Predicting butyrate- and propionate-forming bacteria of gut microbiota from sequencing data.

The bacteria-derived short-chain fatty acids (SCFAs) butyrate and propionate play important (distinct) roles in health and disease, and understanding the ecology of respective bacteria on a community-wide level is a top priority in microbiome research. Applying sequence data (metagenomics and 16S rRNA gene) to predict SCFAs production in vitro and in vivo, a clear split between butyrate- and propionate-forming bacteria was detected with only very few taxa exhibiting pathways for the production of both SCFAs. After in vitro growth of fecal communities from distinct donors (n = 8) on different substrates (n = 7), abundances of bacteria exhibiting pathways correlated with respective SCFA concentrations, in particular in the case of butyrate. For propionate, correlations were weaker, indicating that its production is less imprinted into the core metabolism compared with butyrate-forming bacteria. Longitudinal measurements in vivo (n = 5 time-points from 20 subjects) also revealed a correlation between abundances of pathway-carrying bacteria and concentrations of the two SCFAs. Additionally, lower bacterial cell concentrations, together with higher stool moisture, promoted overall bacterial activity (measured by flow cytometry and coverage patterns of metagenome-assembled genomes) that led to elevated SCFA concentrations with over-proportional levels of butyrate. Predictions on pathway abundances based on 16S rRNA gene data using our in-house database worked well, yielding similar results as metagenomic-based analyses. Our study indicates that stimulating growth of butyrate- and propionate-producing bacteria directly leads to more production of those compounds, which is governed by two functionally distinct bacterial groups facilitating the development of precision intervention strategies targeting either metabolite.

Camphorquinone alters the expression of extracellular proteases in a 3D co-culture model of the oral mucosa.

OBJECTIVE: Objective of our investigation was to determine the influence of CQ on the expression of antioxidant proteins and extracellular proteases in a 3D co-culture model (3DCCM) of the oral mucosa and to analyze the distribution and stability of CQ within 3D-CCMs. METHODS: 3D-CCMs consist of confluent keratinocytes (OKF6/TERT2) on cell culture inserts on top of human gingival fibroblasts (HGFs) in collagen. The treatment was carried out by adding CQ to the cell culture inserts at two time points with declining concentrations. Mass spectrometry was used to analyze the CQ concentration above and underneath the OKF6/TERT2-layer. The expression of antioxidant genes was analyzed by qRT-PCR and western blot. The regulation of extracellular proteases from different families was analyzed by qRT-PCR and Proteome Profiler arrays. RESULTS: GC/MS analysis showed that CQ was evenly distributed within the model. Heme oxygenase-1, NAD(P)H quinone dehydrogenase 1 (NQO1), and superoxide dismutase 1 were induced on the mRNA and protein level in OKF6/TERT2 cells. In HGFs, only the transcription of NQO1 was induced. The transcription of extracellular proteases was increased mainly in OKF6/TERT2 cells 72 h after the initial treatment. The quantity of ten out of 25 analyzed extracellular proteases in the cell culture supernatant above and six underneath the keratinocyte-layer were modulated by CQ. SIGNIFICANCE: Despite its high reactivity, CQ is able to penetrate a dense keratinocyte-layer, presumably across plasma membranes. CQ initially induced the cellular defense machinery against oxidative stress and altered the expression of extracellular proteases. We assume a relationship between both processes.

Effects of HEMA on Nrf2-related gene expression using a newly developed 3D co-culture model of the oral mucosa.

OBJECTIVE: 2-Hydroxyethyl methacrylate (HEMA) is a component of many resin-modified materials and elutes from dental restorations into the oral cavity. Objective of our investigation was to determine the impact of HEMA on oral keratinocytes (OKF6/TERT2) and gingival fibroblasts (HGFs) in a newly established 3D co-culture model (3D-CCM) and to analyze the permeability of OKF6/TERT2 cells for HEMA. METHODS: Well-characterized 3D-CCMs, consisting of confluent OKF6/TERT2 cells on cell culture inserts above HGF-containing collagen gels, were treated supra-epithelial with HEMA. Mass spectrometry was used to measure the supra- and sub-epithelial distribution of HEMA after 24 h. The impact of HEMA on nuclear factor erythroid 2-related factor 2 (Nrf2) target genes was measured by qRT-PCR and western blot analysis. RESULTS: Mass spectrometry showed that HEMA was evenly distributed above and below the keratinocyte layer after 24 h. Analyzed target genes of Nrf2 were induced in both cell types on the mRNA-level but less pronounced in HGFs. On the protein-level, both cell types showed similar effects: At 5 mM HEMA, heme oxygenase-1 was induced 5.1-fold in OKF6/TERT2 cells and 4.1-fold in HGFs. NAD(P)H quinone dehydrogenase-1 was approximately induced 1.85-fold in both cell types. SIGNIFICANCE: Our 3D-CCM is suitable to analyze the biocompatibility of dental materials due to an improved simulation of the oral mucosa compared to monolayer cultures. Our results indicate that HEMA is able to penetrate a dense layer of keratinocytes and to activate the cellular oxidative defense response. This may be due to the activation of the Nrf2-pathway in both cell types.

GC-MS and GC-MS/MS measurement of ibuprofen in 10-µL aliquots of human plasma and mice serum using [α-methylo-2H3]ibuprofen after ethyl acetate extraction and pentafluorobenzyl bromide derivatization: Discovery of a collision energy-dependent H/D isotope effect and pharmacokinetic application to inhaled ibuprofen-arginine in mice.

GC-MS and GC-MS/MS methods were developed and validated for the quantitative determination of ibuprofen (d0-ibuprofen), a non-steroidal anti-inflammatory drug (NSAID), in human plasma using α-methyl-2H3-4-(isobutyl)phenylacetic acid (d3-ibuprofen) as internal standard. Plasma (10µL) was diluted with acetate buffer (80µL, 1M, pH 4.9) and d0- and d3-ibuprofen were extracted with ethyl acetate (2×500µL). After solvent evaporation d0- and d3-ibuprofen were derivatized in anhydrous acetonitrile by using pentafluorobenzyl (PFB) bromide and N,N-diisopropylethylamine as the base catalyst. Under electron-capture negative-ion chemical ionization (ECNICI), the PFB esters of d0- and d3-ibuprofen readily ionize to form their carboxylate anions [M-PFB]- at m/z 205 and m/z 208, respectively. Collision-induced dissociation (CID) of m/z 205 and m/z 208 resulted in the formation of the anions at m/z 161 and m/z 164, respectively, due to neutral loss of CO2 (44 Da). A collision energy-dependent H/D isotope effect was observed, which involves abstraction/elimination of H- from d0-ibuprofen and D- from d3-ibuprofen and is minimum at a CE value of 5eV. Quantitative GC-MS determination was performed by selected-ion monitoring of m/z 205 and m/z 208. Quantitative GC-MS/MS determination was performed by selected-reaction monitoring of the mass transitions m/z 205 to m/z 161 for d0-ibuprofen and m/z 208 to m/z 164 for d3-ibuprofen. In a therapeutically relevant concentration range (0-1000µM) d0-ibuprofen added to human plasma was determined with accuracy (recovery, %) and imprecision (relative standard deviation, %) ranging between 93.7 and 110%, and between 0.8 and 4.9%, respectively. GC-MS (y) and GC-MS/MS (x) yielded almost identical results (y=4.00+0.988x, r2=0.9991). In incubation mixtures of arachidonic acid (10µM), d3-ibuprofen (10µM) or d0-ibuprofen (10µM) with ovine cyclooxygenase (COX) isoforms 1 and 2, the concentration of d3-ibuprofen and d0-ibuprofen did not change upon incubation at 37°C up to 60min. The trough pharmacokinetics of an inhaled arginine-containing ibuprofen preparation in mice was studied after once-daily treatment (0.0, 0.07, 0.4 and 2.5mg/kg body weight) for three days. A linear relationship between ibuprofen concentration in serum (10µL) and administered dose 24h after the last drug administration was observed.

Simultaneous GC-MS/MS measurement of malondialdehyde and 4-hydroxy-2-nonenal in human plasma: Effects of long-term L-arginine administration.

Here, we report the simultaneous derivatization and quantification of malondialdehyde (MDA) and 4-hydroxy-2-nonenal (HNE) in human plasma by GC-MS/MS using [1,3-2H2]-MDA (d2-MDA) and [9,9,9-2H3]-HNE (d3-HNE) as the internal standards, respectively. MDA, d2-MDA, HNE and d3-HNE were converted to their pentafluorobenzyl oximes (PFBOX) by pentafluorobenzyl hydroxylamine. Subsequently, the hydroxyl groups of the PFBOX of HNE and d3-HNE were trimethylsilylated with N,O-bis(trimethylsilyl)trifluoroacetamide/1% trimethylchlorosilane. GC-MS/MS analyses were performed in the electron-capture negative-ion chemical ionization mode. Quantification was performed by selected-reaction monitoring the mass transitions m/z 442 to m/z 243 for MDA, m/z 444 to m/z 244 for d2-MDA, m/z 403 → m/z 283 for HNE and m/z 406 → m/z 286 for d3-HNE. The method was applied to measure MDA and HNE in plasma of patients suffering from coronary artery disease (CAD) or peripheral artery occlusive disease (PAOD) before and after oral supplementation of L-arginine (3 g/day) or placebo for 3 (CAD and PAOD) and 6 months (PAOD). All plasma samples were analyzed after completion of the studies. Our results revealed that storage of plasma samples (at -80 °C) leads to lower MDA and HNE plasma concentrations in the plasma samples that were collected at the end of the studies as compared to those collected at the begin of the studies. Based on MDA and HNE measurements in plasma, L-arginine did not influence lipid peroxidation in CAD and PAOD patients. Long-term studies on lipid peroxidation are best performed by measuring oxidative stress biomarkers such as MDA and/or HNE in plasma samples immediately after their collection. Long-term storage of plasma samples even at -80 °C is not recommended.

Neurotransmitter and their metabolite concentrations in different areas of the HPRT knockout mouse brain.

Lesch-Nyhan syndrome (LNS) is characterized by uric acid overproduction and severe neurobehavioral symptoms, such as recurrent self-mutilative behavior. To learn more about the pathophysiology of the disease, we quantified neurotransmitters and their metabolites in the cerebral hemisphere, cerebellum and the medulla oblongata of HPRT knockout mice, an animal model for LNS, in comparison to the corresponding wild-type. Our analyses included l-glutamate, 4-aminobutanoic acid (GABA), acetylcholine, serotonin, 5-hydroxyindoleacetic acid (5-HIAA), norepinephrine, l-normetanephrine, epinephrine and l-metanephrine and were conducted via high performance liquid chromatography (HPLC) coupled to tandem mass spectrometry (MS/MS). Among these neurotransmitter systems, we did not find any abnormalities in the HPRT knockout mouse brains. On one side, this might indicate that HPRT deficiency most severely affects dopamine signaling, while brain functioning based on other neurotransmitters is more or less spared. On the other hand, our findings may reflect a compensating mechanism for impaired purine salvage that protects the brain in HPRT-deficient mice but not in LNS patients.

Altered histamine neurotransmission in HPRT-deficient mice.

Lesch-Nyhan syndrome (LNS) is an X-chromosomal disorder with congenital deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) as underlying defect. We determined the concentrations of dopamine, histamine and their metabolites in brains of HPRT knockout mice, which serve as an animal model for LNS, and compared the results to those obtained from wild-type controls. Analyses were performed by high performance liquid chromatography (HPLC)-coupled tandem mass spectrometry (MS/MS). Besides a decrease of dopamine and 3-methoxytyramine (3-MT) concentrations in the cerebral hemisphere, HPRT-deficient mice also exhibited significantly reduced 1-methylhistamine (1-MH) and 1-methylimidazole-4-acetic acid (1-MI4AA) concentrations in the brain hemisphere and medulla. Moreover, the amount of 1-MI4AA was significantly decreased in the cerebellum. Our findings show that neuronal perturbations caused by HPRT deficiency are not restricted to the dopamine system but also affect histaminergic neurotransmission. These new insights into the brain metabolism of an LNS mouse model may help to find new therapeutic strategies to improve the quality of life of LNS patients.

GC-MS and GC-MS/MS measurement of the cardiovascular risk factor homoarginine in biological samples.

L-Homoarginine (hArg) has recently emerged as a novel cardiovascular risk factor and to herald a poor prognosis in heart failure patients. Here, we report on the development and thorough validation of gas chromatography-mass spectrometry (GC-MS) and gas chromatography-tandem mass spectrometry (GC-MS/MS) methods for the quantitative determination of hArg in biological samples, including human plasma, urine and sputum. For plasma and serum samples, ultrafiltrate (10 µL; cutoff, 10 kDa) was used. For urine samples, native urine (10 µL) was used. For sputum, protein precipitation by acetone was performed. hArg is derivatized to its methyl ester tri(N-pentafluoropropionyl) derivative; de novo synthesized trideutero-methyl ester hArg is used as the internal standard (IS). Alternatively, [guanidino-(15)N2]-arginine can be used as an IS. Quantitative analyses were performed after electron-capture negative-ion chemical ionization by selected-ion monitoring in GC-MS and selected-reaction monitoring in GC-MS/MS. We obtained very similar hArg concentrations by GC-MS and GC-MS/MS, suggesting that GC-MS suffices for accurate and precise quantification of hArg in biological samples. In plasma and serum samples of the same subjects very close hArg concentrations were measured. The plasma-to-serum hArg concentration ratio was determined to be 1.12 ± 0.21 (RSD, 19 %), suggesting that blood anticoagulation is not a major preanalytical concern in hArg analysis. In healthy subjects, the creatinine-corrected urinary excretion of hArg varies considerably (0.18 ± 0.22 µmol/mmol, mean ± SD, n = 19) unlike asymmetric dimethylarginine (ADMA, 2.89 ± 0.89 µmol/mmol). In urine, hArg correlated with ADMA (r = 0.475, P = 0.040); in average, subjects excreted in the urine about 17.5 times more ADMA than hArg. In plasma of healthy humans, the concentration of hArg is of the order of 2 µM. hArg may be a low-abundance constituent of human plasma proteins. The GC-MS and GC-MS/MS methods we report in this article are useful to study the physiology and pathology of hArg in experimental and clinical settings.

GC-MS/MS and LC-MS/MS studies on unlabelled and deuterium-labelled oleic acid (C18:1) reactions with peroxynitrite (O=N-O-O⁻) in buffer and hemolysate support the pM/nM-range of nitro-oleic acids in human plasma.

Oleic acid (cis-9,10-octadecenoic acid) is the most abundant monounsaturated fatty acid in human blood. Peroxynitrite (ONOO(-)) is a short-lived species formed from the reaction of nitric oxide (NO) and superoxide (O2(-)). Peroxynitrite is a potent oxidizing and moderate nitrating agent. We investigated reactions of unlabelled and deuterium labelled oleic acid in phosphate buffered saline (PBS) and lysed human erythrocytes with commercially available sodium peroxynitrite (Na(+)ONOO(-)). Non-derivatized reaction products were analyzed by spectrophotometry, HPLC with UV absorbance detection, and LC-MS/MS electrospray ionization in the negative-ion mode. Reaction products were also analyzed by GC-MS/MS in the electron capture negative-ion chemical ionization mode after derivatization first with pentafluorobenzyl (PFB) bromide and then with N,O-bis(trimethylsilyl)trifluoroacetamide. Identified oleic acid reaction products in PBS and hemolysate include cis-9,10-epoxystearic acid and trans-9,10-epoxystearic acid (about 0.1% with respect to oleic acid), threo- and erythro-9,10-dihydroxy-stearic acids. Vinyl nitro-oleic acids, 9-nitro-oleic acid (9-NO2OA) and 10-nitro-oleic acid (10-NO2OA), or other nitro-oleic acids were not found to be formed from the reaction of oleic acid with peroxynitrite in PBS or hemolysate. Our in vitro study suggests that peroxynitrite oxidizes but does not nitrate oleic acid in biological samples. Unlike thiols and tyrosine, oleic acid is not susceptible to peroxynitrite. GC-MS/MS analysis of PFB esters is by far more efficient than LC-MS/MS analysis of non-derivatized oleic acid and its derivates. Our in vitro results support our previous in vivo findings that nitro-oleic acid plasma concentrations of healthy and diseased subjects are in the pM/nM-range.

UPLC-MS/MS measurement of S-nitrosoglutathione (GSNO) in human plasma solves the S-nitrosothiol concentration enigma.

We developed and validated a fast UPLC-MS/MS method with positive electrospray ionization (ESI+) for the quantitative determination of S-nitrosoglutathione (GSNO) in human plasma. We used a published protocol for the inactivation of plasma γ-glutamyltransferase (γGT) activity by using the γGT transition inhibitor serine/borate and the chelator EDTA for the stabilization of GSNO, and N-ethylmaleimide (NEM) to block SH groups and to avoid S-transnitrosylation reactions which may diminish GSNO concentration. S-[(15)N]Nitrosoglutathione (GS(15)NO) served as internal standard. Fresh blood was treated with NEM/serine/borate/EDTA, plasma spiked with GS(15)NO (50nM) was ultrafiltered (cut-off 10kDa) and 10µL aliquots of the ultrafiltrate were analyzed by UPLC-MS/MS. Five HILIC columns and an Acquity UPLC BH amide column were tested. The mobile phase was acetonitrile-water (70:30, v/v), contained 20mM ammonium formate, had a pH value of 7, and was pumped isocratically (0.5mL/min). The Nucleoshell column allowed better LC performance and higher MS sensitivity. The retention time of GSNO was about 1.1min. Quantification was performed by selected-reaction monitoring the mass transition m/z 337 ([M+H](+))→m/z 307 ([M+H(14)NO](+)) for GSNO (i.e., GS(14)NO) and m/z 338 ([M+H](+))→m/z 307 ([M+H(15)NO](+)) for GS(15)NO. NEM/serine/borate/EDTA was found to stabilize GSNO in human plasma. The method was validated in human plasma (range, 0-300nM) using 50nM GS(15)NO. Accuracy and precision were in generally acceptable ranges. A considerable matrix effect was observed, which was however outweighed by the internal standard GS(15)NO. In freshly prepared plasma from heparinized blood donated by 10 healthy subjects, no endogenous GSNO was determined above 2.8nM, the limit of quantitation (LOQ) of the method. This study challenges previously reported GSNO plasma concentrations being far above the present method LOQ value and predicts that the concentration of low-molecular-mass and high-molecular-mass S-nitrosothiols are in the upper pM- and lower nM-range, respectively.

A validated, rapid UPLC-MS/MS method for simultaneous ivabradine, reboxetine, and metoprolol analysis in human plasma and its application to clinical trial samples.

A recent clinical trial assessing human autonomic cardiovascular regulation applied pacemaker channel inhibition with ivabradine, norepinephrine transporter blockade with reboxetine, and beta-adrenoreceptor blockade with metoprolol. To verify patient adherence, we developed and validated a fast UPLC-MS/MS assay measuring all three compounds simultaneously. Deuterium-labeled drugs, d3-ivabradine, d5-reboxetine and d7-metoprolol, served as internal standards. Sample preparation of 200µL human plasma consisted of a single liquid-liquid extraction step by means of ethyl acetate. Chromatographic separation was performed on a 50-mm long BEH C18 column with gradient elution using a mixture of water and methanol each containing 2mM ammonium acetate over 4.5min. The mass spectrometer was operated in the positive electrospray ionization (ESI+) mode. Characteristic product ions resulting from collision-induced dissociation of unlabeled and deuterium-labeled drugs with argon were used for quantification in the selected-reaction monitoring mode. We validated the method according to the European Medicines Agency (EMA) guideline on bioanalytical method validation over the range from 1ng/mL to 500ng/mL for all three analytes. Linear responses with correlation coefficients>0.99 over that range were acquired. The LOQ value was 1ng/mL for each drug. Regulatory criteria for accuracy (80-120%) and precision (RSD<15%) were met for all drugs. The internal standard-normalized matrix factor was close to 1 for low and high analyte concentrations. We successfully measured ivabradine, reboxetine, and metoprolol concentrations in 107 human plasma samples from a clinical trial. Quality control samples processed in parallel confirmed the method's reliability in a clinical setting.

Glutathione promotes prostaglandin H synthase (cyclooxygenase)-dependent formation of malondialdehyde and 15(S)-8-iso-prostaglandin F2α.

Prostaglandin (PG) H synthases (PGHS) or cyclooxygenases (COX) catalyse the peroxidation of arachidonic acid (AA) to PGG(2) and PGH(2) which are further converted to a series of prostaglandins and thromboxane A(2). Here, we report that GSH promotes concomitant formation of the current oxidative stress biomarkers malondialdehyde (MDA) and 15(S)-8-iso-prostaglandin F(2α) from AA via PGHS. This illustrates an uncommon interplay of enzymatic and chemical reactions to produce species that are considered to be exclusively produced by free-radical-catalysed reactions. We propose mechanisms for the PGHS/AA/GSH-dependent formation of MDA, 15(S)-8-iso-prostaglandin F(2α) and other F(2)-isoprostanes. These mechanisms are supported by clinical observations.

Stable isotope gas chromatography-tandem mass spectrometry determination of aminoethylcysteine ketimine decarboxylated dimer in biological samples.

Aminoethylcysteine ketimine decarboxylated dimer (AECK-DD; systematic name: 1,2-3,4-5,6-7,8-octahydro-1,8a-diaza-4,6-dithiafluoren-9(8aH)-one) is a previously described metabolite of cysteamine that has been reported to be present in mammalian brain, urine, plasma, and cells in culture and vegetables and to possess potent antioxidative properties. Here, we describe a stable isotope gas chromatography-tandem mass spectrometry (GC-MS/MS) method for specific and sensitive determination of AECK-DD in biological samples. (13)C(2)-labeled AECK-DD was synthesized and used as the internal standard. Derivatization was carried out by N-pentafluorobenzylation with pentafluorobenzyl bromide in acetonitrile. Quantification was performed by selected reaction monitoring of the mass transitions m/z 328 to 268 for AECK-DD and m/z 330 to 270 for [(13)C(2)]AECK-DD in the electron capture negative ion chemical ionization mode. The procedure was systematically validated for human plasma and urine samples. AECK-DD was not detectable in human plasma above approximately 4nM but was present in urine samples of healthy humans at a maximal concentration of 46nM. AECK-DD was detectable in rat brain at very low levels of approximately 8pmol/g wet weight. Higher levels of AECK-DD were detected in mouse brain (∼1nmol/g wet weight). Among nine dietary vegetables evaluated, only shallots were found to contain trace amounts of AECK-DD (∼6.8pmol/g fresh tissue).

Stable-isotope dilution GC-MS method for ethanol in vapour ethanol and microdialysis systems based on carbonate-catalyzed extractive pentafluorobenzoylation.

Common ethanol detection methods are not applicable to cell culture media and microdialysates due to interference with medium constituents including amino acids and pH indicators. We present a novel GC-MS method for the accurate and precise analysis of ethanol in cell cultures and microdialysates. The method is based on the carbonate-catalyzed extractive pentafluorobenzoylation of ethanol and deuterium-labelled ethanol serving as the internal standard and on their GC-MS analysis in the electron-capture negative-ion chemical ionization mode. The method was used to optimize experimental conditions in a custom-made ethanol vapour system utilized for studies examining ethanol influences on neuronal cell lines and in microdialysis.

Renal carbonic anhydrases are involved in the reabsorption of endogenous nitrite.

Nitrite (ONO(-)) exerts nitric oxide (NO)-related biological actions and its concentration in the circulation may be of particular importance. Nitrite is excreted in the urine. Hence, the kidney may play an important role in nitrite/NO homeostasis in the vasculature. We investigated a possible involvement of renal carbonic anhydrases (CAs) in endogenous nitrite reabsorption in the proximal tubule. The potent CA inhibitor acetazolamide was administered orally to six healthy volunteers (5 mg/kg) and nitrite was measured in spot urine samples before and after administration. Acetazolamide increased abruptly nitrite excretion in the urine, strongly suggesting that renal CAs are involved in nitrite reabsorption in healthy humans. Additional in vitro experiments support our hypothesis that nitrite reacts with CO(2), analogous to the reaction of peroxynitrite (ONOO(-)) with CO(2), to form acid-labile nitrito carbonate [ONOC(O)O(-)]. We assume that this reaction is catalyzed by CAs and that nitrito carbonate represents the nitrite form that is actively transported into the kidney. The significance of nitrite reabsorption in the kidney and the underlying mechanisms, notably a direct involvement of CAs in the reaction between nitrite and CO(2), remain to be elucidated.

Measurement of nitrite in urine by gas chromatography-mass spectrometry.

Nitric oxide (NO) is enzymatically produced from L-arginine and has a variety of biological functions. Autoxidation of NO in aqueous media yields nitrite (O = N-O(-)). NO and nitrite are oxidized in erythrocytes by oxyhemoglobin to nitrate (NO(3)(-)). Nitrate reductases from bacteria reduce nitrate to nitrite. Nitrite and nitrate are ubiquitous in nature, they are present throughout the body and they are excreted in the urine. Nitrite in urine has been used for several decades as an indicator and measure of bacteriuria. Since the identification of nitrite as a metabolite of NO, circulating nitrite is also used as an indicator of NO synthesis and is considered an NO storage form. In contrast to plasma nitrite, the significance of nitrite in the urine beyond bacteriuria is poorly investigated and understood. This chapter describes a gas chromatography-mass spectrometry (GC-MS) protocol for the quantitative determination of nitrite in urine of humans. Although the method is useful for detection and quantification of bacteriuria, the procedures described herein are optimum for urinary nitrite in conditions other than urinary tract infection. The method uses [(15)N]nitrite as internal standard and pentafluorobenzyl bromide as the derivatization agent. Derivatization is -performed on 100-µL aliquots and quantification of toluene extracts by selected-ion monitoring of m/z 46 for urinary nitrite and m/z 47 for the internal standard in the electron-capture negative-ion chemical ionization mode.

Measurement of 3-nitro-tyrosine in human plasma and urine by gas chromatography-tandem mass spectrometry.

Reaction of reactive nitrogen species (RNS), such as peroxynitrite and nitryl chloride with soluble tyrosine and tyrosine residues in proteins produces soluble 3-nitro-tyrosine and 3-nitro-tyrosino-proteins, respectively. Regular proteolysis of 3-nitro-tyrosino-proteins yields soluble 3-nitro-tyrosine. 3-Nitro-tyrosine circulates in plasma and is excreted in the urine. Both circulating and excretory 3-nitro-tyrosine are considered suitable biomarkers of nitrative stress. Tandem mass spectrometry coupled with gas chromatography (GC-MS/MS) or liquid chromatography (LC-MS/MS) is one of the most reliable analytical techniques to determine 3-nitro-tyrosine. Here, we describe protocols for the quantitative determination of soluble 3-nitro-tyrosine in human plasma and urine by GC-MS/MS.

Simultaneous UPLC-MS/MS quantification of the endocannabinoids 2-arachidonoyl glycerol (2AG), 1-arachidonoyl glycerol (1AG), and anandamide in human plasma: minimization of matrix-effects, 2AG/1AG isomerization and degradation by toluene solvent extraction.

Analysis of the endocannabinoid (EC) system's key molecules 2-arachidonoyl glycerol (2AG) and arachidonoyl ethanolamide (anandamide, AEA) is challenging due to several peculiarities. 2AG isomerizes spontaneously to its biologically inactive analogue 1-arachidonoyl glycerol (1AG) by acyl migration and it is only chromatographically distinguishable from 1AG. Matrix-effects caused primarily by co-extracted phospholipids may further compromise analysis. In addition, 2AG and 1AG are unstable under certain conditions like solvent evaporation or reconstitution of dried extracts. We examined effects of different organic solvents and their mixtures, such as toluene, ethyl acetate, and chloroform-methanol, on 2AG/1AG isomerisation, 2AG/1AG stability, and matrix-effects in the UPLC-MS/MS analysis of 2AG and AEA in human plasma. Toluene prevented, both, 2AG isomerisation to 1AG and degradation of 2AG/1AG during evaporation. Toluene extracts contain only 2% of matrix-effect-causing plasma phospholipids compared to extracts from the traditionally used solvent mixture chloroform-methanol. Toluene and all other tested organic solvents provide comparable 2AG and AEA extraction yields (60-80%). Based on these favourable toluene properties, we developed and validated a UPLC-MS/MS method with positive electrospray ionization (ESI+) that allows for simultaneous accurate and precise measurement of 2AG and AEA in human plasma. The UPLC-MS/MS method was cross-validated with a previously described fully-validated GC-MS/MS method for AEA in human plasma. A close correlation (r(2)=0.821) was observed between the results obtained from UPLC-MS/MS (y) and GC-MS/MS (x) methods (y=0.01+0.85x). The UPLC-MS/MS method is suitable for routine measurement of 2AG and AEA in human plasma samples (1 mL) in clinical settings as shown by quality control plasma samples processed over a period of 100 days. The UPLC-MS/MS method was further extended to human urine. In urine, AEA was not detectable and 2AG was detected in only 3 out of 19 samples from healthy subjects at 160, 180 and 212 pM corresponding to 12.3, 14.5 and 9.9 pmol/mmol creatinine, respectively.

¹8O-Labeled nitrous acid and nitrite: Synthesis, characterization, and oxyhemoglobin-catalyzed oxidation to ¹8O-labeled nitrate.

We describe a simple laboratory method for specific labeling of nitrite with ¹8O for use in chemical and biochemical studies in the area of nitric oxide research. NaNO2 (0.1 mmol) is diluted in H2¹8O (45 µl) and acidified with HCl (1 µl, 5 M), and the solution is allowed to equilibrate. Subsequently, the sample is mixed by vortexing with ethyl acetate (500 µl), and the organic phase is dried over anhydrous Na2SO(4). Ethyl acetate is evaporated to dryness, and the residue is reconstituted in phosphate-buffered saline. In human blood hemolysate, oxyhemoglobin (HbFe¹6O2) was shown to oxidize N¹8O2⁻ to ¹6ON¹8O2⁻.

Quantification of endocannabinoids in biological systems by chromatography and mass spectrometry: a comprehensive review from an analytical and biological perspective.

The endocannabinoids anandamide (arachidonoyl ethanolamide, AEA) and 2-arachidonoyl glycerol (2AG) are physiologically occurring, biologically active compounds on CB(1) and CB(2) receptors with multiple physiological functions. AEA and 2AG have been identified and quantified in many mammalian biological fluids and tissues, such as human plasma, adipocytes, tissues and tissue microdialysates, at concentrations in the picomolar-to-nanomolar range under basal conditions. In this article, recently published chromatographic and mass spectrometric analytical methods, i.e., HPLC with fluorescence or ultraviolet detection, LC-MS, LC-MS/MS, GC-MS and GC-MS/MS, are reviewed and discussed, notably from the quantitative point of view. We focus on and emphasize the particular importance of blood sampling, sample storage and work-up including solvent and solid-phase extraction and derivatization procedures, matrix-effects, and stability of analytes. As 2AG spontaneously isomerizes to its CB(1)/CB(2) receptors biologically inactive 1-arachidonoyl glycerol (1AG) by acyl migration, this phenomenon and its particular importance for accurate quantification of 2AG are discussed in detail. Due to the electrical neutrality of AEA and 2AG their solvent extraction by toluene offers the least matrix-effect and minimum isomerization. LC-MS/MS is the most frequently used analytical technique for AEA and 2AG. At present, the utility of the GC-MS/MS methodology seems to be limited to AEA measurement in human plasma, bronchoalveolar liquid (BAL) and microdialysate samples. Despite great instrumental advances in the LC-MS/MS methodology, sampling and sample treatment remains one of the most crucial analytical steps in 2AG analysis. Extension of the LC-MS/MS methodology, for instance to microdialysate and BAL samples from clinical studies, is a big analytical challenge in endocannabinoid analysis in clinical settings. Currently available LC-MS/MS and GC-MS/MS methods should be useful to investigate the metabolism of AEA and 2AG beyond hydrolysis, i.e., by ß- and ω-oxidation pathways.