Failure to apply standard limit-of-detection or limit-of-quantitation criteria to specialized pro-resolving mediator analysis incorrectly characterizes their presence in biological samples

Specialized pro-resolving mediators (SPM) derived from oxygenation of long chain polyunsaturated fatty acids (PUFA) were originally described by Serhan and colleagues and have been proposed as mediators of inflammation resolution. Families of SPM described in the literature include lipoxins, resolvins, maresins, protectins and their peptide conjugates. Gomez and co-authors reported that levels of plasma SPM from patients with early rheumatoid arthritis predict response to biologic therapy after 6 months. SPM were measured in this study using liquid chromatography tandem mass spectrometry (LC-MS/MS). On reviewing the methods, supplementary analytical data, and the online peer review file, we note serious concerns, regarding both analytical methods and experimental conclusions. Application of this flawed methodology to SPM analysis brings into question the very occurrence of many of these lipids in biological samples, their proposed impact on inflammatory processes, and claims of their utility as biomarkers

Nitrated fatty acids: synthesis and measurement

Nitrated fatty acids are the product of nitrogen dioxide reaction with unsaturated fatty acids. The discovery of peroxynitrite and peroxidase-induced nitration of biomolecules led to the initial reports of endogenous nitrated fatty acids. These species increase during ischemia/reperfusion, but concentrations are often at or near the limits of detection. Here, we describe multiple methods for nitrated fatty acid synthesis and sample extraction from complex biological matrices and a rigorous method of qualitative and quantitative detection of nitrated fatty acids by liquid chromatography–mass spectrometry. In addition, optimized instrument conditions and caveats regarding data interpretation are discussed.Fil: Woodcock, Steven R.. University of Pittsburgh; Estados UnidosFil: Bonacci, Gustavo Roberto. University of Pittsburgh; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Gelhaus, Stacy L.. University of Pittsburgh; Estados UnidosFil: Schopfer, Francisco J.. University of Pittsburgh; Estados Unido

Rapid purification of RNA secondary structures

A new method for rapid purification and structural analysis of oligoribonucleotides of 19 and 20 nt is applied to RNA hairpins SL3 and SL2, which are stable secondary structures present on the ψ recognition element of HIV-1. This approach uses ion-pairing reversed-phase liquid chromatography (IP-RPLC) to achieve the separation of the stem–loop from the transcription mix. Evidence is presented that IP-RPLC is sensitive to the different conformers of these secondary structures. The purity of each stem–loop was confirmed by mass spectrometry and PAGE. IP-RPLC purification was found to be superior to PAGE in terms of time, safety and, most importantly, purity

Stable Isotope Labeling by Essential Nutrients in Cell Culture for Preparation of Labeled Coenzyme A and Its Thioesters

Stable isotope dilution mass spectrometry (MS) represents the gold standard for quantification of endogenously formed cellular metabolites. Although coenzyme A (CoA) and acyl-CoA thioester derivatives are central players in numerous metabolic pathways, the lack of a commercially available isotopically labeled CoA limits the development of rigorous MS-based methods. In this study, we adapted stable isotope labeling by amino acids in cell culture (SILAC) methodology to biosynthetically generate stable isotope labeled CoA and thioester analogues for use as internal standards in liquid chromatography/multiple reaction monitoring mass spectrometry (LC/MRM-MS) assays. This was accomplished by incubating murine hepatocytes (Hepa 1c1c7) in media in which pantothenate (a precursor of CoA) was replaced with [¹³C₃¹⁵N₁]-pantothenate. Efficient incorporation into various CoA species was optimized to >99% [¹³C₃¹⁵N₁]-pantothenate after three passages of the murine cells in culture. Charcoal−dextran-stripped fetal bovine serum (FBS) was found to be more efficient for serum supplementation than dialyzed or undialyzed FBS, due to lower contaminating unlabeled pantothenate content. Stable isotope labeled CoA species were extracted and utilized as internal standards for CoA thioester analysis in cell culture models. This methodology of stable isotope labeling by essential nutrients in cell culture (SILEC) can serve as a paradigm for using vitamins and other essential nutrients to generate stable isotope standards that cannot be readily synthesized

Regulation of Benzo[<i>a</i>]pyrene-Mediated DNA- and Glutathione-Adduct Formation by 2,3,7,8-Tetrachlorodibenzo-<i>p</i>-dioxin in Human Lung Cells

Environmental carcinogens, such as polycyclic aromatic hydrocarbons (PAHs), require metabolic activation to DNA-reactive metabolites in order to exert their tumorigenic effects. Benzo[<i>a</i>]pyrene (B[<i>a</i>]P), a prototypic PAH, is metabolized by cytochrome P450 (P450) 1A1/1B1 and epoxide hydrolase to (−)-B[<i>a</i>]P-7,8-dihydro-7,8-diol (B[<i>a</i>]P-7,8-dihydrodiol). B[<i>a</i>]P-7,8-dihydrodiol then undergoes further P4501A1/1B1-mediated metabolism to the ultimate carcinogen, (+)-<i>anti</i>-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydro-B[<i>a</i>]P (B[<i>a</i>]PDE), which forms DNA-adducts primarily with 2′-deoxyguanosine (dGuo) to form (+)-<i>anti</i>-<i>trans</i>-B[<i>a</i>]PDE-N²-dGuo (B[<i>a</i>]PDE-dGuo) in DNA. Pretreatment of cells with 2,3,7,8-tetrachlorodibenzo-<i>p</i>-dioxin (TCDD) is known to induce P4501A1/1B1 mRNA expression through the aryl hydrocarbon receptor (AhR) pathway. This causes increased B[<i>a</i>]PDE-dGuo formation in liver cells. In contrast, TCDD induction of H358 lung cells surprisingly caused a decrease in (−)-B[<i>a</i>]P-7,8-dihydrodiol-mediated (+)-B[<i>a</i>]PDE-dGuo adduct formation when compared with the non-TCDD-induced cells. Furthermore, treatment of the TCDD-induced cells with (±)-B[<i>a</i>]PDE also resulted in decreased (+)-B[<i>a</i>]PDE-dGuo adduct formation when compared with the non-TCDD-induced cells. These data suggested that it was a detoxification pathway that had been up-regulated rather than an activation pathway that had been down-regulated. LC-MS was used to analyze B[<i>a</i>]PDE-dGuo and B[<i>a</i>]PDE-GSH-adducts in H358 lung and HepG2 liver cells. There was a significant increase in the (−)-B[<i>a</i>]PDE-GSH-adduct with high enantiomeric excess after treatment of the TCDD-induced H358 cells with (±)-B[<i>a</i>]PDE when compared with the noninduced cells. This could explain why increased activation of (−)-B[<i>a</i>]P-7,8-dihydrodiol through TCDD up-regulation of P4501A1/1B1 did not lead to increased (+)-B[<i>a</i>]PDE-dGuo adducts in the H358 lung cells. In addition, TCDD did not induce B[<i>a</i>]PDE-GSH-adduct formation in HepG2 liver cells. (±)-B[<i>a</i>]PDE-GSH-adducts were formed at much lower levels in both TCDD-induced and noninduced HepG2 cells when compared with (−)-B[<i>a</i>]PDE-GSH-adducts in the H358 lung cells. Therefore, our study has revealed that there is a subtle balance between activation and detoxification of B[<i>a</i>]P in lung-derived compared with liver-derived cells and that this determines how much DNA damage occurs