In Vitro and In Vivo Digestibility of Soybean,Fish, and Microalgal Oils,and Their Influences on Fatty Acid Distribution in Tissue Lipid of Mice

The digestion rates of microalgal (docosahexaenoic acid, DHA, 56.8%; palmitic acid, 22.4%), fish (DHA, 10.8%; eicosapentaenoic acid, EPA, 16.2%), and soybean oils (oleic, 21.7%; linoleic acid, 54.6%) were compared by coupling the in vitro multi-step and in vivo apparent digestion models using mice. The in vitro digestion rate estimated based on the released free fatty acids content was remarkably higher in soybean and fish oils than in microalgal oil in 30 min; however, microalgal and fish oils had similar digestion rates at longer digestion. The in vivo digestibility of microalgal oil (91.49%) was lower than those of soybean (96.50%) and fish oils (96.99%). Among the constituent fatty acids of the diet oils, docosapentaenoic acid (DPA) exhibited the highest digestibility, followed by EPA, DHA, palmitoleic, oleic, palmitic, and stearic acid, demonstrating increased digestibility with reduced chain length and increased unsaturation degree of fatty acid. The diet oils affected the deposition of fatty acids in mouse tissues, and DHA concentrations were high in epididymal fat, liver, and brain of mice fed microalgal oil. In the present study, microalgal oil showed lower in vitro and in vivo digestibility, despite adequate DHA incorporation into major mouse organs, such as the brain and liver.     

Mapping of surrogate markers of cellular components and structures using laser desorption/ionization mass spectrometry

Laser desorption/ionization mass spectrometry (LDI-MS) has been used to assess the potential of using surrogate markers, bound to cellular structures containing nucleic acids, to image or map the position of these structures within biological samples. In this study, organic dyes were used as markers because of their established use in the histochemical marking of nucleic acids, and also because they are amenable to LDI-MS. Eight cationic dyes were tested and all could be desorbed from nucleic acid samples without additional matrix after specifically binding to these molecules. Methylene Blue was the best of these based on its sensitivity to detection by LDI-MS and the fact that it can be washed from the tissue in areas where it was not specifically bound to provide low-intensity background signals. Experiments are reported which characterize the M(+) ion signal obtained from Methylene Blue with regard to sensitivity, reproducibility and possible use for quantitation. This dye was used to map (with a lateral resolution of 25 microm) several nucleic acid-containing samples spotted on prepared surfaces, and to image the location of nucleic acids in two model tissues, retinal vertical sections and thyroid whole mount sections.     

A novel derivatization reagent possessing a bromoquinolinium structure for biological carboxylic acids in HPLC‐ESI‐MS/MS

A novel bromoquinolinium reagent, i.e. 1‐(3‐aminopropyl)‐3‐bromoquinolinium bromide (APBQ), was synthesized for the analysis of carboxylic acids. A simple and practical precolumn derivatization procedure using the APBQ in RP chromatography and MS (HPLC‐MS) has been developed using bile acids and free fatty acids, as the representative carboxylic acids in biological samples. The APBQ efficiently reacted with carboxylic acids at 60°C for 60 min in the presence of N,N‐dicyclohexylcarbodiimide and pyridine as the activation reagents. Because the APBQ possesses a bromine atom in the structure, the identification of a series of carboxylic acids was easily achieved due to the characteristic bromine isotope pattern in the mass spectra. The APBQ also has a quaternary amine structure, thus the positively charged derivatives are predominate for the highly sensitive detection of carboxylic acids. The APBQ was successfully applied to the selective determination of biological carboxylic acids in human plasma. The bile acids (chenodeoxycholic acid and deoxycholic acid) and several saturated (stearic acid and palmitic acid) and unsaturated free fatty acids (oleic acid and linoleic acid) were reasonably determined by HPLC‐MS under the proposed procedure. Based on the results of analyses of human plasma and saliva, the proposed procedure using APBQ seems to be applicable for the qualitative and quantitative analyses of a series of carboxylic acids in biological samples.     

Infusion mass spectrometry as a fingerprint to characterize varnishes in oil pictorial artworks

Mass spectrometry methodology to characterize drying oil used as binding media and varnishes in pictorial artworks, prior to conservation or restoration treatment, is proposed. The analytical treatment requires prior basic hydrolysis of the samples to release the fatty acids: caprylic, pelargonic, capric, sebacic, azelaic, suberic, eicosanoic, lauric, mirystic, palmitic, linolenic, linoleic, oleic and stearic, followed by separation from the matrix by a hexane/water extraction. After removing the solvent, the remaining solid is dissolved in potassium hydroxide, propanol and methanol. The mixture is directly infused into a mass spectrometer without any previous derivatization or separation steps. The detector is operated in electrospray negative ion mode and the [M-H](-) ions of the fatty acids enable identification of the acids. Obtained data for fatty acid ion abundances are analyzed by linear discriminant analysis. The drying oils studied (linseed, poppy seed and walnut) were satisfactorily distinguished. The analytical method shows adequate sensitivity, reproducibility, speed and ease. The proposed methodology has been successfully applied to samples from artistic samples belonging to the Cultural Heritage of Valencia (Spain).     

Molecular histology analysis by matrix‐assisted laser desorption/ionization imaging mass spectrometry using gold nanoparticles as matrix

Gold nanoparticles (AuNPs) were applied and optimized as matrix for matrix‐assisted laser desorption/ionization mass spectrometry analysis of animal tissues, and enabled histological analysis of animal tissues at molecular level by imaging mass spectrometry (IMS). AuNPs were coated on animal tissue in a solvent‐free manner via argon ion sputtering. Metabolites, including neurotransmitters, fatty acids and nucleobases, were directly detected from mouse brain tissue. Based on region‐specific chemical profiles, fine histological features of mouse brain tissue and heterogeneous regions of tumor tissue were both revealed. Copyright © 2011 John Wiley & Sons, Ltd.     

An argon-ion laser fluorometer optimal for concentration detection of 5-bromomethyl fluorescein derivatized carboxylic acids

A continuous wave, argon ion laser-induced fluorescence detector was designed for use with conventional high performance liquid chromatography. Palmitic acid was selected as a model analyte to evaluate the performance of the detector. Using the intense 488.0 nm emission line, a concentration detection limit of 7.5 × 10^–10 M (38 femtomoles on-column) was established for palmitic acid at a signal to noise ratio of three. The linear dynamic range extended over two orders of magnitude with a correlation coefficient of 0.9998. The contribution of scattered radiation to the analytical signal was minimal. The concentration detection limit achieved with the constructed laser fluorometer was superior to other reports for fatty acids.     

6-oxy-(acetyl piperazine) fluorescein as a new fluorescent labeling reagent for free fatty acids in serum using high-performance liquid chromatography

A new fluorescein-based fluorescent derivatizating reagent, 6-oxy-(acetyl piperazine) fluorescein (APF), has been designed, synthesized and developed for carboxylic acid labeling. It was used as a pre-column derivatizing reagent for the determination of seven free fatty acids (lauric acid, myristic acid, arachidonic acid, linoleic acid, palmitic acid, oleic acid, and stearic acid) with high-performance liquid chromatography (HPLC). The derivatization reaction of APF with seven fatty acids was completed at 60 degrees C for 1 h using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) as the condensing reagent. On a C18 column, the derivatives of APF with seven free fatty acids could be separated completely in 22 min using a mobile phase of methanol-water (88:12, v/v) containing 7 mmol L(-1) pH 6.5 Na2HPO4-H3Cit3 buffer with fluorescence detection at lambdaex/lambdaem=467/512 nm. The detection limits could reach 0.1-6.4 nmol L(-1) (signal-to-noise=3). This reagent was applied to the determination of the free fatty acids in human serum samples with satisfying recovery efficiencies varying from 93 to 105%.     

Analysis of deprotonated acids with silicon nanoparticle-assisted laser desorption/ ionization mass spectrometry

Chemically modified silicon nanoparticles were applied for the laser desorption/negative ionization of small acids. A series of substituted sulfonic acids and fatty acids was studied. Compared to desorption ionization on porous silicon (DIOS) and other matrix-less laser desorption/ionization techniques, silicon nanoparticle-assisted laser desorption/ionization (SPALDI) mass spectrometry allows for the analysis of acids in the negative ion mode without the observation of multimers or cation adducts. Using SPALDI, detection limits of many acids reached levels down to 50 pmol/μl. SPALDI of fatty acids with unmodified silicon nanoparticles was compared to SPALDI using the fluoroalkyl silylated silicon powder, with the unmodified particles showing better sensitivity for fatty acids, but with more low-mass background due to impurities and surfactants in the untreated silicon powder. The fatty acids exhibited a size-dependent response in both SPALDI and unmodified SPALDI, showing a signal intensity increase with the chain length of the fatty acids (C12-C18), leveling off at chain lengths of C18-C22. The size effect may be due to the crystallization of long chain fatty acids on the silicon. This hypothesis was further explored and supported by SPALDI of several, similar sized, unsaturated fatty acids with various crystallinities. Fatty acids in milk lipids and tick nymph samples were directly detected and their concentration ratios were determined by SPALDI mass spectrometry without complicated and time-consuming purification and esterification required in the traditional analysis of fatty acids by gas chromatography (GC). These results suggest that SPALDI mass spectrometry has the potential application in fast screening for small acids in crude samples with minimal sample preparation.     

Identification of glycerophospholipid fatty acid remodeling by using mass spectrometry imaging in bisphenol S induced mouse liver

Exposure to BPS induced glycerophospholipid fatty acid remodeling, which might be useful in toxicity evaluation for bisphenols-induced hepatic diseases.     

Rapid and Sensitive LC–MS/MS Analysis of Fatty Acids in Clinical Samples

Free fatty acids (FFAs), major cellular metabolites, play an important role during tumor pathogenesis. Enhanced de novo fatty acid synthesis in tissues is a characteristic feature of cancer. Therefore, measurement of FFA concentration in biological samples is beneficial for cancer research and clinical diagnosis. Herein, a rapid, stable, and sensitive detection methodology was established to simultaneously quantify 22 FFAs using high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI–MS/MS). The HPLC–MS/MS system was run in negative ion mode for 15 min using multiple reaction monitoring. The lipids were extracted from colon tissues of colon cancer patients and then injected into the HPLC–MS/MS system for analysis. Colon samples were analyzed by inter-day repeatability and intra-day repeatability, with less than 5 % deviation for most fatty acids. This approach is successful to determine low picogram concentrations of each FFA molecule using milligrams of tissue, and provides a promising method for FFA microanalysis in clinical samples.

     

Monitoring cellular responses upon fatty acid exposure by Fourier transform infrared spectroscopy and Raman spectroscopy

We investigated the applicability of FTIR-spectroscopy as a high throughput screening method for detection of biochemical changes in intact liver cells in bulk upon fatty acid exposure. HepG2 cells adapted to serum free (HepG2-SF) growth were exposed to four different fatty acids, three octadecenoic acids, differing in cis/trans-configuration or double bond position (oleic acid, elaidic acid and vaccenic acid) as well as palmitic acid in three days. High throughput FTIR spectroscopic measurements on dried films of intact cells showed spectra with high signal-to-noise ratio and great reproducibility. When applying principal component analysis (PCA) a clear discrimination between fatty acid exposures was observed. Higher levels of triacylglycerides were accumulated in cells exposed to elaidic acid than when exposed to the other fatty acids; the least accumulation appeared to be in cells exposed to palmitic acid. An increased absorption at ~966 cm(-1) corresponding to trans-double bond was detected upon elaidic acid exposure but not upon vaccenic acid exposure. Instead, upon vaccenic acid exposure two new absorption bands were observed at 981 and 946 cm(-1) due to the presence of double bond conjugation. Raman spectroscopy on single cells, with and without treatment by vaccenic acid, confirmed the presence of conjugation. By fatty acid composition analysis, the conjugation was further specified to be conjugated linoleic acid (CLA) isomers. Thus, instead of being preserved as a monounsaturated fatty acid, vaccenic acid was converted into CLA in HepG2 cells. The results demonstrate the applicability of high-throughput FTIR spectroscopy as an explorative method in in vitro systems from which biologically relevant hypotheses can be generated and further investigated.     

Capillary column gas chromatography-mass spectrometry for the determination of the fatty acid composition of human adipose tissue

A procedure for determining the fatty acid composition of human adipose tissue using gas chromatography-mass spectrometry was developed. Adipose tissue was obtained from the lateral upper aspect of the right thigh by needle biopsy and prepared for analysis by lyophilisation, total lipid extraction and base-catalysed transesterification of the complexed fatty acids to form fatty acid methyl esters. Capillary column gas chromatography resolved thirty different peaks, ranging in carbon length from 12 to 24. Provisional identification of the peaks was by cochromatography with authentic standards and confirmed by gas chromatography-mass spectrometry using electron-impact ionisation. Fatty acid methyl esters were quantified in absolute amounts with respect to dry tissue weight and as a percentage of the total fat. Statistical analysis of the results from twenty healthy subjects using the two-tailed unpaired Student's t-test demonstrated women had significantly higher levels of myristoleic and palmitoleic acids (p less than 0.001) and lower levels of palmitic acid (p less than 0.05) in adipose tissue when compared with the male group. Similarly total saturated fatty acids was lower (p less than 0.05) and total monounsaturated fatty acids was higher in women than in men.     

Identification of lipid binders in old oil paintings by separation of 4-bromomethyl-7-methoxycoumarin derivatives of fatty acids by liquid chromatography with fluorescence detection

A HPLC-fluorescence method for identification of drying oils from binding media or protective film used in pictorial works of art prior to conservation or restoration is proposed. Fluorescence derivatization of fatty acids released by hydrolysis of structural drying oils is studied. The derivatization reagent was 4-(bromomethyl)-7-methoxycoumarin with 18-crown-6 as catalyst. Mobile phase was programmed from methanol-water (90:10 v/v) to methanol-water (100:0 v/v) in 25 min. The excitation and emission wavelengths were 325 and 395 nm, respectively. Under these chromatographic conditions, coumarin derivatives of myristic, palmitic, oleic and stearic acids were satisfactorily resolved. The method shows good sensitivity, with a detection limit of 6.0 x 10(-8) mmol, and good linearity between 1.0 x 10(-7) and 1.8 x 10(-4) mmol of each analyte. Peak area ratios among fatty acids derivatives, especially the stearic acid/palmitic acid peak area ratio, are useful to identify the drying oils. The proposed method has been successfully applied to artistic samples from items of the cultural heritage of Valencia (Spain).     

Direct, sensitive and selective detection of free fatty acids by high-performance liquid chromatography with post-column ion-pair extraction and absorbance detection

Free fatty acids (C8-C18) are separated by reversed-phase liquid chromatography and detected using a simple post-column dynamic extraction system in which the acids are extracted as ion pairs with chloroform from the aqueous acetonitrile (gradient: 79-99% acetonitrile) mobile phase after the post-column addition of aqueous Methylene Blue solution. The chloroform phase containing the ion pairs is monitored with an absorbance detector at 651 nm. The detection limits ranged from 26 to 83 ng, depending upon the acid, with coefficients of variation of 1.2-14%. Application of the method to butter and margarine samples permitted detection of free fatty acids down to 35 ppm and in orange juice, down to 0.5 ppm using only an organic solvent extraction without further sample clean-up for isolation of the fatty acids.     

Separation of Hydroxylated Metabolites of Fatty Acids (C10-C18) on a μPorasil Silica Column Using an Isocratic HPLC System

Abstract

A simple, versatile, and rapid normal-phase isocratic HLPC system is described for the analysis of the major (omega and omega-1) metabolites of C₁₀-C₁₈ chain length fatty acids formed upon incubation with rat liver microsomes and NADPH. Quantitation was achieved by radiometric detection. Chromatographic separation was performed by elution of the fatty acids and their omega and omega-1 metabolites from a 10μ silica column (μPorasil) with hexane:2-propanol:acetic acid (96.5:2.5:1.0). Retention times for these metabolites ranged from 10 to 13 minutes for stearic acid and from 16 to 21 minutes for capric acid. Recovery of the fatty acids and their metabolites from the column was greater than 95 percent. Relative quantitative conversion of the fatty acid substrates to omega and omega-1 metabolites was in the following order: myristic acid > capric acid=lauric acid=palmitic acid ? stearic acid. The omega products were formed preferentially over the omega-1 products of all the fatty acids except lauric acid. The method proved suitable for routine determination of NADPH-dependent fatty acid hydroxylase activities in rat liver microsomes.     

Precise and global identification of phospholipid molecular species by an Orbitrap mass spectrometer and automated search engine Lipid Search

In the present research, we have established a new lipidomics approach for the comprehensive and precise identification of molecular species in a crude lipid mixture using a LTQ Orbitrap mass spectrometer (MS) and reverse-phase liquid chromatography (RPLC) combination with our newly developed lipid search engine “Lipid Search”. LTQ Orbitrap provides high mass accuracy MS spectra by Fourier-transform (FT) mass spectrometer mode and can perform rapid MSⁿ by ion trap (IT) mass spectrometer mode. In this study, the negative ion mode was selected to detect fragment ions from phospholipids, such as fatty acid anions, by MS2 or MS3. We selected the specific detection approach by neutral loss survey-dependent MS3, for the identification of molecular species of phosphatidylcholine, sphingomyelin and phosphatidylserine. Identification of molecular species was performed by using both the high mass accuracy of the mass spectrometric data obtained from FT mode and structural data obtained from fragments in IT mode. Some alkylacyl and alkenylacyl species have the same m/z value as molecular-related ions and fragment ions, thus, direct acid hydrolysis analysis was performed to identify alkylacyl and alkenylacyl species, and then the RPLC–LTQ Orbitrap method was applied. As a result, 290 species from mouse liver and 248 species from mouse brain were identified within six different classes of phospholipid, only those in manually detected and confirmed. Most of all manually detected mass peaks were also automatically detected by “Lipid Search”. Adding to differences in molecular species in different classes of phospholipids, many characteristic differences in molecular species were detected in mouse liver and brain. More variable number of saturated and monounsaturated fatty acid-containing molecular species were detected in mouse brain than liver.     

Desorption electrospray ionization imaging mass spectrometry of lipids in rat spinal cord

Imaging mass spectrometry allows for the direct investigation of tissue samples to identify specific biological compounds and determine their spatial distributions. Desorption electrospray ionization (DESI) mass spectrometry has been used for the imaging and analysis of rat spinal cord cross sections. Glycerophospholipids and sphingolipids, as well as fatty acids, were detected in both the negative and positive ion modes and identified through tandem mass spectrometry (MS/MS) product ion scans using collision-induced dissociation and accurate mass measurements. Differences in the relative abundances of lipids and free fatty acids were present between white and gray matter areas in both the negative and positive ion modes. DESI-MS images of the corresponding ions allow the determination of their spatial distributions within a cross section of the rat spinal cord, by scanning the DESI probe across the entire sample surface. Glycerophospholipids and sphingolipids were mostly detected in the white matter, while the free fatty acids were present in the gray matter. These results show parallels with reported distributions of lipids in studies of rat brain. This suggests that the spatial intensity distribution reflects relative concentration differences of the lipid and fatty acid compounds in the spinal cord tissue. The “butterfly” shape of the gray matter in the spinal cord cross section was resolved in the corresponding ion images, indicating that a lateral resolution of better than 200 μm was achieved. The selected ion images of lipids are directly correlated with anatomic features on the spinal cord corresponding to the white and the gray matter.     

Changes of phospholipid composition within the dystrophic muscle by matrix-assisted laser desorption/ionization mass spectrometry and mass spectrometry imaging

Duchenne muscular dystrophy (DMD) is a neuromuscular disease linked to the lack of the dystrophin, a submembrane protein, leading to muscle weakness and associated with a defect of the lipid metabolism. A study of the fatty acid composition of glycerophosphatidylcholines by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF-MS) and tandem mass spectrometry (MS/MS) enabled us to characterize a change of the lipid composition of dystrophic cells at the time of the differentiation. This modification has been used as a marker to identify with profiling and imaging MALDI-ToF MS regenerating areas in sections of an mdx mouse leg muscle. It is the first time that such a slight change in fatty acid composition has been observed directly on tissue slices using mass spectrometry. This approach will be useful in monitoring the treatment of muscular regeneration.     

Simultaneous detection of phosphatidylcholines and glycerolipids using matrix‐enhanced surface‐assisted laser desorption/ionization‐mass spectrometry with sputter‐deposited platinum film

Matrix‐assisted laser desorption/ionisation (MALDI) imaging mass spectrometry (IMS) allows for the simultaneous detection and imaging of several molecules in brain tissue. However, the detection of glycerolipids such as diacylglycerol (DAG) and triacylglycerol (TAG) in brain tissues is hindered in MALDI‐IMS because of the ion suppression effect from excessive ion yields of phosphatidylcholine (PC). In this study, we describe an approach that employs a homogeneously deposited metal nanoparticle layer (or film) for the detection of glycerolipids in rat brain tissue sections using IMS. Surface‐assisted laser desorption/ionisation IMS with sputter‐deposited Pt film (Pt‐SALDI‐IMS) for lipid analysis was performed as a solvent‐free and organic matrix‐free method. Pt‐SALDI produced a homogenous layer of nanoparticles over the surface of the rat brain tissue section. Highly selective detection of lipids was possible by MALDI‐IMS and Pt‐SALDI‐IMS; MALDI‐IMS detected the dominant ion peak of PC in the tissue section, and there were no ion peaks representing glycerolipids such as DAG and TAG. In contrast, Pt‐SALDI‐IMS allowed the detection of these glycerolipids, but not PC. Therefore, using a hybrid method combining MALDI and Pt‐SALDI (i.e., matrix‐enhanced [ME]‐Pt‐SALDI‐IMS), we achieved the simultaneous detection of PC, PE and DAG in rat brain tissue sections, and the sensitivity for the detection of these molecules was better than that of MALDI‐IMS or Pt‐SALDI alone. The present simple ME‐Pt‐SALDI approach for the simultaneous detection of PC and DAG using two matrices (sputter‐deposited Pt film and DHB matrix) would be useful in imaging analyses of biological tissue sections. Copyright © 2015 John Wiley & Sons, Ltd.     

Comparative evaluation of labelling patterns and turnover of lipids,tagged by 15 /p-123I-phenyl-/ pentadecanoic and 1-14C-palmitic acid

Uptake and turnover of chloroform/methanol extractable tissue lipids labelled in vivo simultaneously with 15/p-¹²³I-phenyl-/pentadecanoic and l-¹⁴C-palmitic acid were compared. Lipid turnover studies were performed in fasted pentobarbital-anaesthetized Wistar rats in tissues with highly varying free fatty acid turnover rates. In all tissues investigated, i.e. heart, lung, liver, spleen and kidneys, both tracers labelled nearly identical lipid fraction. Main tracer uptake was found in free fatty acids, phospholipids, diglycerides and triglycerides. A highly significant correlation of uptake and turnover in main tissue lipid fraction indicated an essentially identical metabolic pathway of both tracers in intermediary tissue lipid metabolism. Concordant tracer uptake and turnover patterns in tissue of lipids with highly varying free fatty acid metabolic rates suggested that intrinsic metabolic activity of the tissue and respective lipid fraction was the major determinant of metabolic handling of both iodophenyl fatty- and palmitic acid. Thus, the feasibility of iodophenylpentadecanoic acid as free fatty acid tracer for studying tissue lipid metabolism is demonstrated.