days 4 and 5 only ( p<0.05 for both days). There was no correlation between TBSA and the measured urinary nitrate levels. This study confirms that the biosynthesis of NO is increased during the first week following burn trauma and establishes that the renal elimination of the by-products of NO metabolism is not increased during the first three days after injury. Notwithstanding the potential effects of burns on nitrate distribution, our findings may reflect a delay in the release of NO following the initial insult.

Prospective study of serum and urinary nitrate levels in patients with systemic lupus erythematosus.

To study prospectively whether serum and urinary nitrate levels are related to lupus activity. Fifty patients with systemic lupus erythematosus (SLE) were studied prospectively for 2 yr. Every 4 months, the SLE Disease Activity Index (SLEDAI) was administered to the patients, and blood and 24 h urine samples were obtained; 88 healthy controls were also studied. Nitrate levels were measured by the Greiss method. Statistical analyses were performed using standard parametric and non-parametric tests, and analysis of serial measurements. Twelve patients suffered infections, 12 active nephritis and 17 episodes of non-renal activity. By analysis of serial measurements, serum and urinary nitrate levels did not correlate with SLEDAI. C-Reactive protein (CRP) levels, presence of infection and creatinine clearance weakly influenced nitrate levels. In SLE, serum and urinary nitrate levels do not parallel lupus activity. Other variables, related or not to SLE, seem to affect these levels.

Protective role of nitric oxide in Staphylococcus aureus infection in mice.

This study was carried out to determine the role of nitric oxide (NO) in Staphylococcus aureus infection in mice. NO production in spleen cell cultures was induced by heat-killed S. aureus. Expression of mRNA of the inducible isoform of NO synthase (iNOS) was induced in the spleens and kidneys of S. aureus-infected mice. When mice were treated with monoclonal antibodies (MAbs) against tumor necrosis factor alpha (TNF-alpha) or gamma interferon (IFN-gamma) before S. aureus infection, the induction of iNOS mRNA expression in the kidneys was inhibited. These MAbs also inhibited NO production in spleen cell cultures stimulated with heat-killed S. aureus. NO production in the spleen cell cultures and levels of urinary nitrate plus nitrite were suppressed by treatment with aminoguanidine (AG), a selective inhibitor of iNOS. The survival rates of AG-treated mice were significantly decreased by either lethal or sublethal S. aureus infections. However, an effect of AG administration on bacterial growth was not observed in the spleens and kidneys of mice during either type of infection. Production of TNF-alpha and IFN-gamma was not affected by AG treatment in vitro and in vivo. These results suggest that NO plays an important role in protection from lethality by the infection, but the protective role of NO in host resistance against S. aureus infection was not proved. Moreover, our results show that TNF-alpha and IFN-gamma regulate NO production while NO may not be involved in the regulation of the production of these cytokines during S. aureus infection.

Transdermal delivery of nitric oxide from diazeniumdiolates

Adverse physiological effects can often interfere with the use of nitric oxide (NO) as a therapeutic agent, especially when it is systemically generated from prodrugs. NO which is generated and delivered site-specifically by transdermal donors may be useful in the treatment of parasitic, bacterial or viral skin infections without causing systemic side effects. Three diazeniumdiolates (formerly “NONOates”), including two water soluble compounds, (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]-diazen-1-ium-1,2-diolate (DETA-NO) and (Z)-1-[N-(3-aminopropyl)-N-(3-ammoniopropyl)amino]-diazen-1-ium-1,2-diolate (DPTA-NO), and one insoluble compound, DPTA-NO grafted to dextran microspheres (DPTA-NO–g-dextran) were used to transdermally deliver NO to rats. Dextran microspheres were obtained by simultaneously grafting DPTA-NO to dextran and cross-linking dextran with CNBr in an oil–water emulsion. Suspended in hydrogel, DETA-NO, DPTA-NO, and DPTA-NO–g-dextran were applied three times to depilated rats at 4 day intervals. Results show that metabolic urinary nitrate levels increase with time (24–48 h), reach a maximum, and return to baseline by the fourth day. DPTA-NO applications produced an average maximum nitrate level of 94.2 μmol/day±34.2 μmol S.D. compared to the average maximum nitrate level of 39.8 μmol/day±8.6 μmol S.D. obtained from treatment with DETA-NO. These results suggest that DPTA-NO delivered NO more efficiently than DETA-NO. When DPTA-NO–g-dextran microspheres were used as the NO donor, results comparable to DPTA-NO were observed with an average maximum nitrate level of 14.9 μmol/day±3.0 μmol S.D.. These and other conclusive data indicate that, via these diazeniumdiolates, NO can be delivered effectively through rat skin.

Formation of nitrosamines during consumption of nitrate- and amine-rich foods, and the influence of the use of mouthwashes.

We studied the formation of carcinogenic nitrosamines during consumption of food rich in nitrate and amines, and its possible inhibition by use of an antibacterial mouthwash. Twelve volunteers were fed a diet containing the high-nitrate vegetables lettuce or spinach during two periods of four consecutive days, in combination with fish products containing high levels of amines as nitrosatable precursors. During the two periods, the subjects used an antibacterial mouthwash containing chlorhexidine or a control mouthwash without antibacterial activity. Twenty-four-hour urine samples were collected after consumption of the meals, and saliva samples were collected 1 h after each meal. The nitrate and nitrite contents of the urine and saliva samples were determined by spectrophotometry (for nitrite) and HPLC (for nitrate). The concentrations of volatile nitrosamines in the urine samples were determined by gas chromatography-mass spectrometry. Significant increases in mean urinary nitrate levels (from 59 to 135 mg/24 h) and in mean salivary nitrate levels (from 10 to 56 microg/ml) and salivary nitrite levels (from 2 to 11 microg/ml) were observed during the consumption of food rich in nitrate and amines, as well as a significant increase in the mean urinary excretion of total examined volatile nitrosamines (from 2 to 7 nmol/24 h) and of N-nitrosodimethylamine (from 1.2 to 2.9 nmol/24 h). Use of the antibacterial mouthwash resulted in a decrease in mean salivary nitrite levels from 16 to 3 microg/ml and a decrease in mean urinary excretion of N-nitrosomorpholine (from 7.0 to 0.3 nmol/24 h). For the whole data set, significant correlations were observed between nitrate intake in food and urinary nitrate (p = 0.01; r2 = 0.07) and between urinary nitrate and urinary N-nitrosodimethylamine (p = 0.002; r2 = 0.11). In conclusion, consumption of a diet rich in nitrate and amines increases the risk of formation of carcinogenic nitrosamines. Use of an antibacterial mouthwash containing chlorhexidine can result in inhibition of nitrosamine formation.

INCREASED URINARY NITRIC OXIDE (NO) OXIDATION PRODUCTS IN CHILDREN WITH ACTIVE COELIAC DISEASE. † 711

NO production catalyzed by INOS (inducible NO synthase) is thought to take place mainly in macrophages after activation by inflammatory mediators. In the body NO is oxidized to nitrite and nitrate which is excreted in the urine. The concentration of inflammatory mediators in small bowel biopsy specimens in coeliac disease is increased. The latter could induce increase NO production by stimulation of intestinal macrophage INOS, resulting in high levels of urinary NO oxidation products, nitrite and nitrate (NOx). The aim of the present study was to evaluate the urinary NOx/creatinine ratios in children with active coeliac disease (n=12), in healthy control children (n=11) and in sick control children (n=18). The griess reagent method was used for the measurement of urinary NOx. Mean and standard deviation for the NOx/creatinine ratios in coeliac disease patients was 2,01 ± 1,37 (range 0,57-5,11). These values were 0,11 ± 0,07 (range 0,03-0,25) for the healthy control children and 0,20 ± 0,20 (range 0,03-0,71) for the sick control patients. Values for coeliac disease patients were significantly different from those of both healthy controls (p<0.0001) and sick controls(p<0.0001). All active coeliac disease patients showed increased NOx/creatinine ratios. We conclude that urinary NOx/creatinine ratios could be used as an indicator of mucosal immune activation in coeliac disease patients.

Measurement of Nitrate and Nitrite in Extracellular Fluids: A Window to Systemic Nitric Oxide Metabolism

There is a growing body of both clinical and experimental data that demonstrates that activation of the immune system, whether locally or systemically, is associated with an increased production of nitric oxide (NO) as measured by increases in plasma and/or urinary levels of nitrate (NO−3) and nitrite (NO−2). Because NO may mediate some of the tissue injury and dysfunction observed in certain pathophysiologic conditions associated with immune system activation (e.g., arthritis, colitis, sepsis) a simple, sensitive, and inexpensive method to measure NO−3 and NO−2 levels in the plasma and urine may provide an important tool for monitoring NO production and possibly disease activity in vivo. Therefore, the objective of this article is to outline the methods used in our laboratory to quantify NO−3 and NO−2 in extracellular fluids such as plasma, urine, and/or lymph. These same methods may also be used for other extracellular fluids including saliva, tears, sweat, and possibly bile. We describe two methods that require that NO−3 first be reduced to NO−2 and then NO−2 determined spectrophotometrically by the Griess reaction. We have found that the enzymatic reduction of NO−3 to NO−2 using either a commercially available preparation of nitrate reductase (Aspergillus nitrate reductase; Boehringer-Mannheim) or the nitrate reductase associated with Escherichia coli which have been grown anaerobically in the presence of large amounts of nitrate to be the most satisfactory methods. Both methods are relatively inexpensive, simple, and fairly sensitive. Studies from our laboratory have determined that heparin should be removed from plasma (or lymph) samples prior to addition of the Griess reagent because we have found that acidification of dilute heparinized plasma may result in substantial precipitation of the sample.

Nitric oxide produced during murine listeriosis is protective.

Nitric oxide (NO) has been shown to be important for intracellular microbiostasis in vitro. To determine the role of NO in immune function in vivo, groups of C57BL/6 mice were given a sublethal intravenous inoculum of Listeria monocytogenes EGD, and their urine was monitored daily for nitrate, the mammalian end product of NO metabolism. Urinary nitrate levels peaked at 5 to 10 times the basal level on days 5 to 6, when spleen and liver Listeria counts declined most steeply, and decreased thereafter, when spleens and livers were nearly sterile. Peritoneal macrophages explanted from Listeria-infected mice produced nitrite spontaneously, whereas macrophages from uninfected mice did not. The inducible NO synthase mRNA was detectable in the spleens of infected mice on days 1 to 4 of infection. When Listeria-infected mice were treated orally throughout the infection with NG-monomethyl-L-arginine (NMMA), a specific NO synthase inhibitor they showed no detectable rise in urinary nitrate excretion. Mean Listeria counts in the livers and spleens NMMA-treated mice were 1 to 3 orders of magnitude greater than counts in control mice on days 4 through 8 of infection. Compared with control mice, NMMA-treated mice also showed worse clinical signs of infection, namely, weight loss, hypothermia, decreased food and water intake, and decreased urine output. Histologically NMMA-treated mice had many more inflammatory foci in their livers and spleens than control mice. The histologic observation that mononuclear cells are present at sites of infection suggests that inhibiting NO production did not block the flux of macrophages into infected viscera. As controls for possible drug toxicity, a group of uninfected mice given NMMA orally showed no detrimental effects on weight, temperature, and food and water intake. These experiments demonstrate that inhibition of NO production in Listeria-infected mice results in an exacerbated infection and thus that NO synthesis is important for immune defense against Listeria infection in mice.

Arginine-free diet suppresses nitric oxide production in wounds

To determine the effects of dietary arginine on nitric oxide production during wound healing, we fed male Sprague-Dawley rats a low nitrate diet containing 0% or 3% l-arginine. Nitrate from daily urine samples was reduced to nitric oxide and measured with a chemiluminescence analyzer to indirectly assess nitric oxide synthesis. Once baseline nitrate levels were established, rats received circular, full thickness dermal wounds. The results demonstrate that dietary arginine is not necessary for the sudden increase in nitrate output immediately after wounding, although removal of arginine appears to limit the maximum amount of nitric oxide produced. Arginine-deficient rats had significantly lower ( P < 0.001) urinary nitrate levels than the corresponding 3%-arginine group over the entire experiment. Despite lower nitrate output in the arginine-free animals, the early response to wounding was similar in both the arginine-free and 3%-arginine groups, as nitrate output doubled within 48 hours of wounding (+ 112% and + 104%, respectively). However, endogenous arginine was not sufficient to maintain increased nitrate output, which returned to baseline levels in arginine-free animals within 11 days after wounding, while the nitrate output of the 3%-arginine group remained significantly elevated (+ 30–60%, P < 0.001) through day 30 and wound closure. In addition, animals on the arginine-free diet increased nitrate output 14-fold in response to lipopolysaccharide challenge. These results suggest that removing dietary arginine does not prevent the cytotoxic activity of macrophages during the inflammatory response, but suppresses nitric oxide synthesis during later tissue repair. This may help explain previous evidence that supplemental arginine improves wound healing, although video image analysis of wounds was inconclusive.

Urinary markers for exposures to alkylating or nitrosating agents.

Investigation of urinary markers as indices of endogenous nitrosation and of gastric cancer etiology has been a major focus of our work. As part of this effort, studies have been carried out on a Colombian population at high risk for gastric cancer. In this group, nitrosoproline excretion was highly correlated with nitrate excretion in the subpopulation with advanced gastric pathology, but not in control subpopulations with more normal stomachs. Neither urinary 7-methylguanine nor 3-methyladenine was strongly related to gastric pathology or to urinary nitrate or nitrosoproline levels. More recently, as evidence has accumulated concerning the importance of nitric oxide as a cellular messenger, we have begun research toward developing markers for the presence of nitric oxide and for endogenous nitrosation via this compound. Nitric oxide is formed from arginine by activated endothelial cells as a messenger for vasodilation. We have shown that prolonged exercise leads to increased urinary nitrate and that when 15N-arginine is ingested by humans, 15N-nitrate levels increase in 24-hr urine collections. Nitrosohydroxyethylglycine and 3-nitrotyrosine were evaluated as indices for the formation of N-nitrosomorpholine and for the nitration of protein, respectively, under experimental conditions (e.g., immunostimulation) expected to enhance nitric oxide formation. Nitrotyrosine has not proved useful as a biomarker for nitration/nitrosation reactions in immunostimulated rats. Immunostimulation of rats following administration of morpholine led to increases in urinary nitrate and nitrosohydroxyethylglycine. This procedure, however, would not be appropriate for humans due to the toxicity of morpholine and the carcinogenicity of N-nitrosomorpholine.

Increased endogenous N-nitrosamine and nitrate formation by induction of nitric oxide synthase in rats with acute hepatic injury caused by Propionibacterium acnes and lipopolysaccharide administraction

In rats treated i.v. with heat-killed Propionibacterium acnes (100 mg/kg body wt), followed 5 days later by an i.v. dose of Escherichia coli lipopolysaccharide (LPS, 1 mg/kg body wt), acute hepatic cell necrosis was accompanied by significant induction of nitric oxide (NO) synthase activity in the liver. Endogenous nitrosation of thiazolidine 4-carboxylic acid (TCA, 50 mumol/rat) administered by three different routes (i.v., i.p. and p.o.) 5 h after LPS injection to the P. acnes-treated rats was assessed by analysing its nitrosated product (NTCA) excreted in 24 h urine. The amounts of NTCA formed in vivo after i.v., i.p. and p.o. administration of TCA were 4.07 +/- 1.00, 5.79 +/- 2.15 and 58.3 +/- 20.7 nmol/rat (n = 5-10) respectively, which were about 5-, 10- and 8-fold greater than those excreted by rats which had not been treated with P.acnes and LPS but received TCA by the same route. Nitrate concentration in plasma and NO synthase activity in the liver started to increase within 2.5 h after LPS injection, reached a maximum at 7.5 h and remained at high levels for several further hours. Levels of nitrite and nitrate in gastric contents were also increased significantly after LPS administration. The co-administration of N omega-nitro-L-arginine (an inhibitor of NO synthase) and LPS resulted in a marked reduction of urinary levels of nitrate and NTCA, indicating that nitrosation is mediated by NO synthase. These results together suggest that induction of NO synthase by infection with bacteria, parasite and viruses could result in increased endogenous nitrosation not only in the infected tissues but also in the stomach, where nitrosamines would be formed more rapidly under acidic conditions.

Urinary excretion of N-nitrosamino acids and nitrate by inhabitants of high- and low-risk areas for stomach cancer in Poland.

Urine samples were collected from 96 inhabitants of a high-risk rural area and a low-risk urban area for stomach cancer in Poland, according to the following protocol: (1) when they were undosed; (2) after ingestion of proline 3 times a day; and (3) after ingestion of proline together with vitamin C 3 times a day. The samples were analyzed for N-nitrosamino acids and nitrates, as indices of exposure to preformed and endogenously formed N-nitrosamines. The median values of N-nitrosoproline (NPRO) and N-nitrosothiazolidine 4-carboxylic acid (NTCA) excreted in the urine of undosed subjects were not different between the two areas; but N-nitrososarcosine and 3-(N-nitroso-N-methylamino)propionic acid levels were 3- to 4-fold higher in subjects of the high-risk area. After intake of proline, the NPRO level increased (p less than 0.02) only in subjects in the high-risk area; intake of vitamin C tended to inhibit this increase in NPRO and lowered the levels of other nitrosamino acids. The urinary level of nitrates was 1.4-fold, but significantly higher among subjects in the high-risk area than among those in the low-risk area; nitrate levels were not correlated with the amounts of cured meat or types of vegetables consumed. Urinary nitrate levels and excretion of NPRO, NTCA and the sum of all nitrosamino acids analyzed showed positive, though modest, correlations. These results indicate a higher potential for endogenous nitrosamine formation, possibly by intragastric nitrosation among subjects in the high-risk rural area.

Levels of mutagens in the urine of smokers of black and blond tobacco correlate with their risk of bladder cancer.

Levels of urinary mutagens, thioethers, N-nitrosamino acids, nitrate, nicotine, cotinine and creatinine were measured in 21 non-smokers, 26 smokers of blond tobacco, 9 smokers of black tobacco and 5 smokers of both types of tobacco, all eating a similar diet. Results were expressed either per 24 h urine or per mmol creatinine. The sum of urinary nicotine and cotinine levels (N + C) was used as a measure of exposure to the number of cigarettes smoked. Statistically significant positive dose-effect relationships were obtained between the urinary N + C levels and (i) the number of revertants (Salmonella typhimurium TA98, with a metabolic activation system); (ii) the concentration of thioethers; (iii) the levels of N-nitrosoproline or the sum of all nitrosamino acids excreted and (iv) the amount of urinary nitrate. No such correlation was found between N + C levels and induction potency in the SOS chromotest. A linear dose-effect relationship between urinary mutagenicity (i.e. log revertants of S. typhimurium TA98) and N + C levels or number of cigarettes per day was established for smokers of blond tobacco. After adjustment for N + C levels, the urine of smokers of black tobacco contained twice as much mutagenic material as did the urine of blond tobacco smokers (P = 0.02). For other exposure markers, no statistically significant difference was found between the two types of smokers. Epidemiological studies have shown that the risk of urinary bladder cancer is 2.5 times higher in smokers of black tobacco than in blond tobacco. Therefore, our findings on urinary mutagenicity provide experimental evidence that the type of tobacco is the factor responsible for the observed difference in risk and that smoking of black as compared to blond tobacco results in a higher exposure of the urinary bladder to genotoxic hence potentially carcinogenic substances.