Edited by: Mario Diaz, University of La Laguna, Spain
Reviewed by: Stefano Piotto, University of Salerno, Italy; Lillian DeBruin, Wilfrid Laurier University, Canada
*Correspondence: Michael Cascio
This article was submitted to Membrane Physiology and Membrane Biophysics, a section of the journal Frontiers in Physiology
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Recognition of the contributions of lipids to cellular physiology, both as structural components of the membrane and as modulatory ligands for membrane proteins, has increased in recent years with the development of the biophysical and biochemical tools to examine these effects. Their modulatory roles in ion channels and transporters function have been extensively characterized, with the molecular mechanisms of these activities being the subject of intense scrutiny. The physiological significance of lipids in biochemistry is expanding as numerous fatty acyls are discovered to possess signaling properties. These bioactive lipids are often found in quantities of pmol/g of tissue and are co-extracted with numerous lipophilic molecules, making their detection and identification challenging. Common analytical methodologies involve chromatographic separation and mass spectrometric techniques; however, a single chromatographic step is typically ineffective due to the complexity of the biological samples. It is, therefore, essential to develop approaches that incorporate multiple dimensions of separation. Described in this manuscript are normal phase and reversed phase separation strategies for lipids that include detection of the bioactive primary fatty acid amides and N-acyl glycines via tandem mass spectrometry. Concerted utilization of these approaches are then used to separate and sensitively identify primary fatty acid amides extracted from homogenized tissue, using mouse brains as a test case.
Lipids are classically defined as hydrophobic or amphiphilic small molecules with limited solubility in aqueous solutions where they typically aggregate non-covalently. The macromolecular complexes are constituents of cellular membranes or comprise relatively inert complexes used for energy storage. In considering the former, the lipid composition of the bilayer alters its physicochemical properties, and this local environment for receptors and other membrane proteins affect their structure and function (Coskun and Simons,
In addition to these indirect mutual effects of lipids and signaling, many lipids exert bioactive properties directly as cellular signals and 2nd messengers (for reviews, see Hannun and Obeid,
Lipid milieus of biological samples are often very complex mixtures. Even the most common lipid extraction methods, such as Folch-Pi (Folch et al.,
Multidimensional liquid chromatography (MDLC) analysis is the process of separating samples with at least, but most typically, two different chromatographic separation schemes (columns or dimensions)(Giddings,
Fortuitously, straight chain fatty acyls have a well-predicted elution order by reversed phase chromatography where retention is directly proportional to the length of the acyl chain. The degrees of unsaturation reduce the retention such that it is similar to acyl chains 2 carbons shorter per each degree of unsaturation. For example, a C18:1 fatty acyl would have an elution order closer to a C16:0 fatty acyl; this is known as a critical pair (Gutnikov,
Normal phase separations of fatty acyls have been achieved with thin layer chromatography and adapted to solid phase extraction columns (Sultana and Johnson,
Silver ion, or argenation, chromatography has been used extensively for separation of lipid samples with a high variation of unsaturation numbers and geometrical configuration, such as are commonly found in triacylglycerols (Dobson et al.,
Multidimensional liquid chromatography (MDLC) separations have been demonstrated for triacylglycerols in plant and animal samples (Dugo et al.,
To our knowledge, MDLC methods have not yet been published for PFAMs or NAGs, although our laboratory has utilized an SPE method prior to GC-MS analyses (Sultana and Johnson,
Methanol (Optima grade), formic acid (Optima grade), ammonium acetate, hexane, acetonitrile, and ammonium hydroxide were purchased from Fisher Scientific (Fair Lawn, NJ, USA). Oxalyl chloride, oleic acid, erucic acid, petroselaidic acid, heptane HPLC grade, methyl-tert-butyl-ether HPLC grade, isopropanol HPLC grade, acetic acid, and anhydrous dichloromethane were from Sigma Aldrich (St Louis, MO, USA). N, N-dimethylformamide, heptadecanoic acid, and eicosanoic acid were purchased from Aldrich Chemical Company (Milwaukee, WI, USA). Lauric acid, myristic acid, palmitic acid, stearic acid, and docosanoic acid were purchased from Acros Organics (New Jersey, USA). Elaidic acid was from MP Biomedical Inc. (Solon, OH, USA) and linoleamide was purchased from Enzo Life Sciences (Ann Arbor, MI, USA). Stearoyl ethanolamine, oleoylglycine, linoleoylglycine, palmitoylglycine, arachidonoylglycine, and arachidoylglycine were purchased from Cayman Chemicals (Ann Arbor, MI, USA). 1 monopalmitoyl-rac-glycerol (MAG) and tristearin (TAG) were from Sigma (St. Louis, MO) 99% purity and 1,2-dipalmitoyl-rac-glycerol (DAG) was from MP Biomedicals (Solon, Ohio).
PFAM and NAG standards were prepared in a mixture at 1 mM concentration of each standard. The mixture was separated via normal phase chromatography utilizing a YMC PVA-Sil column (4.6 × 250 mm, 5 μm particle size). Gradient elution is carried out starting at 95% mobile phase A (heptane with 0.5% v/v methyl-tert-butyl-ether) and increasing linearly to 50% mobile phase B (methyl-tert-butyl-ether with 10% v/v 2-propanol and 0.2% v/v acetic acid) over 40 min with a flow rate of 1 mL/minute. Fractions were collected at 1 min intervals with an injection volume of 200 μL and the times corresponding to NAG and PFAM elution were determined by reversed phase chromatography and MS/MS detection.
Palmitoylglycine, linoleoylglycine, oleoylglycine, stearidonoylglycine, arachidonoylglycine, and arachidoylglycine were analyzed on an Agilent Technologies 1200 Liquid Chromatography system with a 6460 Triple Quadrupole Mass Spectrometry Detector. Mobile phase A was methanol and mobile phase B water, with 10 mM ammonium acetate in both phases. Separations were carried out on a YMC Cartenoid column (4.6 × 150 mm, 5 μm particle size) with a linear gradient of 90 to 100% mobile phase A over 15 min with a 15 min hold time. An additional separation method was developed on a Phenomenex C18 column (4.6 × 100 mm, 2.6 μm particle size) with a linear gradient of 80 to 100% mobile phase A over 5 min and a 2 min hold time. A second gradient method was used and consisted of 70% mobile phase A hold for 7 min, a linear gradient increase to 80% for 7 min, a 1 min hold at 80% before increasing to 100% mobile phase A over 5 min, and a final 5 min hold for a total analysis time of 25 min.
Ionization was achieved with an ESI source operated in negative mode with optimized parameters: fragmentor voltage 135 V, sheath gas flow 11 L/minute, nebulizer pressure 55 psi, nozzle voltage 500 V, capillary voltage 3500 V, drying gas flow 9 L/minute, drying gas temperature 275°C, and dwell time of 500 ms. Multiple reaction monitoring parameters were set-up to analyze the [M–H]− parent ions and 74
All primary fatty acid amide standards were synthesized in house at greater than 98% purity. Briefly, PFAMs were synthesized by a modified procedure described by Philbrook (
Purity of all synthesized amides was verified by GCMS on a Varian CP-3800 GC with Varian Saturn 2000 Ion Trap Mass Spectrometer. Gas chromatography was performed on a Varian Factor Four Capillary Column (VF-5 ms, 30 m × 0.25 mm ID) with a flow of 1 mL/min helium carrier gas. Injector temperature was held at 250°C with split injection (ratio 10). Temperature gradient started at 55°C and ramped 40°C/min to 150°C with a hold of 3.62 min before ramping 10°C/min to 275°C and holding 6.50 min. The total run time was 25 min. Eluted fatty amides were ionized by chemical ionization with methanol and analyzed in selected ion mode. The peak area of fatty acid substrate and PFAM product from GC-MS runs were used to determine purity. All PFAMs were found to be of 98% purity or greater. Lauramide (C12:0), myristamide (C14:0), linoleamide (C18:29,12), palmitamide (C16:0), oleamide (C18:19), elaidamide (C18:19trans), petroselaidamide (C18:16trans), heptadecanoamide (C17:0), stearamide (C18:0), arachidamide (C20:0), erucamide (C22:113), and behenamide (C22:0) were separated on a Agilent RP C18 column (2.0 × 50 mm, 1.8 μm particle size) with a gradient elution of methanol and water, both containing 0.3% formic acid.
PFAMs were detected using an Agilent 6460 Triple Quadrupole Mass Spectrometer equipped with an atmospheric pressure chemical ionization (APCI) source. Optimized detection parameters are as follows: gas temperature 325°C, vaporization temperature 325°C, gas flow 4 L/minute, nebulizer pressure 22 psi, capillary voltage 3500 V, corona 4 μA, and fragmentor 125 V. Multiple reaction monitoring was used to detected the [M+H]+ parent ions and product ions of 55 and 43
Extraction of polar lipids from biological samples was achieved by a modified Folch-Pi method (Folch et al.,
Samples of mouse brain (a generous gift from Dr. S. Amara, Univ. of Pittsburgh School of Medicine) were weighed and frozen at 20°C. The tissue sample was homogenized in a 2:1 chloroform:methanol solvent mixture containing 1 mM indomethancin with a volume of 20 times the sample weight. Heptadecanoamide was added as an internal standard to a final concentration of 10 μM. The insoluble material was removed by centrifugation and the supernatant was vortexed with an aqueous 10% KCl solution to remove salts, proteins, and water soluble components. The organic phase was dried under a stream of nitrogen and further separated by the normal phase separation method outlined in a previous section.
A 975 nmol lipid mixture of heptanoamide (FAs), tristearin (TAGs), 1,2-dipalmitoyl-rac-glycerol (DAGs), 1-monopalmitoyl-rac-glycerol (MAGs), N-oleoylglycine (NAGs), palmitamide (PFAMs), and stearoyl ethanolamine (NAEs) single representative standards from each lipid class were separated by normal phase chromatography (Figure
Palmitoylglycine (C16:0), oleoylglycine (C18:19), linoleoylglycine (C18:29,12), stearidonoyolglyicne (C18:46,9,12,15), arachidonoylglycine (C20:45,8,11,14), and arachidoylglycine (C20:0) were separated utilizing a C30 YMC carotenoid column and a fused-core Phenomenex C18 column. Separation of palmitoylglycine (C16:0), oleoylglycine (C18:19), linoleoylglycine (C18:29,12), and arachidoylglycine (C20:0) was achieved on a C30 YMC carotenoid column (4.6 × 150 mm, 5 μm particles size) with gradient elution of methanol and water. Both mobile phases were modified with 10 mM ammonium acetate to aid in ionization. Elution was achieved by linear increase in methanol from 90 to 100% over 15 min, followed by a 15 min hold. Elution of each component was determined by identification of the parent mass ion (Figure
In an effort to reduce the analysis time, a fused-core Phenomenex Kinetix C18 column (4.6 × 100 mm, 2.6 μm particles size) was employed using methanol/water gradient elution (Figure
A second elution method was developed (Figure
Utilizing the method developed in Figure
Stearidonoylglycine | C18:46,9,12,15 | 0.475 | 1.00 | 0.999 |
Linoleoylglycine | C18:29,12 | 0.499 | 1.00 | 0.999 |
Arachidonoylglycine | C20:45,8,11,14 | 0.484 | 1.00 | 0.999 |
Palmitoylglycine | C16:0 | 1.00 | 1.00 | 0.994 |
Oleoylglycine | C18:29 | 1.00 | 1.00 | 0.999 |
Arachidoylglycine | C20:0 | 1.00 | 1.00 | 0.999 |
Very long chain PFAMs (C12 to C22) were separated via reversed phase chromatography employing a sub 2 μm particle size column (Agilent RP C18 2.1 × 50 mm, 1.8 μm particle size). A gradient elution was established for a select group of PFAMs (Figure
Utilizing the separation method developed in Figure
Lauramide | C12:0 | 50 | 100 | 0.999 |
Myristamide | C14:0 | 50 | 50 | 0.999 |
Linoleamide | C18:29,12 | 10 | 50 | 0.999 |
Palmitamide | C16:0 | 20 | 50 | 0.999 |
Oleamide | C18:19 | 400 | 400 | 0.999 |
Elaidamide | C18:19trans | 40 | 400 | 0.998 |
Petroselaidamide | C18:16trans | 40 | 400 | 0.999 |
Stearamide | C18:0 | 50 | 50 | 0.997 |
Erucamide | C22:113 | 50 | 100 | 0.998 |
Arachidamide | C20:0 | 10 | 50 | 0.999 |
behenamide | C22:0 | 20 | 20 | 0.999 |
The developed extraction, normal phase, and reversed phase separation methods were used serially to analyze PFAM content in a sample of mouse brain (Figure
Brain tissue was chosen due to the documented interaction of oleamide and neuroreceptors, such as 5HTR. Myristamide (C14:0), linoleamide (C18:2), palmitamide (C16:0), oleamide (C18:1), stearamide (C18:0), and behenamide (C22:0) were identified in mouse brain tissue extracts by matching retention time and parent ion mass. An internal standard, heptadecanoamide, which was added prior to homogenization of the tissue samples, was recovered at 72% confirming collection of the correct fraction in the normal phase separation. The blank sample had a similar % recovery of the internal standard with no PFAM peaks present (data not shown).
This work describes various strategies for efficient separation and detection of saturated and unsaturated fatty acyls. Different subclasses of lipids were resolved with a normal phase separation scheme utilizing a heptane and methyl-tert-butyl ether mobile phase and gradient elution. The addition of isopropanol to the mobile phase was necessary to increase the solubility of these lipids, reducing the carry over between injections. Nonetheless, it was still essential to occasionally wash the column with polar solvents, especially if large injection volumes (>100 μL) were used frequently. The fatty acyl subclasses separated via the normal phase method are those commonly extracted with Folch-Pi from biological samples. This method is comparable to previously reported SPE methods (Sultana and Johnson,
Following separation of fatty acyls with normal phase the individual subclasses (e.g., PFAMs or NAGs) can be further separated to determine the distinct analytes present. This was achieved with C18 reversed phase chromatography and detected with tandem MS. The elution order followed the trends observed with fatty acids eluted from reversed phase columns (Gutnikov,
Utilizing the developed methodology, PFAMs were successfully detected in extracts of mouse brain tissue. PFAMs and NAEs have been reported in biological tissues at pmol/g of tissue. Though the utilized methodology has successfully identified PFAMs in brain tissue, the levels are not sufficient for quantitation. It is clear, even with careful method selection and sample processing consideration, that quantitation of trace levels of the PFAM and NAG subclasses pose a challenge for conventional separation and detection methods. Several factors were found to have a profound impact on analysis. When selectively analyzing for fatty amides the use of all plastic containing equipment and sample vessels should be eliminated. Under Folch-Pi extraction conditions, these substrates leach fatty amides, thus, contaminating the sample matrix (Cooper and Tice,
Additionally, rigorous care should be taken to fully control experimental conditions before and after tissue excision. Quantitation studies on fatty acid ethanolamines in biological samples have found similar effects with tissue quality (Epps et al.,
In summary, we developed an off-line MDLC system for analysis of PFAMs and NAGs. Using lipid standards, normal phase separation was capable of automated separation of complex biological lipid matrices with comparable sample recovery to SPE. Each individual subclass of lipids could be successfully sampled from the normal phase and further resolved into each individual component with a secondary reversed phase method. Fused core particle and sub-2 μm column packings enabled high resolution separation of lipid lengths from C12 to C22 within 12 min. This included resolution of several positional isomers of C18:1, some of which have been shown to affect physiological states in vertebrates. The MDLC coupled with MS/MS was then shown to be capable in identifying PFAMs in samples with a more complex lipid composition, homogenized mouse brain. The methodology described herein provides a framework for future analyses aimed at identifying and elucidating the roles and significance of lipids in health and disease.
All experimental studies were conducted by ED, except for the initial normal phase chromatography trials that were conducted by KK. Studies were directed by MC and initial drafts were written by ED and MC.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The authors thank the late Dr. Mitchell E. Johnson for support and inspiration of this work. The authors also gratefully recognize the National Institute of Health (2R15 NS038443) for support of this work and the National Science Foundation (MRIDBI-0821401) for support toward purchase of mass spectrometers used in this study.