Simultaneous determination of multiple aldehydes in biological tissues and fluids using gas chromatography/stable isotope dilution mass spectrometry.

Biological oxidative stress has been associated with various degenerative disorders and disease states, and accurate and sensitive methods are needed to determine the extent of oxidation occurring in vivo. Peroxidation of polyunsaturated fatty acids forms complex mixtures of aldehydes and other breakdown products because various oxidants are involved and lipid composition is not uniform. Quantitative analysis of multiple lipid peroxidation products yields a more complete measure of biological oxidation than measurement of a single aldehyde, particularly when aldehydes exhibit marked differences in reactivity. This report describes extensions of an established gas chromatography/mass spectrometry method to include stable isotope dilution determination in tissue and plasma of three aldehydic products of lipid peroxidation: hexanal, nonanal, and 4-hydroxy-2-nonenal. Use of deuterated internal standards for each analyte improved precision and accuracy compared to a single internal standard for all three aldehydes. Improvements are attributed to differences in extraction and derivatization efficiencies for individual analytes owing to substantial differences in reactivity. Electron ionization of oxime-tert-butyldimethylsilyl derivatives gave greater specificity for detecting all three aldehydes than was possible using electron-capture ionization of O-pentafluorobenzyl oxime derivatives. Exchange of the deuterium label of [2,3-2H]HNE internal standard was determined to be minimal during the analyses.

Quantitative analysis of oligonucleotides by matrix-assisted laser desorption/ionization mass spectrometry.

Quantitative aspects of oligonucleotide analysis by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry remain largely unexplored relative to the efforts that have been devoted to quantitative peptide and protein analysis. The successful quantitation of these other biopolymers coupled with the potential of rapid nucleic acid analysis by desorption/ionization techniques prompted the present investigation into quantifying mixed base oligonucleotides of intermediate molecular weights. This report describes the concentration-dependent desorption/ionization of a 21-base oligonucleotide (MW 6361) using a 36-base oligonucleotide (MW 11 131) as an internal standard. Peak height and peak area ratios (analyte to internal standard) varied linearly as a function of oligonucleotide concentration (R2 = 0.966 and 0.991, respectively). The linearity of response extended over nearly three orders of magnitude, from 0.125 to 100 pmol of analyte applied. The use of an internal standard improved the linearity of the calibration curve and reduced relative standard deviations. These results demonstrate for the first time the quantitation of medium size oligonucleotides using MALDI.

Direct characterization of protein adducts of the lipid peroxidation product 4-hydroxy-2-nonenal using electrospray mass spectrometry.

Oxidative stress and exposures to xenobiotic substances generate reactive substances including the cytotoxic aldehyde 4-hydroxy-2-nonenal. This aldehyde exhibits a variety of biological effects and has been reported as a marker of lipid peroxidation. The toxicity and atherogenicity of 4-hydroxy-2-nonenal have been attributed to the formation of covalent protein adducts. In the current study, two model proteins, beta-lactoglobulin B and human hemoglobin, were exposed to 4-hydroxy-2-nonenal, and the protein adducts were characterized using electrospray ionization mass spectrometry. Our findings provided clear and direct evidence that > 99% of protein modification occurred via Michael addition, and only trace amounts of Schiff base adducts were formed. Confirmation of this result was obtained via quantitative conversion of the modified proteins to oxime and pentafluorobenzyl oxime derivatives as demonstrated by electrospray ionization mass spectrometry, spectrophotometric protein carbonyl assays, and gas chromatography/mass spectrometry determination of 4-hydroxy-2-nonenal released upon treatment with hydroxylamine. These results further demonstrate the availability of the protein-bound aldehyde for subsequent reaction or as a site of molecular recognition. The preponderance of Michael addition products over Schiff base adducts also suggests that most methods for determining 4-hydroxy-2-nonenal in biological tissues or fluids are based on erroneous assumptions that hydrazines or hydroxylamines release 4-hydroxy-2-nonenal from proteins.

Decrease of 12-hydroxyeicosatetraenoic acid production in mouse lungs following dietary oleic anilide consumption: implications for the toxic oil syndrome.

A study was performed to examine the ability of dietary oleic anilide to alter 12-hydroxyeicosatetraenoic acid (12-HETE) production. The structure of oleic anilide, synthesized by reacting oleic acid with aniline, was confirmed by mass spectrometry. The purity of oleic anilide, 75%, was measured by gas chromatography. Oleic acid, which constituted the remaining 25%, is a major component of the rapeseed oil vehicle. Balb/c mice were fed oleic anilide as 0.75% of their diet by weight for three weeks. Their lungs were excised and examined for 12-HETE production in vitro. The 12-HETE levels were significantly (p < 0.01) lower in mice fed oleic anilide than in mice fed the oleic acid control diet. This result illustrates eicosanoid production as a target of fatty acid anilide toxicity. The fatty acid composition, including arachidonic acid, of mouse lungs from both dietary groups was not different. This confirms the availability of substrate for 12-lipoxygenase in both groups. Spleen weights were higher in mice fed oleic anilide than in control mice (p < 0.005). These observations are relevant to immunoregulation and the autoimmune syndromes noted in patients of the Toxic Oil Syndrome (TOS).

Maximum entropy deconvolution of heterogeneity in protein modification: protein adducts of 4-hydroxy-2-nonenal.

To explore the chemistry of the reactions of the cytotoxic aldehyde trans-4-hydroxy-2-nonenal (HNE) with proteins, we incubated this aldehyde in vitro with beta-lactoglobulin B, a model protein of molecular weight 18,277 Da. Direct characterization of reaction products using electrospray ionization mass spectrometry yielded spectra whose complexity suggested extensive product heterogeneity. Spectra were transformed to a true mass scale using both a conventional transform algorithm and a maximum entropy algorithm. Both transformations demonstrated the formation of aldehyde-protein adducts containing from three to nine aldehyde molecules per molecule of protein. Maximum entropy deconvolution resolved Schiff base adducts and/or dehydration products, differing from the Michael addition adducts by 18 Da. The dominant reaction pathway, however, was Michael addition of the aldehyde to nucleophilic functional groups on the protein. The large number of Michael adducts relative to the one available cysteine requires that other amino acids, such as histidine and lysine, also be modified. The data suggest that methods for analysis of HNE that involve displacement of Schiff base groups from proteins will only recover a small fraction of HNE.