Circulating and excretory nitrite and nitrate as indicators of nitric oxide synthesis in humans: methods of analysis

Nitric oxide (NO) is produced in various cells from l-arginine by the catalytical action of NO synthases (NOS). The main metabolic fate of NO includes oxidation to nitrate by oxyhemoglobin in red blood cells and autoxidation to nitrite. Nitrate and nitrite circulate in blood and are excreted in urine. The concentration of these NO metabolites in plasma, serum, and urine can be used to assess NO synthesis in humans. Circulating nitrite reflects constitutive endothelial NOS activity. Excretory nitrate indicates systemic NO production. Nitrite and nitrate can be measured in plasma, serum, and urine of humans by various analytical methods which are based on different analytical principles. These methods include colorimetry, spectrophotometry, fluorescence, chemiluminescence, gas and liquid chromatography, electrophoresis, and mass spectrometry. The present article gives an overview of the most significant currently used quantitative methods of analysis of nitrite and nitrate in human plasma, serum, and urine in the framework of clinical studies and discusses their significance.

     

Effects of specific COX-2-inhibition on renin release and renal and systemic prostanoid synthesis in healthy volunteers

BACKGROUND: The renin-angiotensin system plays a critical role in cardiovascular function, but little is known about the effects of specific cyclooxygenase 2 (COX-2) inhibition on this system in healthy humans under physiologic conditions. METHODS: Twenty-one healthy female volunteers received, in a randomized, double-blind, crossover study, celecoxib 200 mg twice a day, indomethacin 50 mg three times a day, or placebo for 4 days and a single dose, each, on day 5. On day 5 of each treatment, the following parameters were assessed with subjects in an upright position before and after administration of 20 mg furosemide intravenously: plasma renin activity (PRA), plasma aldosterone, serum and urine electrolytes, and creatinine. Index metabolites of prostanoids were analyzed by gas chromatography-tandem mass spectrometry in 24-hour urine on day 4 and in 2-hour urines before and after furosemide administration. RESULTS: Baseline and furosemide-stimulated PRA were reduced to a similar degree by celecoxib and indomethacin. Plasma aldosterone and urinary excretion of potassium showed changes consistent with the alteration of PRA. Urinary excretion rates of prostaglandin E(2), (PGE(2)), 7alpha-hydroxy-5, 11-diketotetranor-prosta-1,16-dioic acid (PGE-M), and 2,3-dinor-thromboxane B(2) (TxB(2)) were not reduced by celecoxib, whereas indomethacin led to a decrease of 40%, 45%, and 80%, respectively. Both active treatments inhibited urinary excretion of 2,3-dinor-6-keto-PGF(1alpha) and 6-keto-PGF(1alpha) by 60% and 40%, respectively. CONCLUSION: Renin-release in healthy humans with normal salt intake is COX-2 dependent. While COX-1 is critical for renal and systemic PGE(2) production, renal prostacyclin synthesis is apparently COX-2 dependent. Finally, the previously demonstrated shift of the thromboxane-prostacyclin balance toward prothrombotic thromboxane by specific COX-2 inhibition is confirmed.     

Assessment of nitric oxide synthase activity in vitro and in vivo by gas chromatography-mass spectrometry

A gas chromatographic-mass spectrometric method for the determination of nitric oxide synthase activity is described. The method is based on the gas chromatographic-mass spectrometric measurement of L-[15N2]arginine-derived [15N]nitrite as its pentafluorobenzyl derivative in the negative-ion chemical ionization mode. Application of the method to the analysis of [15N]nitrite formation by purified neuronal nitric oxide synthase revealed K(M) values of 3.1 microM by Hanes and 4.6 microM by Lineweaver-Burk for L-[15N2]arginine. The corresponding Vmax values were 0.204 and 0.228 micromol [15N]nitrite min(-1) mg(-1) NOS, respectively. N(G)-Nitro-L-arginine and N(G),N(G)-dimethylarginine (asymmetric dimethylarginine) were identified by this method as the most potent enzyme inhibitors. Nitric oxide synthase activity was also assessed in vivo by i.v. injection of L-[15N2]arginine in a rat and determination of plasma [15N]nitrite and [15N]nitrate. The assay described in this work allows for accurate, specific and highly sensitive determination of nitric oxide synthase activity in vitro and in vivo.     

URINARY NO3− EXCRETION AS AN INDICATOR OF NITRIC OXIDE FORMATION IN VIVO DURING ORAL ADMINISTRATION OF l-ARGININE OR l-NAME IN RATS

• 1 Endothelium-derived nitric oxide (NO), a major modulator of vascular tone, is synthesized from the terminal guanidino nitrogen of l -arginine. This reaction is inhibited by analogues of l -arginine, such as N-nitro-l -arginine methyl ester (l -NAME). Many of the biological effects of NO are mediated by the second messenger cGMP. NO is rapidly oxidized to NO₃^? which, like cGMP, is eliminated via excretion into the urine. In a placebo controlled study, we investigated whether oral bolus administration of l -arginine and l -NAME affects the urinary excretion rates of NO₃^? and cGMP in Munich Wistar Frömmter (MWF) rats.
• 2 Twenty MWF rats were kept in metabolic cages and received l -arginine (3 g/kg body weight), l -NAME (50mg/kg), or placebo (0.9% saline) in randomized order. Urine samples were sequentially collected for 10 h and analysed for creatinine, NO₃^? and cGMP.
• 3 l -Arginine induced a slight, but prolonged increase in urine flow, whereas l -NAME induced an early, transient increase in urine flow which was followed by a decrease. Creatinine clearance decreased by 65% after l -NAME, but was not affected by l -arginine or placebo.
• 4 Urinary NO₃^? and cGMP excretion rates transiently increased after l -arginine (NO₃^?: + 29%; cGMP: + 16%) for 4–5h, whereas l -NAME induced an immediate, pronounced and lasting inhibition of urinary NO₃^? and cGMP excretion (NO₃^?:-76%; cGMP:-46%). Urinary NO₃^? and cGMP excretions were significantly correlated (r = 0.755; P< 0.001).
• 5 Urinary excretion rates of NO₃^? and cGMP, expressed as μmol/h, were correlated to urine flow (mL/h; r = 0.617 and 0.649, respectively; both P<0.05), whereas after correction by urinary creatinine (μmol/mmol creatinine) no correlation with urine flow was observed, indicating that these excretion rates were independent of renal excretory function. Thus we conclude that changes in the urinary excretion rates of NO₃^? and cGMP represent changes in NO production rates in vivo when expressed in relation to urinary creatinine. Urinary NO₃^? and cGMP excretion is modulated by acute NO synthase inhibition or substrate provision.

     

Effects of carvedilol on oxidative stress in human endothelial cells and healthy volunteers

Objective Carvedilol is a nonselective - and ₁-receptor antagonist with additional antioxidant properties in vitro. In this study, we assessed the antioxidative potential of carvedilol in cell culture and in antihypertensive doses in healthy men.Methods In vitro, human cultured endothelial cells were treated with native low-density lipoprotein (LDL), oxidized LDL or tumor necrosis factor (TNF) in the absence and in the presence of carvedilol (40 µM); 8-iso-prostaglandin (PG)F₂, as parameter of oxidative stress, was determined in the supernatants. In a double-blind, randomized, cross-over study, 17 healthy men received 25 mg carvedilol b.i.d., 100 mg metoprolol b.i.d. or placebo for 6 days. After each treatment, systemic oxidative stress was assessed by measuring urinary excretion of 8-iso-PGF₂ and 2,3-dinor-5,6-dihydro-8-iso-PGF₂, and the plasma concentration of 3-nitrotyrosine by means of gas chromatography-tandem mass spectrometry. In addition, thiobarbituric acid-reactive substances (TBARS) in plasma were assessed using spectrophotometry.Results Native LDL and oxidized LDL induced 8-iso-PGF₂ production in endothelial cells. Carvedilol significantly reduced this effect (e.g., for oxidized LDL: 2.66±0.22 pg vs 1.46±0.14 pg 8-iso-PGF₂ per µg protein, P<0.05). In healthy volunteers, carvedilol and metoprolol markedly decreased blood pressure and heart rate, but had no statistically significant effect on any indicator of oxidative stress measured. Remarkably, a trend toward reduction of urinary isoprostanes and 3-nitrotyrosine in plasma by both active treatments was observed, suggesting a non-specific antioxidative effect by blockade.Conclusions In vitro, the antioxidative potential of carvedilol was confirmed. In healthy men, antihypertensive doses of carvedilol exert no specific inhibition of oxidative stress.     

Measurement of S-nitrosoalbumin by gas chromatography-mass spectrometry. II. Quantitative determination of S-nitrosoalbumin in human plasma using S-[15N]nitrosoalbumin as internal standard.

A gas chromatographic-mass spectrometric method for the quantitative determination of S-nitrosoalbumin (SNALB) in human plasma is described. The method is based on selective extraction of SNALB and its 15N-labeled SNALB analog (S15NALB) used as internal standard on HiTrapBlue Sepharose affinity columns, Hg2+ -catalysed conversion of the S-nitroso groups to nitrite and [15N]nitrite, respectively, followed by their derivatization to the pentafluorobenzyl derivatives and quantification by GC-MS. Mean recovery of SNALB and S15NALB from plasma was 45%. Mean precision and accuracy within the range 0-10 microM was 95% and 99%, respectively. The limit of quantitation was determined as 100 nM at a precision of 93.8% and an accuracy of 94.8%. Considerable improvement of method sensitivity is possible by eliminating nitrite present in the elution buffer. The limit of detection was 0.2 nM corresponding to 67 amol of S15NALB. In 0.4-ml aliquots of plasma samples from healthy humans, endogenous SNALB was determined at concentrations of 181+/-150 nM (mean +/- SD, n = 23). External addition of SNALB to these plasma samples at 2 microM and 5 microM serving as quality control samples resulted in quantitative recovery of SNALB. Our results show that SNALB occurs in human plasma at concentrations at least one-order of magnitude smaller than those reported in the literature from measurements using chemiluminescence.     

Temporal profiles of cerebrospinal fluid leukotrienes,brain edema and inflammatory response following experimental brain injury

The post-traumatic changes of leukotrienes LTC4, LTD4, LTE4, and LTB4 in cerebrospinal fluid of rats from 10 min to 7 days were investigated after controlled cortical impact in relation to brain edema and cellular inflammatory response. LTC4 increased five-fold at 4 h, normalized at 24 h, and showed another four-fold increase at 7 days. The same pattern was observed for LTD4 and LTE4. LTB4 however, behaved differently: concentrations were lower and levels peaked two-fold at 24 h. Edema in the injured hemisphere increased continuously up to 24 h without change contralaterally. Leukocyte infiltration, macrophage presence and microglia activation were most prominent at 24 h, 7 days and 24 h respectively. Leukotriene changes in CSF seem to reflect those in the affected tissue, with a time delay and in lower concentrations, and were not linearly correlated to brain edema. The initially high leukotriene levels are rather likely to contribute to the cytotoxic edema than to enhance a vasogenic edema component. The profile of LTB4 was parallel to the time course of leukocyte infiltration, indicating initiation of infiltration as well as prolonged production by leukocytes themselves. The second leukotriene peak at 7 days is likely to follow a different pathway and might be related to a production in macrophages or activated glia.     

Tandem mass spectrometric quantification of 8-iso-prostaglandin F2alpha and its metabolite 2,3-dinor-5,6-dihydro-8-iso-prostaglandin F2alpha in human urine

Whole body synthesis of F2-isoprostanes, a family of cyclooxygenase-independent eicosanoids formed by free-radical catalysed peroxidation, should be best assessed by quantifying their urinary metabolites. Two methods for the quantitative determination of F2-isoprostane metabolites in human urine performing either thin-layer chromatography (TLC) (method A) or high-performance liquid chromatography (HPLC) (method B) prior to GC-tandem MS are described. Method A allows for simultaneous quantification of 8-iso-PGF2alpha, one prominent member of the F2-isoprostane family, and its major urinary metabolite, 2,3-dinor-5,6-dihydro-8-iso-PGF2alpha. Mean excretion was found to be 223 and 506 pg/mg creatinine of 8-iso-PGF2alpha and 2,3-dinor-5,6-dihydro-8-iso-PGF2alpha, respectively (n=14). A tight correlation existed between the urinary excretion of these two isoprostanes (r=0.86). Method B enables quantification of dinor-dihydro metabolites of various F2-isoprostanes including 8-iso-PGF2alpha. 2,3-Dinor-5,6-dihydro-8-iso-PGF2alpha was found to be an abundant dinor-dihydro F2-isoprostane metabolite. Validity of method A was proven by a combination of HPLC with TLC prior to GC-tandem MS analysis. A correlation was observed between the urinary concentrations of 2,3-dinor-5,6-dihydro-8-iso-PGF2alpha measured by GC-MS and GC-tandem MS (r=0.84).