Quantification of Interactions between Serum Albumin and Endogenous Free Fatty Acids or Exogenous Chemicals by Stable Isotope-Coded Mass Spectrometry.

As primary endogenous ligands of serum albumin, free fatty acids exert versatile effects on the albumin conformation through cooperative or competitive interactions with exogenous chemicals. Based on equilibrium partition between n-hexane and aqueous phases, we have established three indexes, defined as R A, R V, and R T, for quantitative assessment of the intrinsic binding affinity, the affinitive variation induced by exogenous chemicals, and the topological dependence of albumin affinity, respectively. When albumin molecules in the aqueous phase are in native or denatured forms, or disturbed by exogenous chemicals, corresponding changes of free fatty acids in the n-hexane phase can be quantified by an iFFAM (isotope-coded free fatty acid methylation) approach. Free fatty acids from the control and the sample are differentially derived by d0- or d3-methanol and analyzed by gas chromatography-mass spectrometry. Changes of fatty acids can be revealed by peak ratios of d0- or d3-labeled fragment ions of fatty acid methyl esters.

Effects of co-existed proteins on measurement of pesticide residues in blood by gas chromatography-mass spectrometry.

Accurate measurement of pesticides in biological fluids such as blood is important for quantifying environmental exposures. Beyond sample enrichment and separation, the method presented here is focused on studies of interactions between pesticides and co-existed proteins. It was experimentally demonstrated that entrapped or adsorbed pesticide residues within the folded native structures of proteins were poorly recovered using direct solvent extraction solely. We described here an effective approach termed Enzymatic Digestion-Organic Solvent Extraction (eDOSE) that utilizes the enzymatic approach to disrupt the folded structures of proteins and release entrapped or adsorbed pesticide residues. In this approach, samples were first reduced, alkylated, tryptically digested and then diluted 10 times before the subsequent extraction using an n-hexane solution. Resultant pesticide residues were determined by capillary gas chromatography coupled with a mass spectrometer. Mean recoveries of the 5 organophosphorus pesticides pre-spiked in fish blood including diazinon, parathion-methyl, malathion, parathion-ethyl and ethion were 85%, 95%, 84%, 103%, and 43% respectively using eDOSE strategy but only 24%, 45%, 40%, 27%, and 29% respectively using direct solvent extraction approach. The eDOSE approach was effective for demonstrating the critical role of folded native structure of serum albumin in adsorption of exogenous chemicals. It provides an alterative means for denaturation of proteins when the target analytes are not stable in acidic solution or entrapped within the protein aggregates caused by organic solvents such as acetone that have been applied for protein denaturation. The eDOSE approach should be able to combine with other advanced techniques of enrichment and separation for more efficient and accurate measurement of target compounds present in the context of complex biological systems. This approach can provide wide applications to the analysis of a variety of small molecules including environmental pesticide residues and metabolites as well as other toxins present in cells, tissues and biofluids.

Gas chromatography-mass spectrometric analysis of bonded long chain fatty acids in a single zebrafish egg by ultrasound-assisted one-step transmethylation and extraction.

Changes in the level of lipid composition in a single zebrafish egg have been involved in biological responses to chemical exposures. In this paper, an one-step transmethylation of lipids and simultaneous extraction of resultant fatty acid methyl esters followed by gas chromatography-mass spectrometric analysis was developed for the identification of bonded long chain fatty acids in a single zebrafish egg. The efficiency of transmethylation under different experimental conditions has been investigated. Dried egg homogenates were directly mixed with either 0.5 M NaOH or 1% H2SO4 or 4% HCl solution of methanol and then an n-hexane solution was added on the top. Ultrasonication of this immiscible liquid-liquid-solid system produces high velocity impacts between solid particles and liquid phases and thus promotes mass transfer among phases. It was demonstrated that ultrasound irradiation has strong effect on the alkaline-catalyzed transmethylation of lipids but cannot significantly change the acid-catalyzed transmethylation of lipids. With the aid of ultrasonication, transmethylation can be combined with simultaneous extraction of the resultant fatty acid methyl esters into n-hexane phase. This approach simplifies the sample preparation procedure, shortens the reaction time but improves the efficiency of the transmethylation of lipids and reduces sample losses, especially for small size samples. It has been applied to determine bonded fatty acids in a single zebrafish egg. In total, 28 fatty acids from a single zebrafish egg have been identified reproducibly.

Rapid transmethylation and stable isotope labeling for comparative analysis of fatty acids by mass spectrometry.

Fatty acids covalently bonded with other molecules have been implicated in many important biological processes. We describe here a rapid approach termed isotope-coded fatty acid transmethylation (iFAT) that integrates extraction, transmethylation, and isotopic labeling into a single step with the aid of ultrasonic irradiation for comparative analysis of fatty acids by mass spectrometry. In this approach, samples without any prefractionation were mixed with a methanol solution of 0.5 M NaOH and an n-hexane solution. The intense wave shocks and cavitations generated by ultrasonic irradiation not only speed the alkaline-catalyzed transmethylation reaction but also facilitate the simultaneous mass transfer of fatty acid methyl esters into the top n-hexane extraction phase that was injected into a GC/MS system. By using commercially available d(3)-methanol, we were able to compare the intensity of labeled and unlabeled methyl esters and their corresponding fragment ions. The detection limit can be down to the picogram level. Major advantages of the iFAT strategy are summarized in the following: (1) Efficient heterogeneous reactions. Solid samples such as dried cell lysates or detergent-resistant fractions can be readily transformed and analyzed with the aid of ultrasound irradiation. (2) Accurate quantification of fatty acids. Evaluation of the completeness or losses of transformation reactions across lipid classes has been hampered due to a lack of suitable methods. Isotope labeling can be used as an internal standard for accurate comparison of the fatty acid composition in different cell states. (3) Reduced interferences from complex biological context. The iFAT strategy not only differentially labels fatty acids in different samples, but also volatilizes those molecules, and thus, they are isolated from the bulk background and analyzed by GC/MS. This proposed approach has been applied to quantitatively determine the fatty acid composition in plant oil and in budding yeast cell lysates and detergent-resistant fractions. It should provide a widely applicable means for quantitative comparison of the fatty acid composition in cells and tissues.