Conundrum of dehydroascorbic acid and homocysteine thiolactone reaction products: Structural characterization and effect on peptide and protein N-homocysteinylation.

An excessive blood level of homocysteine (HcySH) is associated with numerous cardiovascular and neurodegenerative disease conditions. It has been suggested that direct S-homocysteinylation, of proteins by HcySH, or N-homosteinylation by homocysteine thiolactone (HTL) could play a causative role in these maladies. In contrast, ascorbic acid (AA) plays a significant role in oxidative stress prevention. AA is oxidized to dehydroascorbic acid (DHA) and if not rapidly reduced back to AA may degrade to reactive carbonyl products. In the present work, DHA is shown to react with HTL to produce a spiro bicyclic ring containing a six-membered thiazinane-carboxylic acid moiety. This reaction product is likely formed by initial imine condensation and subsequent hemiaminal product followed by HTL ring opening and intramolecular nucleophilic attack of the resulting thiol anion to form the spiro product. The reaction product was determined to have an accurate mass of 291.0414 and a molecular composition C10H13NO7S containing five double bond equivalents. We structurally characterized the reaction product using a combination of accurate mass tandem mass spectrometry, 1D and 2D-nuclear magnetic resonance. We also demonstrated that formation of the reaction product prevented peptide and protein N-homocysteinylation by HTL using a model peptide and α-lactalbumin. Furthermore, the reaction product is formed in Jurkat cells when exposed to HTL and DHA.

Maternal inhaled fluticasone propionate intake during pregnancy is detected in neonatal cord blood.

BACKGROUND: Despite recommendations to use inhaled corticosteroids as treatment to control asthma during pregnancy, it is unknown whether inhaled fluticasone propionate (FP) reaches the fetus. Results & methodology: We collected maternal blood on the morning following delivery. FP was detected by ultra-performance LC-MS/MS (UPLC-MS/MS) in 9/17 asthmatic women using FP. Delay between last FP inhalation and maternal blood sampling ranged between 3 and 33 h and FP was detected in a range of 1.572-46.440 pg/ml. Among the nine offspring of these FP users, FP was detected in five cord blood samples. Delay between last predelivery FP inhalation and cord blood sampling ranged from 4 to 20 h and FP was detected in a range of 0.423-4.510 pg/ml. CONCLUSION: Our findings demonstrate placental passage of inhaled FP.

Quantification of ecdysteroids and retinoic acids in whole daphnids by liquid chromatography-triple quadrupole mass spectrometry.

Quantification of ecdysteroids and retinoic acids at picograms per individual is typically achieved with radioimmunoassay methods. However, those methods cannot identify individual types of ecdysteroids or provide an absolute concentration, which poses problems for comparative assays such as the metabolic profiling approach for toxicity testing. The method described in the present paper, based on liquid chromatography-electrospray ionization-triple quadrupole mass spectrometry, was developed to allow the quantification in whole daphnids extracts of ecdysteroids (20-hydroxyecdysone, ecdysone, ponasterone A) and retinoic acid (sum of isomers). This approach avoids having to perform the difficult task of sampling the haemolymph on small organism (<5mm). Recoveries, evaluated at three concentrations in matrix blank fortified samples, ranged from 83 to 119% for ecdysteroids and from 144 to 155% for retinoic acids. Precision (2.4-14.2%) and accuracy (-41.7 to 14.5%) were reproducible and stable over three quality control concentrations. The described liquid chromatography-triple quadrupole mass spectrometry method achieved quantification limits ranging from 210 to 380 pg mL(-1) for ecdysteroids and 5 ng mL(-1) for retinoic acids in spiked matrix blanks. 20-hydroxyecdysone was quantified in Daphnia magna adults (19 ± 8 pg ind(-1)) and juveniles (3.6 ± 1.0 pg ind(-1)), but was below the limit of quantification in neonates (≈ 0.19 pg ind(-1)). Ecdysone was also detected in adult specimens (≈ 1.8 pg ind(-1)).