Biomedical subjects

J C Frölich

Publications and source records attributed to J C Frölich.

At least 37 records · Page 2Linked to original sources

PubMed2000

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.

6-Ketoprostaglandin F1 alpha↗

PubMed2000

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).

Adult↗

PubMed2000

Electrospray ionization mass spectrometry of low-molecular-mass S-nitroso compounds and their thiols.

Low-molecular-mass S-nitroso compounds (R-S-N=O) are potent vasodilators and inhibitors of platelet aggregation. This work describes the electrospray ionization mass spectrometric (ESI-MS) analysis of physiological and synthetic low-molecular-mass S-nitroso compounds and their thiols including S-nitrosoglutathione, S-nitrosocysteine, glutathione and cysteine. Mass spectra of the unlabeled and S-15N-labeled low-molecular-mass S-nitroso compounds investigated are characterized by abundant cations due to [M+H]+, [M+Na]+, [(M+H)-NO]+, [2 M+H]+, and [(2 M+H)-2NO]+. Mass spectra of low-molecular-mass thiols are characterized by abundant cations due to [M+H]+, [M+Na]+ and [2M+H]+. Using off-line electrospray ionization tandem mass spectrometry we unequivocally identified S-[15N]nitrosoglutathione in human red blood cells formed after their incubation with S-[15N]nitrosocysteine. These results suggest that ESI-MS in combination with an appropriate liquid chromatographic system should be a useful analytical approach for the on-line quantitative determination of low-molecular-mass S-nitroso compounds in biological fluids in the presence of their thiols and nitrite. Considerations were made about on-line ESI-MS and quantitative measurements.

Artifacts↗

PubMed2000

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.

Animals↗

PubMed2000

Determination of nitric oxide metabolites by means of the Griess assay and gas chromatography-mass spectrometry in the cavernous and systemic blood of healthy males and patients with erectile dysfunction during different functional conditions of the penis.

Recent research implicated that the relaxation of cavernous arterial and trabecular smooth muscle-- the crucial event in penile erection--is initiated by the release of nitric oxide (NO) from nerve terminals within the cavernous tissue as well as from the endothelia that line the lacunar spaces and the intima of penile arteries. The present study was undertaken to determine whether plasma levels of the NO metabolites nitrate (NO3-) and nitrite (NO2-) in the systemic and cavernous blood of male subjects change during different penile conditions, and whether there is a difference in the NO3- and NO2- levels of normal males and patients with erectile dysfunction (ED). Twenty-four potent adult male volunteers and 15 patients with ED were exposed to visual and tactile erotic stimuli in order to elicit penile tumescence and, in the group of healthy volunteers, rigidity. Whole blood was aspirated from the corpus cavernosum and the cubital vein, and NO3- and NO2- levels were determined in plasma aliquots by means of the Griess reaction and a method combining gas chromatography and mass spectrometry (GC-MS). The mean systemic and cavernous plasma NO3-/NO2- level in blood samples obtained from the healthy volunteers was 25-31 microM when determined by means of the Griess reaction and 37-41 microM when measured by GC-MS. Both approaches revealed that NO3-/NO2- levels in the peripheral and cavernous blood do not change appreciably during developing erection, rigidity and detumescence. Moreover, no significant differences were found between NO3-/ NO2- plasma levels in the systemic and cavernous blood samples taken from the normal subjects and patients during penile flaccidity, tumescence and detumescence. Our results may reflect the fact that NO metabolism in the corpora cavernosa in the phases of penile tumescence and rigidity may account for only a minor fraction of local levels of NO3- and NO2-, which may also derive from exogenous sources. Moreover, the basal levels of NO metabolites in the blood flushing the lacunar spaces of the cavernous body in the state of developing erection could conceal any release of NO that may occur within the penile tissue. Thus, we conclude that the quantification of NO metabolites by means of advanced detection methods, such as GC-MS, is of no use in the workup of ED.

Adult↗

PubMed2000

Nebivolol decreases systemic oxidative stress in healthy volunteers.

AIMS: Nebivolol is a selective, vasodilatory beta1-adrenergic receptor antagonist which has been suggested to possess additional antioxidative properties. The aim of the present study was to assess the actions of nebivolol in antihypertensive doses on systemic oxidative stress in healthy volunteers, reflected by 24 h urinary excretion of 8-iso-PGF2alpha. METHODS: In a double-blind, cross-over study, 12 healthy volunteers received 5 mg nebivolol once daily or placebo for a total of 7 days, separated by a wash out period of 2 weeks. After each treatment period 24 h urinary excretion of 8-iso-PGF2alpha was determined by gas chromatography-tandem mass spectrometry. RESULTS: After the 7 day treatment period nebivolol decreased significantly urinary excretion of 8-iso-PGF2alpha by 24% from 55.3 +/- 5.1 pmol mmol-1 creatinine during the placebo period to 42.3 +/- 4.7 pmol mmol-1 creatinine (mean +/- s.e. mean, P = 0. 01), a mean decrease of 13 pmol mmol-1 creatinine (95% CI: -22.8; -3. 1). CONCLUSIONS: Our data show for the first time that nebivolol decreases systemic oxidative stress in young healthy volunteers.

Adult↗

PubMed2000

COX-2 and the kidneys.

The kidney is the second most frequent target of serious adverse effects of non-steroidal antiinflammatory drugs (NSAIDs). The renal side effects of NSAIDs related to inhibition of cyclooxygenase (COX) comprise reduction in renal blood flow (RBF) and glomerular filtration rate (GFR), sodium/water retention, water intoxication and hyperkalemia. The discovery of two COX-isoenzymes, a constitutive COX-1, serving homeostatic prostanoid synthesis, and an inducible COX-2, responsible for proinflammatory prosta noid production, led to the development of new NSAIDs: Preferential and specific COX-2 inhibitors, promising minimal NSAID-typical toxicity with equivalent efficacy. However, we learned that there is no clear distinction in "physiologic" constitutive COX-1 and "inflammatory" inducible COX-2. This is particular true for the kidney of humans and other mammalians, where COX-2 was found constitutively in meaningful amounts. Animal experiments and clinical trials with preferential and specific COX-2 inhibitors revealed that COX-2 is the critical enzyme for sodium excretion, renin release and likely antagonism of antidiuretic hormone. Additionally, a significant role of COX-2 for nephro genesis is suggested. For renal hemodynamics the given evidence point to COX-1 as the predominant enzyme, but further investigations are required. In summary, the gain of renal safety by use of preferential or specific COX-2 inhibitors is small or negligible with respect to sodium retention, hyperkalemia and probably water intoxication. These drugs may be advantageous regarding renal perfusion, but presently the same precautions as for conventional NSAIDs must be used.

Animals↗

PubMed2000

Role of endogenous nitric oxide in circadian blood pressure regulation in healthy humans and in patients with hypertension or atherosclerosis.

BACKGROUND: Nitric oxide (NO) is involved in the regulation of blood pressure and local blood flow. Its biological activity is impaired in hypertension and atherosclerosis. Because blood pressure undergoes a circadian rhythm, we investigated whether systemic NO production is dependent on a circadian variability, and whether the phasing of diurnal rhythm in NO production corresponds to the one in blood pressure in humans. METHODS: We studied three groups of human subjects: 8 healthy volunteers (HV), 8 patients with essential hypertension (HT), and 8 patients with peripheral arterial occlusive disease (PAOD). Twenty-four-hour ambulatory blood pressure monitoring was performed simultaneously with eight consecutive 3-hour urine collection periods. Urinary nitrate excretion was measured by gas chromatography-mass spectrometry; urinary cyclic GMP excretion was assessed by RIA. RESULTS: Twenty-four-hour mean arterial blood pressure was 119.8 +/- 2.0/75.8 +/- 1.5 mm Hg in HV, 145.0 +/- 6.4/94.9 +/- 2.8 mm Hg in HT (P < 0.05 vs HV), and 137.0 +/- 7.3/81.5 +/- 1.9 mm Hg in PAOD (P = NS vs HV). There was significant circadian variation in blood pressure in all groups, but daily amplitude was lower in HT and PAOD than in HV (P < 0.05); 24-hour mean urinary nitrate excretion was 183.4 +/- 27.2 mumol/mmol creatinine in HV, 102.9 +/- 18.1 mumol/mmol creatinine in HT, and 162.1 +/- 22.2 mumol/mmol creatinine in PAOD (P < 0.05 vs HV and HT). Urinary cyclic GMP excretion was 211.8 +/- 19.0 nmol/mmol creatinine in HV, 108.6 +/- 12.4 nmol/mmol creatinine in HT, and 97.9 +/- 13.4 nmol/mmol creatinine in PAOD (P < 0.05 for HT and PAOD vs HV). Circadian variation was present in urinary nitrate and cyclic GMP excretion in HV but was significantly diminished in HT and PAOD, respectively; 24-hour mean nitrate-to-cyclic GMP ratio was 0.89 +/- 0.05 in HV and 1.10 +/- 0.10 in HT (P = NS). It was increased to 2.02 +/- 0.17 in PAOD (P < 0.05 vs HV and HT). CONCLUSIONS: There is significant circadian variation in urinary nitrate and cyclic GMP excretion rates, two marker molecules for systemic NO production, in healthy humans. NO production is increased in the morning, concomitantly with the morning increase in blood pressure, indicating that NO may buffer blood pressure increase. Diurnal variation in nitrate and cyclic GMP excretion is absent in HT, pointing to impaired NO formation. The major change in PAOD is increased nitrate/cyclic GMP ratio, which points to increased oxidative inactivation of NO in this disease. Disturbed formation and activity of NO may contribute to blood pressure alterations in cardiovascular disease.

Adult↗

PubMed1999

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.

Adult↗

PubMed1999

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.

Fluorobenzenes↗

PubMed1999

Determination of S-nitrosoglutathione in human and rat plasma by high-performance liquid chromatography with fluorescence and ultraviolet absorbance detection after precolumn derivatization with o-phthalaldehyde.

An analytical method is described for the quantification of S-nitrosoglutathione (GSNO), a potent physiological vasodilator and inhibitor of platelet aggregation, in the presence of a high excess of reduced glutathione (GSH). The method is based on the quantitative elimination of GSH by N-ethylmaleimide, the conversion of GSNO by 2-mercaptoethanol to GSH, its reaction with o-phthalaldehyde (OPA) to form a highly fluorescent and UV-absorbing tricyclic isoindole derivative, and subsequent high-performance liquid chromatographic (HPLC) separation with fluorescence and/or UV absorbance detection. The OPA derivatives of GSH and GSNO obtained by this method were found to be identical by mass spectrometry. GSH (up to 50 microM) did not interfere with the analysis of GSNO (up to 1000 nM). The limits of detection of the method for buffered aqueous solutions of GSNO were determined as 3 nM using fluorescence and 70 nM using UV absorbance detection. Isolation of GSNO by HPLC analysis (pH 7.0) of plasma ultrafiltrate samples (200 microl) prior to derivatization allows specific and artifact-free quantification of GSNO in human and rat plasma. Reduced and oxidized glutathione, nitrite, and cysteine did not interfere with the measurement of GSNO in human and rat plasma. The limit of quantitation (LOQ) of the combined method was determined as 100 nM of GSNO in human plasma ultrafiltrate using fluorescence detection. No endogenous GSNO could be detected in ultrafiltrate samples of plasma of 10 healthy humans at concentrations exceeding the LOQ of the method. After iv infusion of GSNO (125 micromol/kg body wt) in a rat for 20 min GSNO and GSH were detected in rat plasma at 60 and 130 microM, respectively. The method should be useful to investigate formation, metabolism, and reactions of GSNO in vitro and in vivo at physiologically relevant concentrations.

Acetylcysteine↗

PubMed1999

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.

Cysteine↗

PubMed1999

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.

Cadmium↗

PubMed1999

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.

Acetylcysteine↗

PubMed1999

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.

Blood↗

PubMed1999

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.

Calibration↗

PubMed1999

High-performance liquid chromatographic analysis of nitrite and nitrate in human plasma as S-nitroso-N-acetylcysteine with ultraviolet absorbance detection.

A rapid HPLC method with UV absorbance detection at 333 nm for the measurement of nitrite and nitrate in ultrafiltrate samples of human plasma is described. The method is based on hydrochloric acid-catalyzed conversion of nitrite by N-acetyl-L-cysteine to S-nitroso-N-acetyl-L-cysteine and isocratic elution using 10 mM NaH2PO4 in acetonitrile-water, pH 2.0 (15:85, v/v). The limit of detection of the method is 50 nM nitrite. The method was validated by gas chromatography-mass spectrometry.

Acetylcysteine↗

PubMed1999

Inhibition of platelet aggregation by S-nitroso-cysteine via cGMP-independent mechanisms: evidence of inhibition of thromboxane A2 synthesis in human blood platelets.

S-Nitroso-cysteine (SNC), a putative endothelium-derived relaxing factor, potently inhibited collagen- and arachidonic acid-induced platelet aggregation (IC50=100 nM) and thromboxane A2 (TxA2) synthesis of human blood platelets. ODQ, a selective inhibitor of the soluble guanylyl cyclase, inhibited SNC-induced formation of cGMP but did not reverse inhibition by SNC of collagen- and arachidonic acid-induced platelet aggregation. Combination of ODQ with SQ-29548, a specific platelet TxA2 receptor antagonist, did not modify the antiaggregatory action of SNC. Our study shows that SNC inhibits platelet aggregation by cGMP-independent mechanisms that may involve inhibition of TxA2 synthesis in human platelets.

Arachidonic Acid↗