Effects of intravenous oxygen on prostacyclin and thromboxane formation in patients with peripheral occlusive arterial disease.

Oxygen infusion is used in complementary medicine for treatment of peripheral occlusive arterial disease. The mechanism of action is unknown. Thus, we determined the effects of oxygen infusion on prostacyclin, thromboxane and nitric oxide synthesis. Twelve patients with peripheral occlusive arterial disease received oxygen 40 ml/d intravenously for 3 weeks. Study parameters, analyzed by gas chromatography-mass spectrometry on day 1, 3, 10, 16, 21: 2,3-dinor-6-oxo-PGF(1alpha), colour invisible 2,3-dinor-TXB2 and nitrate in one-hour-urine before and after oxygen infusion, reflecting prostacyclin, thromboxane and nitric oxide synthesis. Urinary 8-iso-PGF2alpha, indicating oxidative stress, was assessed in one patient. Urinary 2,3-dinor-6-oxo-PGF1alpha rose from baseline more than 4-fold after oxygen infusion. In contrast, urinary 2,3-dinor-TXB2 excretion remained unchanged. Oxygen infusion had no effect on urinary nitrate excretion. Urinary 8-iso-PGF(2alpha) was not influenced by oxygen infusion with and without diclofenac pretreatment. Our data demonstrate a shift of the prostacyclin/thromboxane ratio toward prostacyclin by oxygen infusion. Thus, a mechanism of action is provided and clinical trials with intravenous oxygen find a rational basis.

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).

Solid- and liquid-phase extraction for the gas chromatographic-tandem mass spectrometric quantification of 2,3-dinor-thromboxane B2 and 2,3-dinor-6-oxo-prostaglandin F1 alpha in human urine.

Whole body synthesis of thromboxane A2 is best assessed by quantifying non-invasively its major urinary metabolite, i.e., 2,3-dinor-thromboxane B2 (2,3-dn-TxB2), by gas chromatography-mass spectrometry (GC-MS) or GC-tandem MS. Methods based on these techniques usually require a series of extraction and purification procedures including solid-phase extraction (SPE) and thin-layer chromatography (TLC) or liquid chromatographic separation of authentic or derivatized 2,3-dn-TxB2. Taking advantage of the inherent accuracy of GC-tandem MS and the high selectivity of the extraction of methoximated 2,3-dn-TxB2 on phenylboronic acid SPE cartridges we developed a method that involves only SPE steps prior to quantification by GC-tandem MS. The method was validated by performing in parallel an additional TLC step. Method mean accuracy and precision were of the order of 103% and 95%, respectively. The method allows furthermore co-processing of the same urine sample to quantify accurately and rapidly the major urinary metabolite of prostacyclin, i.e., 2,3-dn-6-oxo-prostaglandin (PG) F1 alpha, by GC-tandem MS. The limit of detection of the method was below each 5 pg of 2,3-dn-TxB2 and 2,3-dn-6-oxo-PGF1 alpha per 5 ml of urine. Our study suggests that dinor metabolites of isothromboxanes and isoprostacyclins are not abundantly present in human urine.

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.

Gas chromatographic-tandem mass spectrometric quantification of free 3-nitrotyrosine in human plasma at the basal state.

A fully validated gas chromatographic-tandem mass spectrometric (GC-tandem MS) method for the accurate and precise quantification of free 3-nitrotyrosine in human plasma at the basal state is described. In the plasma of 11 healthy humans a mean concentration of 2.8 nM (range 1.4-4.2 nM) for free 3-nitrotyrosine was determined by this method. This is the lowest concentration reported for free 3-nitrotyrosine in plasma of healthy humans. The presence of endogenous free 3-nitrotyrosine in human plasma was unequivocally shown by generating a daughter mass spectrum. Various precautions had to be taken to avoid artifactual formation of 3-nitrotyrosine from nitrate during sample treatment. Endogenous plasma 3-nitrotyrosine and 3-nitro-l-[(2)H(3)]tyrosine added for use as internal standard were isolated by high-performance liquid chromatographic (HPLC) analysis of 200-microl aliquots of plasma ultrafiltrate samples (20 kDa cut-off), extracted from a single HPLC fraction by solid-phase extraction, derivatized to their n-propyl ester-pentafluoropropionyl amide-trimethylsilyl ether derivatives, and quantified by GC-tandem MS. Overall recovery was determined as 50 +/- 5% using 3-nitro-l-[(14)C(9)]tyrosine. The limit of detection of the method was 4 amol of 3-nitrotyrosine, while the limit of quantitation was 125 pM using 3-nitro-l-[(14)C(9)]tyrosine. 3-Nitrotyrosine added to human plasma at 1 nM was quantitated with an accuracy of > or = 80% and a precision of > or = 94%. The method should be useful to investigate the utility of plasma free 3-nitrotyrosine as an indicator of nitric oxide ((.)NO)-associated oxidative stress in vivo in humans.

Gas chromatographic-tandem mass spectrometric quantification of human plasma and urinary nitrate after its reduction to nitrite and derivatization to the pentafluorobenzyl derivative.

Gas chromatography-mass spectrometry (GC-MS) of nitrite as its pentafluorobenzyl derivative in the negative-ion chemical ionization mode is a useful analytical tool to quantify accurately and sensitively nitrite and nitrate after its reduction to nitrite in various biological fluids. In the present study we demonstrate the utility of GC-tandem MS to quantify nitrate in human plasma and urine. Our present results verify human plasma and urine levels of nitrite and nitrate measured previously by GC-MS.

Gas chromatographic-mass spectrometric detection of S-nitroso-cysteine and S-nitroso-glutathione.

A gas chromatographic-mass spectrometric (GC-MS) method is described for the quantitative determination of the S-nitroso compounds S-nitroso-cysteine (SNC) and S-nitroso-glutathione (GSNO) using their (15)N-labeled analogs, i.e., S(15)NC and GS(15)NO, as internal standards. The method is based on the specific conversion by HgCl(2) of the unlabeled and (15)N-labeled S-nitroso groups to nitrite and (15)N-nitrite, respectively, and their conversion to the pentafluorobenzyl derivatives. The method was applied to quantify GS(15)NO formed in the cytosol of washed human erythrocytes incubated with S(15)NC. Combination of high-performance liquid chromatography with GC-MS allowed specific and accurate quantification of SNC and GSNO externally added to human plasma ultrafiltrate (range 0-10 microM). Method accuracy and precision for SNC and GSNO were close to 100 and below 9%, respectively. As little as 0.1 nM GS(15)NO corresponding to 30 amol of (15)N-nitrite injected onto the column was precisely detected by the method.

Interference by S-nitroso compounds with the measurement of nitrate in methods requiring reduction of nitrate to nitrite by cadmium.

Various methods suited for the measurement of nitrate require its reduction to nitrite by cadmium under acidic or alkaline conditions. N(G)-Nitroarginine analogs have been shown to interfere with the measurement of nitrate by such assays. In the present work we show by gas chromatography-mass spectrometry that under alkaline reduction conditions the S-nitroso compounds S-nitrosoglutathione and S-nitrosohomocysteine but not S-nitroso-N-acetylcysteine and S-nitroso-N-acetylpenicillamine can considerably contribute to nitrate and thus interfere with its measurement. Our results suggest that S-nitroso compounds may interfere with the measurement of nitrate in methods requiring cadmium-catalyzed reduction of nitrate to nitrite.

Investigations of S-transnitrosylation reactions between low- and high-molecular-weight S-nitroso compounds and their thiols by high-performance liquid chromatography and gas chromatography-mass spectrometry.

S-Transnitrosylation reactions are supposed to be the basic principle by which nitric oxide-related biological activities are regulated in vivo. Mechanisms of S-transnitrosylation reactions are poorly understood and equilibria constants for physiological S-nitroso compounds and thiols are rare. In the present study we investigated S-transnitrosylation reactions of the thiols homocysteine, cysteine, glutathione, N-acetylcysteine, N-acetylpenicillamine, and human plasma albumin and their corresponding S-nitroso compounds SNhC, SNC, GSNO, SNAC, SNAP, and SNALB utilizing high-performance liquid chromatographic and gas chromatographic-mass spectrometric techniques. These methods allowed to study S-transnitrosylation reactions in mixtures of several S-nitroso compound/thiol pairs, to determine equilibria constants, and to elucidate the mechanism of S-transnitrosylation reactions. We obtained the following order for the equilibria constants in aqueous buffered solution at pH 7.4: SNhC approximately SNAC > GSNO approximately SNALB > SNAP > SNC. Our results suggest that the mechanism of S-transnitrosylation reactions of these S-nitroso compounds and their thiols involve heterolytic cleavage of the S&sbond;N bond. Incubation of SNC with human red blood cells resulted in a dose-dependent formation of GSNO in the cytosol through S-transnitrosylation of intracellular GSH by the SNC transported into the cells. This reaction was accompanied with an almost complete disappearance of the SNC fraction transported into the cells. This finding is in full agreement with the equilibrium constant Keq of 1.9 for the reaction SNC + GSH <--> Cys + GSNO in aqueous buffer.

Measurement of S-nitrosoalbumin by gas chromatography-mass spectrometry. I. Preparation, purification, isolation, characterization and metabolism of S-[15N]nitrosoalbumin in human blood in vitro.

S-Nitrosoalbumin (SNALB) and S-[15N]nitrosoalbumin (S[15N]ALB) were prepared by various methods, purified and isolated by a novel selective extraction procedure using HiTrapBlue Sepharose affinity columns and characterized by various techniques including SDS-PAGE electrophoresis, UV-Vis spectroscopy and gas chromatography-mass spectrometry (GC-MS). S-Nitrosylation of albumin in freshly obtained human plasma by unlabeled and 15N-labeled butylnitrite at neutral pH revealed the purest preparations. For GC-MS analysis, SNALB and S[15N]ALB were treated with HgCl2 to obtain nitrite and [15N]nitrite, respectively, which were then analysed as their pentafluorobenzyl derivatives. S[15N]ALB preparations were standardized by GC-MS using nitrite as internal standard. S[15N]ALB was prepared and isolated at concentrations of 188+/-43 microM (mean +/- SD, n = 8) at a final yield of about 45%, an isotopic purity of 98%, and SDS-PAGE electrophoretic purity of 90%. 15N-Labeled SNALB was used to study its metabolism in human blood. The half-life of S[15N]ALB (25 microM) in human heparinized blood in vitro was determined by GC-MS as 5.5 h. The GC-MS method described here could be useful for the quantitative determination of SNALB in human plasma using S[15N]ALB as an internal standard.

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.

Specific and rapid quantification of 8-iso-prostaglandin F2alpha in urine of healthy humans and patients with Zellweger syndrome by gas chromatography-tandem mass spectrometry.

8-iso-Prostaglandin F2alpha (8-iso-PGF2alpha) is currently discussed as a potential index parameter of oxidative stress in vivo. We describe in this article a fully validated gas chromatographic-tandem mass spectrometric method for the quantitative determination of 8-iso-PGF2alpha in human urine. The method is highly specific and requires a single thin-layer chromatographic step for sample purification. Inter- and intraday imprecision were below 8%. Mean inaccuracy was 5.3% for added levels of 8-iso-PGF2alpha up to 2000 pg/ml of urine. We measured highly elevated excretion of 8-iso-PGF2alpha in the urine of children with peroxisomal beta-oxidation deficiency, i.e. Zellweger syndrome, (63.3+/-16.6 ng/mg creatinine) compared to that of healthy children (0.51+/-0.16 ng/mg creatinine) (mean+/-S.D., both n=5). The method could be useful for diagnosing Zellweger syndrome and for investigating the utility of 8-iso-PGF2alpha as a novel marker for oxidative stress in vivo in man.

Measurement of nitrite and nitrate in plasma, serum and urine of humans by high-performance liquid chromatography, the Griess assay, chemiluminescence and gas chromatography-mass spectrometry: interferences by biogenic amines and N(G)-nitro-L-arginine analogs.

In this paper, the HPLC method for the measurement of nitrite and nitrate in serum of humans newly reported by E1 Menyawi et al. is discussed, especially in regard to the extremely low nitrate levels measured in serum of healthy humans. From the discussion, it is concluded that: (1) Biogenic amines at physiological concentrations do not significantly interfere with the batch Griess assay. (2) The HPLC method of E1 Menyawi et al. does not reveal accurate levels for serum nitrate. (3) In serum and plasma of healthy humans, nitrate ranges within 15-70 microM. (4) Exogenous NG-nitro-L-arginine analogs can interfere with the measurement of nitrate in human plasma and urine by the batch Griess assay, chemiluminescence and GC-MS; interferences can be effectively eliminated by solid-phase extraction on cation-exchangers.

Gas chromatographic-tandem mass spectrometric determination of acetylsalicylic acid in human plasma after oral administration of low-dose aspirin and guaimesal.

A fully validated gas chromatographic-tandem mass spectrometric (GC-MS-MS) method is described for the accurate determination of acetylsalicylic acid (ASA) in human plasma after a single low-dose oral administration of aspirin or guaimesal, an ASA releasing prodrug. ASA and the newly prepared O-[2H3]-acetylsalicylic acid (d3-ASA) used as internal standard were determined in 100-microl aliquots of plasma by extractive pentafluorobenzyl (PFB) esterification using PFB bromide and tetrabutylammoniumhydrogen sulphate as the esterifying and ion-pairing agent, respectively, and by GC-MS-MS analysis in the negative-ion chemical ionization mode. The overall relative standard deviations were below 8% for ASA levels in the range 0-1 microg/ml plasma. Mean accuracy was 3.8% for ASA levels within the range 0-100 ng/ml. The limit of quantitation of the method was determined as 200 pg/ml ASA at an accuracy of 5.5% and a precision of 15.2%. The limit of detection was determined as 546 amol of ASA at a signal-to-noise ratio of 10:1.

Measurement of nitrite and nitrate in biological fluids by gas chromatography-mass spectrometry and by the Griess assay: problems with the Griess assay--solutions by gas chromatography-mass spectrometry.

Assay methods based on the Griess reaction are frequently used to measure nitrite and nitrate in urine, plasma, and other biological fluids. With minor exceptions, careful attention has not been paid in extending the Griess assay from aqueous solutions to biological fluids, In the present study, parallel measurements of nitrite and nitrate were performed in urine, plasma, and aqueous solutions with a published batch assay based on the Griess reaction and with gas chromatography-mass spectrometry (GC-MS). We report here further interferences by free reduced thiols, proteins, and other plasma constituents in the Griess assay but not in GC-MS. The best correlation (r2 = 0.985) between the Griess assay and GC-MS was observed for aqueous solutions in the absence of thiols. Unlike GC-MS, the Griess assay was not applicable to whole human plasma and urine samples. For the measurement of nitrate in diluted human urine samples, reduction by cadmium was performed both under acidic (pH 2 or 5) and alkaline (pH 8.8) conditions. The mean recovery rate of nitrate from urine samples was quantitative in the GC-MS but amounted to only 30-80% in the Griess assay. Measurement of nitrate in human urine samples (n = 33) resulted in an excellent correlation between two GC-MS techniques (r2 = 0.979) but only in a poor correlation (r2 < 0.64) between the Griess assay and GC-MS. Unlike GC-MS, the batch Griess assay is associated with many problems in measuring nitrate in biological fluids.

Effect of captopril on prostacyclin and nitric oxide formation in healthy human subjects: interaction with low dose acetylsalicylic acid.

1. Angiotensin converting enzyme inhibitors have been suggested to act in part by potentiating the stimulatory effect of bradykinin on endothelial prostacyclin and/or nitric oxide (NO) formation. This may give rise to interaction with cyclo-oxygenase inhibiting drugs like acetylsalicylic acid, which is most often used in low doses in patients with cardiovascular diseases. 2. We investigated the effects of captopril (2 x 25 mg day-1), or ASA (1 x 100 mg day-1), or the combination of both drugs for 7 days, on blood pressure, prostanoid and NO formation rates in a double-blind, double dummy, randomized crossover study in 13 healthy female subjects. The urinary metabolites of thromboxane A2 (2,3-dinor-TXB2) and prostacyclin (2,3-dinor-6-keto-PGF1 alpha), and PGE2 were measured by gas chromatography/tandem mass spectrometry in urine on days 1, 6 and 7 of each medication. NO formation was assessed using urinary NO3- and cyclic GMP as indicators. 3. Urinary 2,3-dinor-6-keto-PGF1 alpha excretion was not significantly changed by either captopril, ASA, or their combination. Urinary 2,3-dinor-TXB2 excretion was inhibited by > 80% by ASA alone or in combination with captopril (each P < 0.05), but was not affected by captopril alone. Urinary PGE2 excretion was not significantly changed by either of the treatments. Urinary NO3- and cyclic GMP excretion rates were not significantly changed by captopril, ASA, or their combination. 4. Blood pressure was slightly reduced by captopril. ASA had no effect on blood pressure when given alone, nor did it modulate the effect of captopril on blood pressure during co-administration. Angiotensin II/angiotensin I ratio (index of ACE activity) was significantly decreased by captopril alone or in combination with ASA, but was unaffected by ASA alone. 5. Captopril does not stimulate prostacyclin formation in healthy human subjects in a dose sufficient to substantially inhibit ACE activity. Co-administration of ASA significantly inhibits 2,3-dinor-TXB2 excretion, but does not interfere with the blood pressure lowering effect of captopril in healthy human subjects.

Is S-nitroso-N-acetyl-L-cysteine a circulating or an excretory metabolite of nitric oxide (NO) in man? Assessment by gas chromatography-mass spectrometry.

S-Nitroso-L-cysteine has been shown to be a circulating metabolite of the L-arginine-derived nitric oxide (NO.) in mammals. We describe here a highly sensitive gas chromatographic-mass spectrometric (GC-MS) method for the measurement of S-nitroso-N-acetyl-L-cysteine, the potential mercapturic acid of S-nitroso-L-cysteine, in human plasma and urine. For use as internal standard (I.S.) in this method we synthesized S-[15N]nitroso-N-[2H3]acetyl-L-cysteine. In plasma (n = 10) and urine (n = 30) samples of healthy humans no S-nitroso-N-acetyl-L-cysteine was detected (detection limit approximately 1 nM). Injecting the I.S. in the rat showed a good recovery of the I.S. but no endogenous S-nitroso-N-acetyl-L-cysteine. Our results suggest that renal N-acetylation of S-nitroso-L-cysteine does not represent a metabolic pathway in man.