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Sphingolipids

Implications of Shotgun Lipidomics

Shotgun lipidomic research was implemented for characterizing and quantizing sphingosine-1-phosphate (S1P) and dihydrosphingosine-1-phosphate (DHS1P), with the use of precursor ion scanning of m/z 79.0 (corresponding to [PO3]-) in the negative-ion mode. S1P and DHS1P analysis was conducted on crude lipid extracts, in the presence of ammonium hydroxide. This particular approach produced a broad linear dynamic range and a detection limit at low amol/µl concentration for both S1P and DHS1P, and proved to be a simpler and more efficient method for the quantitative analysis of sphingoid base-1-phosphates than any other previously published method. The extensively examined factors that influence sphingoid base-1-phosphate quantization included ion suppression, extraction efficiency, and the potential overlapping with other molecular species. S1P and DHS1P mass levels were determined in samples of human plasma, mouse plasma, and mouse brain tissues. This new methodology is predicted to be an exceedingly useful tool for understanding sphingolipid metabolic pathways, as well as the contributions of sphingoid base-1-phosphates to future physiological and pathological research.

The specific biological analytes for the experiment were human plasma, mouse plasma, and mouse spinal cord and brain tissue extract, which included samples from the cortex, cerebellum and brain stem. Human plasma was sampled from healthy individuals ranging from sixty to seventy years of age. The resultant average yields of mass contents from the plasma of seven humans were 703 and 327 pmol/ml. In addition, researchers claim that their experimental results are the first to indicate the presence of S1P and DHS1P in the mouse tissue analytes. There were also substantial amounts of S1P and DHS1P found in mouse plasma that was believed to reflect sphingoid base-1-phosphate release from platelets, during blood coagulation. S1P and DHS1P levels of distribution were as follows, in the order of decreasing sphingolipid content: spinal cord, brain stem, cerebellum, and the cortex. S1P receptors were reported as being predominantly expressed in brain white matter, rat cerebellum and cerebellar granule cells, and astrocytes. Consequently, the parallel distribution of S1P and DHS1P to S1P receptor levels of expression indicates an interdependence between sphingoid base-1-phosphate ligands and their receptors.

Quantitatively analytical advantages of the experimental method implemented in this experiment include the ability for researchers to avoid using radioisotopes, chromatographic separation, and chemical derivatization, to obtain structural information of sphingoid base-1-phosphates, and to simultaneously detect S1P and DHS1P. Although, there may be some interference of this method if a phosphate-containing lipid molecular species possesses an identical m/z to S1P and/or DHS1P, such interference is already considered negligible, when attributed to overlapping phosphate-containing lipid molecular species that are present in mammalian samples. This unique methodology applied to shotgun lipidomics is expected to provide a myriad of uses for future physiological and pathophysiological research.

References:

http://www.jlr.org/cgi/content/full/47/8/1865#SEC2

Membrane Lipids

KEGG Pathway and MetaCyc Links

Anabolic:

Phospholipid Biosynthesis http://biocyc.org/META/NEW-IMAGE?object=PHOSLIPSYN-PWY http://biocyc.org/META/NEW-IMAGE?object=PHOSLIPSYN2-PWY

Phospholipid desaturation http://biocyc.org/META/NEW-IMAGE?object=PWY-762

Fatty Acid Biosynthesis http://biocyc.org/META/NEW-IMAGE?object=FASYN-INITIAL-PWY

Fatty Acid Elongation in mitochondria http://biocyc.org/META/NEW-IMAGE?object=FASYN-ELONG-PWY

Polyunsaturated fatty acid biosynthesis

Catabolic:

Glycerol degradation IV http://biocyc.org/META/NEW-IMAGE?object=PWY-4261

Phospholipases http://biocyc.org/META/NEW-IMAGE?object=LIPASYN-PWY

Triacylglycerol degradation http://biocyc.org/META/NEW-IMAGE?object=LIPAS-PWY

Metabolism:

Sphingolipid metabolism http://biocyc.org/META/NEW-IMAGE?object=SPHINGOLIPID-SYN-PWY

Lipid metabolism

Glycerolipid metabolism

Glycerophospholipid metabolism

Sphingolipid metabolism

Fatty acid metabolism

Online Source #1:

Lipids and Membrane Structure

Source: http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb1/part2/lipid.htm

Main focus:

This website details the structure of lipid membranes of living cells. It describes the chemical

makeup and the mobility of the lipid bilayer and the factors that affect membrane fluidity. The

site goes on to describe the various types of proteins that can be associated with lipid

membranes and the sugar chains that may extend from them.

Terms:

ceramide - group of amides formed by linkage of a fatty acid to sphingosine, a lipid which has

a long hydrocarbon tail and a polar domain containing an amino group. http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=5497136

phosphoethanolamine - an ethanolamine derivative used to construct sphingomyelins. http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=1015

isoprenoid - a polymer whose carbon skeleton consists wholly or partly of isoprene units

joined end to end.

depalmitoylation - hydrolysis of an ester linkage.

pleckstrin homology domain - a protein domain of approximately 120 amino acids that occurs

in a wide range of proteins involved in intracellular signaling or as constituents of the

cytoskeleton.

oligomer - a polymer that consists of two, three, or four monomers.

Connection:

Like this website discusses, we have studied the lipid bilayer and its chemical makeup in this

course sequence. We focused in on the roles of proteins and lipids in the cell membrane,

something this site also goes into detail about. The website also informs on of membrane

mobility and fluidity and how fluidity is affected by various factors, another topic we discussed.

Online Source #2:

Biochemistry: Fifth Addition; Lipids and Cell Membranes

Source: http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=stryer.chapter.1626

Main Focus:

This site is basically an online biochemistry textbook with a chapter completely devoted to

lipids and cell membranes. The chapter is divided into sections that can be seen with the

following links:

I. The Molecular Design of Life

 12. Lipids and Cell Membranes

http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=stryer.chapter.1626

12.1. Many Common Features Underlie the Diversity of Biological Membranes

http://www.ncbi.nlm.nih.gov/books/bv.fcgi?highlight=12.1&rid=stryer.section.1629

12.2. Fatty Acids Are Key Constituents of Lipids

http://www.ncbi.nlm.nih.gov/books/bv.fcgi?highlight=12.2&rid=stryer.section.1638

12.3. There Are Three Common Types of Membrane Lipids

http://www.ncbi.nlm.nih.gov/books/bv.fcgi?highlight=12.3&rid=stryer.section.1643

12.4. Phospholipids and Glycolipids Readily Form Bimolecular Sheets in Aqueous Media

http://www.ncbi.nlm.nih.gov/books/bv.fcgi?highlight=12.4&rid=stryer.section.1655

12.5. Proteins Carry Out Most Membrane Processes

http://www.ncbi.nlm.nih.gov/books/bv.fcgi?highlight=12.5&rid=stryer.section.1665

12.6. Lipids and Many Membrane Proteins Diffuse Rapidly in the Plane of the Membrane

http://www.ncbi.nlm.nih.gov/books/bv.fcgi?highlight=12.6&rid=stryer.section.1687

12.7. Eukaryotic Cells Contain Compartments Bounded by Internal Membranes

http://www.ncbi.nlm.nih.gov/books/bv.fcgi?highlight=12.7&rid=stryer.section.1698

This source goes into great detail about membrane and membrane structure of both cells and

cellular organelles. It discusses the various types of membrane lipids and how lipids are made

up of fatty acids and even dives into fatty acid nomenclature. The site also describes both

membrane permeability and mobility of its components.

Terms:

amphipathic - molecule containing both polar and nonpolar domains.

sonication - agitation of particles through the use of sound energy.

SDS-polyacrylamide gel electrophoresis - technique used to separate proteins based on

molecular weight.

fluorescence recovery after photobleaching - technique used to visualize the lateral movement

of membrane proteins.

periplasm - the region between the two membranes containing the cell wall.

Connection:

In our course sequence we have talked about membrane components at the molecular level

and described the membrane's fluidity and permeability, which is what this source discusses in

depth. This online textbook also teaches how fluidity is affected by cholesterol and fatty acid

content, which we discussed in class.

Online Source #3:

Membrane Structure and Function

Source: http://cellbio.utmb.edu/cellbio/membrane_intro.htm

Main Focus:

This site starts by introducing the history of how the structure of cell membranes was deduced

and the various models that were proposed. It then discusses the present-day model and the

structure of the fatty acids that make up the membrane bilayer. It describes how fatty acid

structure, cholesterol and temperature all effect the fluidity of membranes. Finally, it goes on to

describe the proteins and glycolipids that can be associated with the membrane.

Terms:

microaggregate - a microscopic particle collection.

microdomain - a lipid raft fortified with cholesterol that occurs in cell membranes.

oligosaccharide - a saccharide polymer made up of 3-10 sugars.

osmium tetroxide - a chemical used to stain and fix lipids.

glycosphingolipids - a sphingolipid containing glucose or galactose. http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=9547206

Connection:

This website describes the makeup of the lipid bilayer, explaining the molecular structure of

the fatty acids it consists of. We talked about the cellular membrane structure in our course

sequence. In class we also talked about the factors that affect the fluidity of the membrane,

something this site goes into immense detail about.

Peer-Reviewed Article #1:

Alteration of viral lipid composition by expression of the phospholipid floppase ABCB4 reduces HIV vector infectivity

Source: http://www.retrovirology.com/content/pdf/1742-4690-5-14.pdf

Main Focus:

This research involved studying the effect that a viral membrane lipid, phosphatidyl choline

(PC), had on HIV infection efficiency. The study was done by using aphosphatidyl choline (PC)

floppase protein that is responsible for transporting PC from the inner to the outer membrane

layer, ABCB4 protein. By utilizing this ability of the ABCB4 protein, the authors were able to

manipulate the lipid composition of the HIV vector and compare its infection efficiency to that

of an unmodulated vector. The results showed that virus made in the presence of ABCB4

showed a large increase in PC membrane composition and a reduced infectivity compared to

the control.

Terms:

lentiviral - genus of slow viruses of the Retroviridae family that have a long incubation period.

transfection - introduction of foreign material into a cell through the use of a virus vector.

Western blot - technique used to detect a specific protein in a tissue sample homogenate or

extract.

sonication - agitation of particles through the use of sound energy.

chemokine - cytokines produced during inflammation that activate white blood cells.

Connection:

This study relates to what we have talked about in class in that it revolves completely around

a specific membrane lipid. Since we have talked about the various lipids that make up a cell

membrane, this study is relevant to what we have learned. We also studied the movement of

lipids within a membrane, which is what was used to set up this experiment.

Peer-Reviewed Article #2:

Seminal Plasma Proteins Regulate the Association of Lipids and Proteins Within Detergent-Resistant Membrane Domains of Bovine Spermatozoa

Source: http://www.biolreprod.org/cgi/rapidpdf/biolreprod.107.066514v1

Main Focus:

This study focused on proteins within sperm membranes and how they direct the

reorganization of lipid rafts during sperm capacitation (the remodeling of sperm plasma

membrane while within female reproductive tract). Several proteins are associated with

cholesterol-enriched detergent-resistant membranes (DRM) domains in spermatozoa. In

capacitated sperm cells these proteins detach from these domains leading to the theory that

these domains may be involved in the reorganization of sperm membranes when cholesterol is

removed. The researchers isolated spermatozoa from bovine epididymis and isolated the DRM

domains and lipids so as to find the lipid:protein ratio. They examined the effects of seminal

plasma on the spermatozoa and found that it lowers the cholesterol concentration in DRM

domains and dissociates proteins from DRM domains. They concluded that seminal plasma

proteins induce lipid efflux and DRM protein dissociation.

Terms:

efflux - a flowing outward.

zona pellucida - thick, solid, transparent outer membrane surrounding a developing ovum.

densitometric - method of measuring the optical density of a material.

dialysis - Method used to separate smaller molecules from larger molecules or of dissolved

substances from colloidal particles in a solution by selective diffusion through a semipermeable

membrane.

acrosome - structure on a spermatozoon that produces enzymes used to penetrate an ovum.

Connection:

This study is relevant to our course in that it discusses factors that can affect plasma

membrane fluidity, specifically cholesterol, and how proteins are integrate into the bilayer. The

study also touches on the topic of lipid rafts, another area we went over in class.

Peer-Reviewed Article #3:

Erythrocyte Glutathione Depletion Impairs Resistance to Haemolysis in Women Consuming Alcohol

Source:

http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=18231625

Main Focus:

This study focused on the effect alcohol has on women erythrocyte membranes. The study

involved taking blood samples from women who drink 200-300mL of alcoholic beverages

every day after work and comparing it to that of women who do not consume alcohol. The

results showed that women who consume alcohol have a decreased phospholipid

concentration and an increased cholesterol concentration resulting in overall decreased

membrane fluidity and a decrease in the ability of the erythrocytes to resist haemolysis.

Peroxidative damage to membranes was also observed.

Terms:

hemolysate - product from the lysis of red blood cells.

haemolysis - breakdown of red blood cells.

heparin - drug used to prevent blood clotting.

Catalase - blood enzyme that decomposes hydrogen peroxide into water and oxygen.

Methemoglobin - brownish-red, crystalline form of hemoglobin occurring when hemoglobin is

oxidized by blood decomposition or toxic agents.

Connection:

This study relates to our course work in that we discussed the effects of cholesterol

concentration on membrane fluidity. We also talked about free radical generation and how it

can lead to damage, something that was shown in the study to increase in women who drink.

Articles and Web Pages for Review and Inclusion