Structural characterization of alpha,beta-unsaturated aldehydes by GC/MS is dependent upon ionization method.

alpha,beta-unsaturated aldehydes are toxic products of lipid peroxidation. Detection and characterization of these aldehydes is important in many human disease states as well as in the food industry. Our study shows that electron ionization-mass spectrometry (EI-MS) and positive-ion chemical ionization-mass spectrometry (PICI-MS), but not electron capture negative ionization-mass spectrometry (ECNI-MS), can be used to detect the C4-hydroxylation state of alpha,beta-unsaturated aldehydes derivatized with pentafluorobenzyl hydroxylamine alone. EI-MS and PICI-MS spectra of 4-hydroxy-2-alkenals contained a fragment with m/z 252, whereas spectra of 2-alkenals contained a fragment with m/z 250. These fragments are consistent with fragmentation between C3 and C4 with transfer of two hydrogens from C4 and the C4 hydroxyl group in the case of 4-hydroxy-2-alkenals. In addition, EI-MS and PICI-MS were able to distinguish 4-hydroxy-2-alkenals and 2-alkenals from 4-keto-2-alkenals and 4-hydroxyalkanals. On the other hand, ECNI-MS provided complex spectra regarding C4-hydroxylation state. Furthermore, the syn- and anti-configurations of PFB-oximes had different resultant spectra using ECNI-MS, but not with EI-MS or PICI-MS. These data indicate that EI-MS and PICI-MS are more amenable for structural analysis of alpha,beta-unsaturated aldehydes than ECNI-MS.

Quantification of trans-4-hydroxy-2-nonenal enantiomers and metabolites by LC-ESI-MS/MS.

Lipid peroxidation is a causal factor in multiple diseases including Alzheimer's disease, atherosclerosis, and alcoholic liver disease. One of the most studied products of lipid peroxidation, trans-4-hydroxy-2-nonenal (HNE), has multiple cell signaling and cytotoxic effects. In this work, we developed an LC-MS/MS method for the quantitation of HNE enantiomers, the metabolite trans-4-hydroxy-2-nonenoic acid, and HNE-glutathione adducts in a single chromatographic run. In this method, (R)-HNE and (S)-HNE are derivatized by (S)-carbidopa to form diastereomers that are separated by a reversed-phase column. This method was successfully validated and tested using respiring rat brain mitochondria that enantioselectively metabolize HNE. Metabolic profiles of HNE biotransformation, including the enantiomeric disposition of HNE, will provide useful biomarker data regarding lipid peroxidation in disease states.

Enantioselective oxidation of trans-4-hydroxy-2-nonenal is aldehyde dehydrogenase isozyme and Mg2+ dependent.

trans-4-Hydroxy-2-nonenal (HNE) is a cytotoxic alpha,beta-unsaturated aldehyde implicated in the pathology of multiple diseases involving oxidative damage. Oxidation of HNE by aldehyde dehydrogenases (ALDHs) to trans-4-hydroxy-2-nonenoic acid (HNEA) is a major route of metabolism in many organisms. HNE exists as two enantiomers, (R)-HNE and (S)-HNE, and in intact rat brain mitochondria, (R)-HNE is enantioselectively oxidized to HNEA. In this work, we further elucidated the basis of the enantioselective oxidation of HNE by brain mitochondria. Our results showed that (R)-HNE is oxidized enantioselectively by brain mitochondrial lysates with retention of stereoconfiguration of the C4 hydroxyl group. Purified rat ALDH5A enantioselectively oxidized (R)-HNE, whereas rat ALDH2 was not enantioselective. Kinetic data using (R)-HNE, (S)-HNE, and trans-2-nonenal in combination with computer-based modeling of ALDH5A suggest that the selectivity of (R)-HNE oxidation by ALDH5A is the result of the carbonyl carbon of (R)-HNE forming a more favorable Bürgi-Duntiz angle with the active site cysteine 293. The presence of Mg2+ ions altered the enantioselectivity of ALDH5A and ALDH2. Mg2+ ions suppressed (R)-HNE oxidation by ALDH5A to a greater extent than that of (S)-HNE. However, Mg2+ ions stimulated the enantioselective oxidation of (R)-HNE by ALDH2 while suppressing (S)-HNE oxidation. These results demonstrate that enantioselective utilization of substrates, including HNE, by ALDHs is dependent upon the ALDH isozyme and the presence of Mg 2+ ions.

Direct and indirect high-performance liquid chromatography enantioseparation of trans-4-hydroxy-2-nonenoic acid.

trans-4-Hydroxy-2-nonenoic acid (HNEA) is a marker of lipid peroxidation resulting from the metabolism of trans-4-hydroxy-2-nonenal (HNE). Direct and indirect RP-HPLC methods for the separation of HNEA enantiomers were developed and compared. The indirect method involved pre-column derivatization with a chiral amino agent, (1S,2S)-(+)-2-amino-1-(4-nitrophenyl)-1,3-propanediol, and subsequent separation of diastereomers on a Spherisorb ODS2 column. The direct separation of HNEA enantiomers was performed using the chiral stationary phase, Chiralpak AD-RH. Validation parameters including limit of quantification, linear range, accuracy and precision were determined. The indirect separation method was successfully applied for the determination of enantiomeric ratio of HNEA in rat brain mitochondrial lysate, and showed that HNEA was formed (R)-enantioselectively from HNE.

Analysis of HNE metabolism in CNS models.

The formation and toxicity of trans-4-hydroxy-2-nonenal in the central nervous system is well documented. However, the metabolism of HNE in the central nervous system (CNS) is not clear. HNE metabolism in the CNS appears to be different from that in other tissues and organs and may be dependent on the cell type and subcellular environment. Our data show that HNE metabolism is affected by the stereocenter of HNE and that oxidation of HNE may be a primary route of metabolism. Further metabolic analysis of HNE disposition is needed to clarify which pathways are truly important in normal and pathological states in the CNS.

Enantioselective metabolism of trans-4-hydroxy-2-nonenal by brain mitochondria.

Trans-4-hydroxy-2-nonenal (HNE) is a product of lipid peroxidation with many cellular effects. HNE possesses a stereogenic center at the C4 carbon that influences the metabolism and alkylation targets of HNE. We tested the hypothesis that rat brain mitochondria metabolize HNE in an enantioselective manner after exposure to racemic HNE. The study of HNE chirality, however, is hindered by the lack of facile methods to chromatographically resolve (R)-HNE and (S)-HNE. We used a chiral hydrazine, (S)-carbidopa, as a derivatization reagent to form diastereomers with (R)-HNE and (S)-HNE that were separated by reverse-phase HPLC. After exposure to racemic HNE, rat brain mitochondria metabolized HNE enantioselectively with a higher rate of (R)-HNE metabolism. By using the purified enantiomers of HNE, we found that this enantioselective metabolism of HNE was the result of higher rates of enzymatic oxidation of (R)-HNE by aldehyde dehydrogenases compared to (S)-HNE. Conjugation of HNE to glutathione was a minor metabolic pathway and was not enantioselective. These studies demonstrate that the chirality of HNE affects its mitochondrial metabolism and potentially other processes in the central nervous system.

Influence of steric hindrance on enantioseparation of Dns-amino acids and pesticides on terguride based chiral selectors in capillary electrophoresis.

Three urea derivatives of ergoline-based chiral selectors (CSs), differing in the size of the urea side chain, i.e. dimethyl- (CSI), diethyl- (CSII), and diisopropylurea (CSIII), were used to study the effect of steric hindrance on the enantioseparation of dansyl amino acids (Dns-AAs), pesticides, and mandelic acid under condition of capillary electrophoresis (CE) in linear polyacrylamide coated capillaries. A mixture of organic modifiers (MeOH/THF, 4:1 v/v) in a BGE consisting of 100 mM beta-alanine-acetate was used to increase the solubility of CSs up to 25 mM. The capillary was filled with CS (high UV absorption), and the inlet and outlet vials contained buffer solutions only. The best enantioseparation of Dns-AAs was achieved on CSI. Increased steric hindrance of the chiral binding site led to reduction of both enantioselectivity and resolution. The opposite pattern was observed for the separation of mandelic acid enantiomers, where the best enantioseparation and resolution was obtained with CSIII. Most of the pesticides studied reached maximum selectivity on the diethylurea ergoline derivative (CSII). Enantioseparation of fenoxaprop was found to be independent of steric hindrance.

l-3,4-Dihydroxyphenyl alanine-extradiol cleavage is followed by intramolecular cyclization in lincomycin biosynthesis.

The LmbB1 protein, participating in the biosynthesis of lincomycin, was heterologously expressed in Escherichia coli, purified in its active form, and characterized as a dimer of identical subunits. Methods for purification and analysis of the LmbB1 reaction product were developed. Molecular mass and fragmentation pattern of the product revealed by capillary electrophoresis-mass spectrometry were in agreement with its proposed structure, 4-(3-carboxy-3-oxo-propenyl)-2,3-dihydro-1H-pyrrole-2-carboxylic acid. The LmbB1 is therefore a dioxygenase catalysing the 2,3-extradiol cleavage of the l-3,4-dihydroxyphenyl alanine aromatic ring. The final LmbB1 reaction product, a unique compound found in biosynthesis of lincomycin and expected in anthramycins, arises through subsequent cyclization of the primary cleavage product, 2,3-secodopa. A possible role of LmbB1 in 2,3-secodopa cyclization and alternative ways of the cyclization in the formation of biosynthetically related compounds, muscaflavin and stizolobinic acid, are discussed.

Ergot alkaloids produced by submerged cultures of Claviceps zizaniae.

Two ergopeptine alkaloids, alpha-ergocryptine (1) and its C(8) epimer alpha-ergocryptinine, have been isolated from the mycelium and fermentation broth of submerged cultures of Claviceps zizaniae CCM 8240. The structure of 1 was determined by HPLC/positive ion APCI MS and NMR analysis. Alkaloid concentrations of 10 microg/mL in 14-day-old fermentation broth and 1 mg/g of dry mycelium mass were found. These results are of considerable biotechnological interest since these were the only detectable alkaloids produced. Toxicity of naturally occurring sclerotia of C. zizaniae cannot be excluded.