Unraveling the link between neuropathy target esterase NTE/SWS, lysosomal storage diseases, inflammation, abnormal fatty acid metabolism, and leaky brain barrier.

Mutations in Drosophila Swiss cheese (SWS) gene or its vertebrate orthologue neuropathy target esterase (NTE) lead to progressive neuronal degeneration in flies and humans. Despite its enzymatic function as a phospholipase is well established, the molecular mechanism responsible for maintaining nervous system integrity remains unclear. In this study, we found that NTE/SWS is present in surface glia that forms the blood-brain barrier (BBB) and that NTE/SWS is important to maintain its structure and permeability. Importantly, BBB glia-specific expression of Drosophila NTE/SWS or human NTE in the sws mutant background fully rescues surface glial organization and partially restores BBB integrity, suggesting a conserved function of NTE/SWS. Interestingly, sws mutant glia showed abnormal organization of plasma membrane domains and tight junction rafts accompanied by the accumulation of lipid droplets, lysosomes, and multilamellar bodies. Since the observed cellular phenotypes closely resemble the characteristics described in a group of metabolic disorders known as lysosomal storage diseases (LSDs), our data established a novel connection between NTE/SWS and these conditions. We found that mutants with defective BBB exhibit elevated levels of fatty acids, which are precursors of eicosanoids and are involved in the inflammatory response. Also, as a consequence of a permeable BBB, several innate immunity factors are upregulated in an age-dependent manner, while BBB glia-specific expression of NTE/SWS normalizes inflammatory response. Treatment with anti-inflammatory agents prevents the abnormal architecture of the BBB, suggesting that inflammation contributes to the maintenance of a healthy brain barrier. Considering the link between a malfunctioning BBB and various neurodegenerative diseases, gaining a deeper understanding of the molecular mechanisms causing inflammation due to a defective BBB could help to promote the use of anti-inflammatory therapies for age-related neurodegeneration.

Quality Control in Targeted GC-MS for Amino Acid-OMICS.

Gas chromatography-mass spectrometry (GC-MS) is suitable for the analysis of non-polar analytes. Free amino acids (AA) are polar, zwitterionic, non-volatile and thermally labile analytes. Chemical derivatization of AA is indispensable for their measurement by GC-MS. Specific conversion of AA to their unlabeled methyl esters (d0Me) using 2 M HCl in methanol (CH3OH) is a suitable derivatization procedure (60 min, 80 °C). Performance of this reaction in 2 M HCl in tetradeutero-methanol (CD3OD) generates deuterated methyl esters (d3Me) of AA, which can be used as internal standards in GC-MS. d0Me-AA and d3Me-AA require subsequent conversion to their pentafluoropropionyl (PFP) derivatives for GC-MS analysis using pentafluoropropionic anhydride (PFPA) in ethyl acetate (30 min, 65 °C). d0Me-AA-PFP and d3Me-AA-PFP derivatives of AA are readily extractable into water-immiscible, GC-compatible organic solvents such as toluene. d0Me-AA-PFP and d3Me-AA-PFP derivatives are stable in toluene extracts for several weeks, thus enabling high throughput quantitative measurement of biological AA by GC-MS using in situ prepared d3Me-AA as internal standards in OMICS format. Here, we describe the development of a novel OMICS-compatible QC system and demonstrate its utility for the quality control of quantitative analysis of 21 free AA and metabolites in human plasma samples by GC-MS as Me-PFP derivatives. The QC system involves cross-standardization of the concentrations of the AA in their aqueous solutions at four concentration levels and a quantitative control of AA at the same four concentration levels in pooled human plasma samples. The retention time (tR)-based isotope effects (IE) and the difference (δ(H/D) of the retention times of the d0Me-AA-PFP derivatives (tR(H)) and the d3Me-AA-PFP derivatives (tR(D)) were determined in study human plasma samples of a nutritional study (n = 353) and in co-processed QC human plasma samples (n = 64). In total, more than 400 plasma samples were measured in eight runs in seven working days performed by a single person. The proposed QC system provides information about the quantitative performance of the GC-MS analysis of AA in human plasma. IE, δ(H/D) and a massive drop of the peak area values of the d3Me-AA-PFP derivatives may be suitable as additional parameters of qualitative analysis in targeted GC-MS amino acid-OMICS.

Impaired Nitric Oxide Synthetase Activity in Primary Ciliary Dyskinesia-Data-Driven Hypothesis.

Low nasal nitric oxide (nNO) is a typical feature of Primary Ciliary Dyskinesia (PCD). nNO is part of the PCD diagnostic algorithm due to its discriminative power against other lung diseases, such as cystic fibrosis (CF). However, the underlying pathomechanisms are elusive. To better understand NO dysregulation in PCD, the L-arginine/NO (Arg/NO) pathway in patients with PCD (pwPCD) and CF (pwCF) and in healthy control (HC) subjects was investigated. In a prospective, controlled study, we measured in 24 pwPCD, 25 age-matched pwCF, and 14 HC the concentrations of the NO precursors Arg and homoarginine (hArg), the arginase metabolite ornithine (Orn), the NO inhibitor asymmetric dimethylarginine (ADMA), and the major NO metabolites (nitrate, nitrite) in sputum, plasma, and urine using validated methods. In comparison to HC, the sputum contents (in µmol/mg) of L-Arg (PCD 18.43 vs. CF 329.46 vs. HC 9.86, p < 0.001) and of ADMA (PCD 0.055 vs. CF 0.015 vs. HC 0.010, p < 0.001) were higher. In contrast, the sputum contents (in µmol/mg) of nitrate and nitrite were lower in PCD compared to HC (nitrite 4.54 vs. 9.26, p = 0.023; nitrate 12.86 vs. 40.33, p = 0.008), but higher in CF (nitrite 16.28, p < 0.001; nitrate 56.83, p = 0.002). The metabolite concentrations in urine and plasma were similar in all groups. The results of our study indicate that PCD, unlike CF, is associated with impaired NO synthesis in the lung, presumably due to mechano-chemical uncoupling.

l-Arginine/nitric oxide pathway and oxidative stress in adults with ADHD: Effects of methylphenidate treatment.

INTRODUCTION: Attention deficit hyperactivity disorder (ADHD) is a mental disorder that was once thought to occur only in children. Meanwhile, it is known that adults can also be affected. The first-line drug in children and adults to treat symptoms of inattention, impulsivity, lack of self-regulation, and hyperactivity is methylphenidate (MPH). Known adverse effects of MPH include cardiovascular problems, such as elevated blood pressure and heart rate. Therefore, biomarkers to monitor potential cardiovascular side effects of MPH are needed. The l-Arginine/Nitric oxide (Arg/NO) pathway is involved in noradrenaline and dopamine release as well as in normal cardiovascular functioning and is therefore a prime candidate for the search of biomarkers. The aim of the present study was to investigate the Arg/NO pathway as well as oxidative stress in adult ADHD patients in plasma and urine and the potential influence of MPH medication. METHODS: In plasma and urine samples of 29 adults with ADHD (39.2 ± 10.9 years) and 32 healthy adults serving as controls (CO) (38.0 ± 11.6 years) the major NO metabolites nitrite and nitrate, Arg, the NO synthesis inhibitor asymmetric dimethylarginine (ADMA) and its major urinary metabolite dimethylamine (DMA) as well as malondialdehyde (MDA) were measured by gas chromatography-mass spectrometry. RESULTS: Of the 29 patients with ADHD 14 were currently without MPH treatment (-MPH) and 15 were treated with MPH (+MPH). Plasma nitrate concentrations were significantly higher in patients not treated with MPH vs. CO (-MPH 60.3 µM [46.2-76.0] vs. CO 44.4 µM [35.0-52.7]; p = 0.002), while plasma nitrite tended to be higher in -MPH patients (2.77 µM [2.26-3.27]) vs. CO (2.13 µM [1.50-2.93]; p = 0.053). Additionally, plasma creatinine concentrations were significantly different, with -MPH showing significantly higher concentrations than the other two groups (-MPH 141 µM [128-159]; +MPH 96.2 µM [70.2-140]; Co 75.9 µM [62.0-94.7]; p < 0.001). Urinary creatinine excretion tended to be lowest in -MPH group vs. +MPH and CO (-MPH 11.4 ± 8.88 mM; +MPH 20.7 ± 9.82 mM; 16.6 ± 7.82 mM; p = 0.076). None of the other metabolites, including MDA, a marker of oxidative stress, showed a difference between the groups. CONCLUSION: Adult patients with ADHD, who are not treated with MPH (-MPH), showed varied Arg/NO pathway, but Arg bioavailability seemed to be consistent over the groups. Our findings imply that urinary reabsorption may be increase and/or excretion of nitrite and nitrate may be decreased in ADHD, resulting in an increase in the plasma concentration of nitrite. MPH seems to partially reverse these effects by not yet known mechanisms, and does not affect oxidative stress.

Pentafluoropropionic Anhydride Derivatization and GC-MS Analysis of Histamine, Agmatine, Putrescine, and Spermidine: Effects of Solvents and Starting Column Temperature.

Gas chromatography-mass spectrometry (GC-MS) is useful for the quantitative determination of the polyamines spermidine (SPD) and putrescine (PUT) and of the biogenic amine agmatine (AGM) in biological samples after derivatization. This GC-MS method involves a two-step extraction with n-butanol and hydrochloric acid, derivatization with pentafluoropropionic anhydride (PFPA) in ethyl acetate, and extraction of the pentafluoropropionic (PFP) derivatives by toluene of SPD, PUT, and AGM. We wanted to extend this GC-MS method for the biogenic amine histamine (HA), but we faced serious problems that did not allow reliable quantitative analysis of HA. In the present work, we addressed this issue and investigated the derivatization of HA and the effects of toluene and ethyl acetate, two commonly used water-insoluble organic solvents in GC-MS, and oven temperature program. Derivatization of unlabelled HA (d0-HA) and deuterium-labelled HA (d4-HA) with PFPA in ethyl acetate (PFPA-EA, 1:4, v/v; 30 min, 65 °C) resulted in the formation of d0-HA-(PFP)2 and d4-HA-(PFP)2 derivatives. d4-HA and 13C4-SPD were used as internal standards for the amines after standardization. Considerable quantitative effects of toluene and ethyl acetate were observed. The starting GC column temperature was also found to influence considerably the GC-MS analysis of HA. Our study shows the simultaneous quantitative analysis of HA as HA-(PFP)2, AGM as AGM-(PFP)3, PUT as PUT-(PFP)2, and SPD as SPD-(PFP)3 derivatives requires the use of ethyl acetate for their extraction and injection into the GC-MS apparatus and a starting GC column temperature of 40 °C instead of 70 °C. The PFP derivatives of HA, AGM, PUT, and SPD were found to be stable in ethyl acetate for several hours at room temperature. Analytically satisfactory linearity, precision, and accuracy were observed for HA, AGM, PUT, and SPD in biologically relevant ranges (0 to 700 pmol). The limits of detection of AGM, PUT, and SPD were about two times lower in ethyl acetate compared to toluene (range, 1-22 fmol). The limits of detection were 1670 fmol for d0-HA and 557 fmol for d4-HA. Despite the improvements achieved in the study for HA, its analysis by GC-MS as a PFP derivative is challenging and less efficient than that of PUT, AGM, and SPD.

Short-Term Supplementation of Sodium Nitrate vs. Sodium Chloride Increases Homoarginine Synthesis in Young Men Independent of Exercise.

The aim of the study was to investigate the effects of short-term oral administration of inorganic nitrate (NaNO3; n = 8) or placebo (NaCl; n = 9) (each 0.1 mmol/kg body weight/d for 9 days) on plasma amino acids, creatinine, and oxidative stress in healthy young men. At baseline, the plasma concentrations of amino acids did not differ between the groups. At the end of the study, the plasma concentrations of homoarginine (hArg; by 24%, p = 0.0001), citrulline and ornithine (Cit/Orn; by 16%, p = 0.015), and glutamine/glutamate (Gln/Glu; by 6%, p = 0.0003) were higher in the NaNO3 group compared to the NaCl group. The plasma concentrations of sarcosine (Sarc; by 28%, p < 0.0001), tyrosine (by 14%, p = 0.0051), phenylalanine (by 8%, p = 0.0026), and tryptophan (by 8%, p = 0.0047) were lower in the NaNO3 group compared to the NaCl group. These results suggest that nitrate administration affects amino-acid metabolism. The arginine/glycine amidinotransferase (AGAT) catalyzes two reactions: (1) the formation of l-homoarginine (hArg) and l-ornithine (Orn) from l-arginine (Arg) and l-lysine (Lys): Arg + Lys <−> hArg + Orn, with equilibrium constant Kharg; (2) the formation of guanidinoacetate (GAA) and Orn from Arg and glycine (Gly): Arg + Gly <−> GAA + Orn, with equilibrium constant Kgaa. The plasma Kgaa/KhArg ratio was lower in the NaNO3 group compared to the NaCl group (1.57 vs. 2.02, p = 0.0034). Our study suggests that supplementation of inorganic nitrate increases the AGAT-catalyzed synthesis of hArg and decreases the N-methyltransferase-catalyzed synthesis of GAA, the precursor of creatine. To our knowledge, this is the first study to demonstrate elevation of hArg synthesis by inorganic nitrate supplementation. Remarkably, an increase of 24% corresponds to the synthesis capacity of one kidney in healthy humans. Differences in the association between plasma concentrations of amino acids in the NaNO3 and NaCl groups suggest changes in amino-acid homeostasis. Plasma concentrations of the oxidative stress marker malondialdehyde (MDA) did not change after supplementation of NaNO3 or NaCl over the whole exercise time range. Plasma nitrite concentration turned out to be a more discriminant marker of NaNO3 ingestion than plasma nitrate (area under the receiver operating characteristic curve: 0.951 vs. 0.866, p < 0.0001 each).

Possible role of SIRT1 and SIRT3 in post-translational modifications in human breast milk during the neonatal period.

We measured free and proteinic concentrations of native and modified amino acids from post-translational modifications (PTMs) and correlated them with the activity of SIRT1 and SIRT3 in the pellet and aqueous phases of human breast milk samples of ten lactating women during the neonatal period. SIRT1 and SIRT3 correlated directly with citrullination, asymmetric dimethylation and glycation of L-arginine, hydroxylation and glycation of L-lysine. SIRT1 and SIRT3 correlated inversely with the hydroxylation of L-proline. SIRT1 and SITR3 tended to correlate inversely with oxidative stress measured as malondialdehyde. Our study suggests that SIRT1 and SIRT3 may modulate PTMs in human breast milk cells.

Characterization of the L-Arginine/Nitric Oxide Pathway and Oxidative Stress in Pediatric Patients with Atopic Diseases.

INTRODUCTION: L-Arginine (Arg) is a semi-essential amino acid. Constitutive and inducible nitric oxide synthase (NOS) isoforms convert Arg to nitric oxide (NO), a potent vaso- and bronchodilator with multiple biological functions. Atopic dermatitis (AD) and bronchial asthma (BA) are atopic diseases affecting many children globally. Several studies analyzed NO in airways, yet the systemic synthesis of NO in AD and BA in children with BA, AD or both is elusive. METHODS: In a multicenter study, blood and urine were obtained from 130 of 302 participating children for the measurement of metabolites of the Arg/NO pathway (BA 31.5%; AD 5.4%; AD + BA 36.1%; attention deficit hyperactivity disorder (ADHD) 12.3%). In plasma and urine amino acids Arg and homoarginine (hArg), both substrates of NOS, asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA), both inhibitors of NOS, dimethylamine (DMA), and nitrite and nitrate, were measured by gas chromatography-mass spectrometry. Malondialdehyde (MDA) was measured in plasma and urine samples to evaluate possible effects of oxidative stress. RESULTS: There were no differences in the Arg/NO pathway between the groups of children with different atopic diseases. In comparison to children with ADHD, children with AD, BA or AD and BA had higher plasma nitrite (p < 0.001) and nitrate (p < 0.001) concentrations, suggesting higher systemic NO synthesis in AD and BA. Urinary excretion of DMA was also higher (p = 0.028) in AD and BA compared to patients with ADHD, suggesting elevated ADMA metabolization. DISCUSSION/CONCLUSION: The Arg/NO pathway is activated in atopic diseases independent of severity. Systemic NO synthesis is increased in children with an atopic disease. Plasma and urinary MDA levels did not differ between the groups, suggesting no effect of oxidative stress on the Arg/NO pathway in atopic diseases.

GC-MS Studies on Derivatization of Creatinine and Creatine by BSTFA and Their Measurement in Human Urine.

In consideration of its relatively constant urinary excretion rate, creatinine (2-amino-1-methyl-5H-imidazol-4-one, MW 113.1) in urine is a useful endogenous biochemical parameter to correct the urinary excretion rate of numerous endogenous and exogenous substances. Reliable measurement of creatinine by gas chromatography (GC)-based methods requires derivatization of its amine and keto groups. Creatinine exists in equilibrium with its open form creatine (methylguanidoacetic acid, MW 131.1), which has a guanidine and a carboxylic group. Trimethylsilylation and trifluoroacetylation of creatinine and creatine are the oldest reported derivatization methods for their GC-mass spectrometry (MS) analysis in human serum using flame- or electron-ionization. We performed GC-MS studies on the derivatization of creatinine (d0-creatinine), [methylo-2H3]creatinine (d3-creatinine, internal standard) and creatine (d0-creatine) with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) using standard derivatization conditions (60 min, 60 °C), yet in the absence of any base. Reaction products were characterized both in the negative-ion chemical ionization (NICI) and in the positive-ion chemical ionization (PICI) mode. Creatinine and creatine reacted with BSTFA to form several derivatives. Their early eluting N,N,O-tris(trimethylsilyl) derivatives (8.9 min) were found to be useful for the precise and accurate measurement of the sum of creatinine and creatine in human urine (10 µL, up to 20 mM) by selected-ion monitoring (SIM) of m/z 271 (d0-creatinine/d0-creatine) and m/z 274 (d3-creatinine) in the NICI mode. In the PICI mode, SIM of m/z 256, m/z 259, m/z 272 and m/z 275 was performed. BSTFA derivatization of d0-creatine from a freshly prepared solution in distilled water resulted in formation of two lMate-eluting derivatives (14.08 min, 14.72 min), presumably creatinyl-creatinine, with the creatininyl residue existing in its enol form (14.08 min) and keto form (14.72 min). Our results suggest that BSTFA derivatization does not allow specific analysis of creatine and creatinine by GC-MS. Preliminary analyses suggest that pentafluoropropionic anhydride (PFPA) is also not useful for the measurement of creatinine in the presence of creatine. Both BSTFA and PFPA facilitate the conversion of creatine to creatinine. Specific measurement of creatinine in urine is possible by using pentafluorobenzyl bromide in aqueous acetone.

GC-MS measurement of spermidine and putrescine in serum of elderly subjects: intriguing association between spermidine and homoarginine.

Gas chromatography-mass spectrometry (GC-MS) methods were developed, validated and used to measure serum spermidine (SPD) and putrescine (PUT) in 9 seropositive Helicobacter pylori (Hp +) and 18 seronegative Helicobacter pylori (Hp -) subjects (31-105 years). Homoarginine (hArg) was also measured by GC-MS. There were no statistical differences (unpaired t test) between the Hp + and Hp - subjects with respect to the serum concentrations of SPD (67.6 ± 40.3 vs. 93.7 ± 37.7 nM, P = 0.109), PUT (220 ± 139 vs. 236 ± 85 nM, P = 0.708) and hArg (1.60 ± 0.64 µM vs. 1.83 ± 0.74 µM, P = 0.554). Serum SPD and hArg concentrations correlated with each other (r = 0.426, P = 0.026, n = 27). The PUT/SPD molar ratio correlated inversely with the hArg concentration (r = - 0.406, P = 0.034, n = 27) and proteinic citrulline (r = - 0.487, P = 0.01, n = 27). These results suggest that SPD and PUT synthesis is associated with hArg formation and protein citrullination in healthy elderly subjects. The mechanisms underlying these associations and their significance remain to be elucidated.

Synthesis of homoagmatine and GC-MS analysis of tissue homoagmatine and agmatine: evidence that homoagmatine but not agmatine is a metabolite of pharmacological L-homoarginine in the anesthetized rat.

Low L-homoarginine (hArg) concentrations in human blood and urine are associated with renal and cardiovascular morbidity and mortality, yet the underlying mechanisms and the biological activities of hArg are elusive. In humans and rats, hArg is metabolized to L-lysine. The aim of the present work was to study hArg metabolism to agmatine (Agm) and homoagmatine (hAgm) in the anesthetized rat. Using a newly developed and validated GC-MS method and a newly synthesized and structurally characterized hAgm we investigated the metabolism of i.p. administered hArg (0, 20, 220, 440 mg/kg) to hAgm and Agm in lung, kidney, liver and heart in anesthetized rats. Our study provides unequivocal evidence that hArg is metabolized to hAgm but not to Agm. Whether hAgm derived from hArg's metabolism may contribute to the pathophysiological significance of endogenous hArg and for the favoured effects of pharmacological hArg remains to be demonstrated. The biology of hArg warrants further investigations.

GC-MS quantification of urinary symmetric dimethylarginine (SDMA), a whole-body symmetric l-arginine methylation index.

Circulating and excretory NG,N´G-dimethyl-l-arginine (symmetric dimethylarginine, SDMA) and NG,NG-dimethyl-l-arginine (asymmetric dimethylarginine, ADMA) are cardiovascular risk factors. Despite close chemical structures, the gas chromatography-mass spectrometry (GC-MS) measurement of SDMA is remarkably more difficult than that of ADMA for as yet unknown reasons. Here, we describe an improved GC-MS method for the quantitative determination of SDMA in human urine using commercially available NG,N´G-di-[2H3]methyl-l-arginine (d6-SDMA) as internal standard. The method is based on a single derivatization step with pentafluoropropionic anhydride (PFPA) in ethyl acetate (30 min, 65 °C) to N,N,N,O-tetrakis-pentafluoropropionyl derivatives, electron-capture negative-ion chemical ionization and selected-ion monitoring of the mass-to-charge (m/z) ions of m/z 456 for SDMA and m/z 462 for d6-SDMA.

Adipokine concentrations in lipoaspirates may have a role in wound healing.

OBJECTIVES: In addition to its use as a volume filler, fat grafting may have a potential role in wound healing based on the concentration of growth factors in the lipoaspirate. In this study, we compare the quantitative and qualitative concentration of the various growth factors and adipokines using the Shippert or the Coleman techniques to prepare the lipoaspirate. METHODS: We measured leptin, adiponectin and the growth factors, i.e., acidic fibroblast growth factor (aFGF), basic FGF (bFGF), keratinocyte growth factor (KGF), bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF) by ELISA in solid and liquid fractions obtained with both techniques in human fat obtained with Coleman technique and Shippert technique. RESULTS: All of these peptides, except BMP-2, were detected in relevant quantities in the solid fraction. The Coleman but not the Shippert technique resulted in statistically higher adiponectin concentrations in the solid tissue fraction. The other four growth factors occurred in significantly higher concentrations in the solid fractions compared to the liquid fractions, independent of the processing technique. CONCLUSION: In summary, we demonstrated that KGF, aFGF, bFGF and VEGF, as well as leptin and adiponectin, are contained in fat suspensions obtained by liposuction and in the supernatant. Only the concentration of adiponectin was in the range reported to contribute to wound healing.

Comprehensive analysis of the L-arginine/L-homoarginine/nitric oxide pathway in preterm neonates: potential roles for homoarginine and asymmetric dimethylarginine in foetal growth.

L-Arginine (Arg) and L-homoarginine (hArg) are precursors of nitric oxide (NO), a signalling molecule with multiple important roles in human organism. In the circulation of adults, high concentrations of asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) and low concentrations of hArg emerged as cardiovascular risk factors. Yet, the importance of the Arg/hArg/NO pathway, especially of hArg and ADMA, in preterm neonates is little understood. We comprehensively investigated the Arg/hArg/NO pathway in 106 healthy preterm infants (51 boys, 55 girls) aged between 23 + 6 and 36 + 1 gestational weeks. Babies were divided into two groups: group I consisted of 31 babies with a gestational age of 23 + 6 - 29 + 6 weeks; group II comprised 75 children with a gestational age of 30 + 0 - 36 + 1 weeks. Plasma and urine concentrations of ADMA, SDMA, hArg, Arg, dimethylamine (DMA) which is the major urinary ADMA metabolite, as well as of nitrite and nitrate, the major NO metabolites, were determined by GC-MS and GC-MS/MS methods. ADMA and hArg plasma levels, but not the hArg/ADMA molar ratio, were significantly higher in group II than in group I: 895 ± 166 nM vs. 774 ± 164 nM (P = 0.001) for ADMA and 0.56 ± 0.04 µM vs. 0.48 ± 0.08 µM (P = 0.010) for hArg. There was no statistical difference between the groups with regard to urinary ADMA (12.2 ± 4.6 vs 12.8 ± 3.6 µmol/mmol creatinine; P = 0.61) and urinary SDMA. Urinary hArg, ADMA, SDMA correlated tightly with each other. Urinary excretion of DMA was slightly higher in group I compared to group II: 282 ± 44 vs. 247 ± 35 µmol/mmol creatinine (P = 0.004). The DMA/ADMA molar ratio in urine was tendentiously higher in neonates of group I compared to group II: 27 ± 13 vs. 20 ± 5 (P = 0.065). There were no differences between the groups with respect to Arg in plasma and to nitrite and nitrate in plasma and urine. In preterm neonates, ADMA and hArg biosynthesis increases with gestational age without remarkable changes in the hArg/ADMA ratio or NO biosynthesis. Our study suggests that ADMA and hArg are involved in foetal growth.

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 pentafluorobenzyl derivatization and GC-ECNICI-MS measurement of nitrite and malondialdehyde in human urine: Close positive correlation between these disparate oxidative stress biomarkers.

Urinary nitrite and malondialdehyde (MDA) are biomarkers of nitrosative and oxidative stress, respectively. At physiological pH values of urine and plasma, nitrite and MDA exist almost entirely in their dissociated forms, i.e., as ONO- (ONOH, pKa=3.4) and -CH(CHO)2 (CH2(CHO)2, pKa=4.5). Previously, we reported that nitrite and MDA react with pentafluorobenzyl (PFB) bromide (PFB-Br) in aqueous acetone. Here, we report on the simultaneous derivatization of nitrite and MDA and their stable-isotope labeled analogs O15NO- (4µM) and CH2(CDO)2 (1µM or 10µM) with PFB-Br (10µL) to PFBNO2, PFB15NO2, C(PFB)2(CHO)2), C(PFB)2(CDO)2 by heating acetonic urine (urine-acetone, 100:400µL) for 60min at 50°C. After acetone evaporation under a stream of nitrogen, derivatives were extracted with ethyl acetate (1mL). A 1-µL aliquot of the ethyl acetate phase dried over anhydrous Na2SO4 was injected in the splitless mode for simultaneous GC-MS analysis in the electron capture negative-ion chemical ionization mode. Quantification was performed by selected-ion monitoring (SIM) the anions [M-PFB]-m/z 46 for ONO-, m/z 47 for O15NO-, m/z 251 for -C(PFB)(CHO)2, and m/z 253 for -C(PFB)(CDO)2. The retention times were 3.18min for PFB-ONO2/PFB-O15NO2, and 7.13min for -C(PFB)(CHO)2/-C(PFB)(CDO)2. Use of CH2(CDO)2 at 1µM but not at 10µM was associated with an unknown interference with the C(PFB)2(CDO)2 peak. Endogenous MDA can be quantified using O15NO- (4µM) and CH2(CDO)2 (10µM) as the internal standards. The method is also useful for the measurement of nitrate and creatinine in addition to nitrite and MDA. Nitrite and MDA were measured by this method in urine of elderly healthy subjects (10 females, 9 males; age, 60-70 years; BMI, 25-30kg/m2). Creatinine-corrected excretion rates did not differ between males and females for MDA (62.6 [24-137] vs 80.2 [52-118]nmol/mmol, P=0.448) and for nitrite (102 [71-174] vs. 278 [110-721]nmol/mmol P=0.053). We report for the first time a close correlation (r=0.819, P<0.0001) between MDA and nitrite in human urine. This correlation is assumed to be due to involvement of myeloperoxidase which catalyzes the formation of hypochlorite (-OCl) from chloride and hydrogen peroxide. In turn, hypochlorite reacts both with nitrite and with polyunsaturated fatty acids such as arachidonic acid, with the later reaction generating MDA. The proposed mechanisms are supported by the literature but remain to be fully explored.

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.

Cross-validated stable-isotope dilution GC-MS and LC-MS/MS assays for monoacylglycerol lipase (MAGL) activity by measuring arachidonic acid released from the endocannabinoid 2-arachidonoyl glycerol.

2-Arachidonoyl glycerol (2AG) is an endocannabinoid that activates cannabinoid (CB) receptors CB1 and CB2. Monoacylglycerol lipase (MAGL) inactivates 2AG through hydrolysis to arachidonic acid (AA) and glycerol, thus modulating the activity at CB receptors. In the brain, AA released from 2AG by the action of MAGL serves as a substrate for cyclooxygenases which produce pro-inflammatory prostaglandins. Here we report stable-isotope GC-MS and LC-MS/MS assays for the reliable measurement of MAGL activity. The assays utilize deuterium-labeled 2AG (d8-2AG; 10µM) as the MAGL substrate and measure deuterium-labeled AA (d8-AA; range 0-1µM) as the MAGL product. Unlabelled AA (d0-AA, 1µM) serves as the internal standard. d8-AA and d0-AA are extracted from the aqueous buffered incubation mixtures by ethyl acetate. Upon solvent evaporation the residue is reconstituted in the mobile phase prior to LC-MS/MS analysis or in anhydrous acetonitrile for GC-MS analysis. LC-MS/MS analysis is performed in the negative electrospray ionization mode by selected-reaction monitoring the mass transitions [M-H]-→[M-H - CO2]-, i.e., m/z 311→m/z 267 for d8-AA and m/z 303→m/z 259 for d0-AA. Prior to GC-MS analysis d8-AA and d0-AA were converted to their pentafluorobenzyl (PFB) esters by means of PFB-Br. GC-MS analysis is performed in the electron-capture negative-ion chemical ionization mode by selected-ion monitoring the ions [M-PFB]-, i.e., m/z 311 for d8-AA and m/z 303 for d0-AA. The GC-MS and LC-MS/MS assays were cross-validated. Linear regression analysis between the concentration (range, 0-1µM) of d8-AA measured by LC-MS/MS (y) and that by GC-MS (x) revealed a straight line (r2=0.9848) with the regression equation y=0.003+0.898x, indicating a good agreement. In dog liver, we detected MAGL activity that was inhibitable by the MAGL inhibitor JZL-184. Exogenous eicosatetraynoic acid is suitable as internal standard for the quantitative determination of d8-AA produced from d8-2AG by hepatic MAGL activity. The formation of d8-prostaglandin E2 by the consecutive catalytic action of recombinant MAGL on d8-2AG and recombinant cyclooxygenase-2 (COX) on d8-AA was demonstrated by GC-MS/MS.

Comments on Boelaert et al. Determination of Asymmetric and Symmetric Dimethylarginine in Serum from Patients with Chronic Kidney Disease: UPLC-MS/MS versus ELISA. Toxins 2016, 8, 149.

n/a.

Airborne aldehydes in cabin-air of commercial aircraft: Measurement by HPLC with UV absorbance detection of 2,4-dinitrophenylhydrazones.

This paper presents the strategy and results of in-flight measurements of airborne aldehydes during normal operation and reported "smell events" on commercial aircraft. The aldehyde-measurement is a part of a large-scale study on cabin-air quality. The aims of this study were to describe cabin-air quality in general and to detect chemical abnormalities during the so-called "smell-events". Adsorption and derivatization of airborne aldehydes on 2,4-dinitrophenylhydrazine coated silica gel (DNPH-cartridge) was applied using tailor-made sampling kits. Samples were collected with battery supplied personal air sampling pumps during different flight phases. Furthermore, the influence of ozone was investigated by simultaneous sampling with and without ozone absorption unit (ozone converter) assembled to the DNPH-cartridges and found to be negligible. The method was validated for 14 aldehydes and found to be precise (RSD, 5.5-10.6%) and accurate (recovery, 98-103 %), with LOD levels being 0.3-0.6 µg/m(3). According to occupational exposure limits (OEL) or indoor air guidelines no unusual or noticeable aldehyde pollution was observed. In total, 353 aldehyde samples were taken from two types of aircraft. Formaldehyde (overall average 5.7 µg/m(3), overall median 4.9 µg/m(3), range 0.4-44 µg/m(3)), acetaldehyde (overall average 6.5 µg/m(3), overall median 4.6, range 0.3-90 µg/m(3)) and mostly very low concentrations of other aldehydes were measured on 108 flights. Simultaneous adsorption and derivatization of airborne aldehydes on DNPH-cartridges to the Schiff bases and their HPLC analysis with UV absorbance detection is a useful method to measure aldehydes in cabin-air of commercial aircraft.