Sphingolipids in intestine and liver: How to analyze?

Abstract

Identification and quantification of lipids, in particular sphingolipids from intestine and liver, using multidimensional mass spectrometry has dramatically improved our understanding of lipid-based molecular pathways and signaling. The editorial gives a short overview about basic technical approaches to characterize lipids from intestine and liver.

Key Words: Intestine, Lipids, Liver, Mass spectrometry

INTRODUCTION

The molecular class of lipids includes a highly divers group of molecules with hydrophobic or amphiphilic character. Important molecule sub-groups are fatty acids, glycerolipids, and sphingolipids which are widely distributed in intestinal tissues. Lipids are involved in several signaling cascades as pure molecules or protein modifying agents. In addition, lipids are important as structural components and in energy storage.

The family of sphingolipids includes ceramides and sphingosines which are of high importance in cellular signaling and addressing regulation of cellular permeability, cell survival, and transformation. The complex sphingolipids are molecular described and sub-classified by backbones and additional groups including sugars[1,2]. The sphingoid base backbone is available by two biochemical pathways: (1) de novo or (2) cleavage of sphingomyelin[3]. The interested reader is referred to more specific articles describing the biochemistry of sphingolipid metabolism in more detail[4].

Sphingolipids are target molecules for coupling of highly diverse sugar residues. The term complex sphingolipids describes this structural feature. Such molecules are abundantly found in liver and intestine and essential for structural integrity, barrier function, and inflammation, where they act as bioactive messengers[5,6]. In the last decade, molecular analysis of lipids from liver tissues has become a very important topic, because the incidence of fatty liver associated disorders, i.e., fibrosis and carcinogenesis, is dramatically increasing.

Standard algorithms are established for extraction of lipids from liver and intestinal tissues[7]. Dissection of target areas from fresh intestinal or liver tissues followed by homogenization without cryo-conservation is essential. Prior to the extraction of lipids for measurements an internal lipid marker must be added. After the extraction step cryoconservation and storing of the mix is possible.

In following a short overview of mass spectrometry-based lipid analysis and profiling of sphingolipids from intestinal and liver sources is given.

MASS SPECTROMETRY-BASED SPHINGOLIPID ANALYSIS

Lipidomics, the large-scale analysis of cellular lipid pathways and networks, is an analytical approach to distinguish different lipid species that are structurally similar and often metabolically interconvertible[5]. In this setting, comprehensive mass spectrometry-based techniques are established for elucidation of physiological properties and functionality of lipids in a single sample[2,6].

From the technical point of view, mass spectrometers include an ion source, a mass analyzer (separation of ions by mass to charge ratio), and a detector module. Electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) mass spectrometry are the basic technical systems. MALDI mass spectrometry is a laser-based soft ionization method preferentially used for protein analysis, but useful in lipid research too. A special organic matrix component is mixed-up with the lipid containing sample and irritated with a laser to ionize probe molecules. Because generation of intact molecular ions with MALDI is possible, this technique has been successfully used to analyze complex lipids. Tissue imaging mass spectrometry, another interesting application for MALDI, has the advantage to identify lipid quantities and their distribution and sub-cellular localization in a tissue. In this setting extraction of lipids from their biological sources is not necessary[7,8].

In contrast to MALDI, ESI based technologies are soft ionization methods, where lipid containing solutions continuously infused through a small capillary into an electric field generating very fine charged droplets. The droplets rapidly evaporate and divide into individual charged ions, best analyzed with a triple quadrupole mass analyzer[9-12].

Liquid chromatography-mass spectrometry (LC-MS) and tandem mass spectrometry (LC-MS/MS) are further important technologies and very popular in lipid research[2]. In addition, electron crystallography has been established to study membrane proteins in a lipid-rich environment. The shotgun lipidomics approach, another mass spectrometric technique, works without direct coupling of any chromatography for lipid separation. This infusion-based lipidomic technique allows quantification of a high number of lipid species in small probes from intestine and liver. The approach is hampered by the fact that the method does not distinguish isomeric and isobaric lipid species[13-15].

Concerning sphingolipids, LC-MS- or LC-/MS/MS, shotgun-lipidomics, and MALDI imaging mass spectrometers are established as basic technical approaches. Using LC-MS/MS identification, quantification, and structural analysis of free sphingoid bases/phosphates, ceramides, monohexosylceramides, lactosylceramides, sphingomyelins and other glycosphingolipids are possible[16-19]. Combination of normal-phase HPLC and ESI-MS analysis is another interesting approach to identify sphingolipids in heterogeneous lipid containing solutions and materials[20]. Although direct infusion shotgun lipidomics has some limitations, the method is useful and very efficient to quantify lipid species from any unknown sample including detection of low-abundance sphingolipid metabolites e.g., ceramide-phosphates and sphinganine[21].

CONCLUSION

Lipids and in particular sphingolipids are highly distributed throughout intestinal and liver. They have diverse functions in cellular systems including molecular signaling. In-detail analysis of sphingolipids is assumed as promising for better understanding of intestinal and liver physiology and pathology. At present, several types of mass spectrometry-based measurements are established as powerful tools in lipid analysis. Advantages and disadvantages of the different technologies have to been critically proofed to find the adequate method answering an experimental working hypothesis.

ACKNOWLEDGEMENTS

The comments of Dr. Reinartz A and Dr. Henkel C to improve the manuscript as well as the help of P Akens in typing and proofreading the manuscript are highly acknowledged.