Lipids
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Lipids are extracted from tissues using organic solvents. Adsorption chromatography separates lipids by polarity. Gas-liquid chromatography analyzes fatty acid methyl esters. Specific enzymatic hydrolysis and mass spectrometry are used to deduce lipid structures. Minor lipids serve as essential cofactors, while phospholipids generate signaling molecules. Membrane lipids include glycerophospholipids and sphingolipids.
Lipids
- 1. Chapter 10 - Lipids • 10.1 Storage Lipids • 10.2 Structural Lipids in Membranes • 10.3 Lipids as Signals, Cofactors and Pigments • 10.4 Working with Lipids
- 2. 10.1 Storage Lipids • Fatty Acids - Structure • Fatty Acids - Physical Properties • Triacylglycerols - the simplest Lipids • Triacylglytcerols in Energy Storage Thermal insulation • Triacylglycerols in food • Waxes
- 3. Fatty Acid Structure • Carboxylic Acids (COOH is C1) • hydrocarbon tails (C4 - C36) • Saturated fatty acids N:0 • Unaturated Fatty acids • Double bonds specified by (Δn ) • Branches
- 4. Fatty Acids - Physical Properties • Solubility • Longer chains • more hydrophobic, less soluble • Double bonds increase solubility • Melting points • Depend on chain length and saturation • Double bonds lead acyl chain disorder and low melting temps • Unsaturated FAs are solids at Room Temp
- 6. Triacylglycerols • Glycerol head group HO-CH2- CH(OH)-CH2-OH • Ester linkage from each hydroxyl to Fatty acid • Carboxylate charge is lost • TAGs more hydrophobic than FAs
- 7. Triacylglycerols in Energy Storage & Thermal insulation • Concentrated source of energy – Energy derived from oxidation reactions – More completely reduced state yields 2x the energy/g as Carbohydrates • Pure non-aqueous phase – Lipases hydrolize the ester linkages to release Fatty Acids
- 8. Triacylglycerols in food • Vegetable Oils – unsaturated - catalytic hydrogenation reduces double bonds - less specific than enzymatic methods makes some trans-fats
- 9. Waxes • Esters of long chain fatty Acids with long chain alcohols • Higher melting points • Hydrophobic
- 10. 10.1 Summary • Lipids are water insoluble • Common Fatty Acids have 12-24 carbon atoms (an even number) • Triacyl glycerols primary storage fats in food.
- 11. 10.2 Structural Lipids in Membranes
- 12. Glycerophospholipids • Phosphorylation of Glycerol creates a stereocenter on C2 – L glycerol 3 phosphate head group adds a negative charge – Phospho diesters with: • Ethanolamine, Choline, Serine, Glycerol, Inositol Phosphate, Phosphatidyl Glycerol
- 14. 2 Fatty Acid chains esterified to remaining hydroxyls (diacyl glycerol) • Common Glycerophospholipids have – 16:0 or 18:0 at C1 – 18:1 (Δ 2 ) or 20:1 (Δ 2 ) at C2
- 15. Ether Lipids • Ether linkage instead of ester at C1 • Plasmalogens have a C1=C2 double bond • Platelet activating factor is a signalling lipid with: – saturated acyl chain at CI (ether linkage) – acetyl ester at C2 – phospho-choline on C3
- 16. Galactolipids and Sulfolipids in Chloroplasts • Galactose (or DiGal) attached to Glycerol C3 • Sulpho lipids contain sulphonate on sugar • Sulphonate charge replaces typical phosphate charge
- 17. Archael "Extremophile" Lipids • Longer acyl chains and dual head groups can can replace 2 normal phospholipids – Replace a bilayer with a monolayer • Ether linkages • More stable at high temperatures, Acid Environments
- 18. Sphingolipid s • Sphingosine is a combination acyl chain & Head group (HO-1 CH-CH=CH-(CH2)12-CH3 2 CH-NH3 3 CH2-OH • Acyl chain in amide linkage at C2 makes a ceramide
- 19. Head groups at C3 • Sphingomyelins – Phosphocholine or Phosphoethanolamine • Glycosphingolipids – Cerebrosides have Glucose, galactose – Globosides have simple neutral Oligosaccharides, – Gangliosides have more complicated anionic oligosaccharides
- 20. Glyco- sphingolipids specify cell identitity • Blood Groups –O, –A (+GalNac), –B (+Gal)
- 21. Lipid Degradation in Lysosomes • Phospholipase A1 cleaves ester linkage at C1 – glycerophospholipid => lysophospholipid + FA1 • Phospholipase A2 is a lysophospholipase – cleaves ester linkage at C2 – lysophospholipid => Glycerophosphate head group + FA2 • Phospholipase C cleaves phospho-glycerol ester linkages – glycerophospholipid => diacyl glyceral + phospho head group
- 22. Sterols • Have 4 fused rings • Cholesterol is the major sterol in vertebrates • Steroid Hormones – Testosterone, Estrogen
- 23. 10.2 Summary • Polar Lipids major constituent of membranes • Glycerophospholipids are charged depending on constituents • Galactolipids are abundant in chloroplasts • Archaebacteria have extreme lipids • Sphingolipids are built on a sphingosine framework • Sterols have a polycyclic aromatic ring structure
- 24. 10.3 Lipids as Signals, Cofactors and Pigments • Phosphatidylinositol signalling • Sphinogosine • Eicosanoids • Steroid Hormones • Plant signalling • Vitamin A and D • Vitamin E and K - Redox Cofactors • Dolichols
- 25. Phosphatidylinositol signaling • PI is phosphorylated to give PI 4,5 bisphosphate • On the inner (cytoplasmic) leaflet of the cell membrane • Phospholipase C cleaves PI 4,5 bisphosphate to give – IP3 and DAG – both molecules active in signalling through protein kinase C • Pleckstrin homology domains bind to inositol 3,4,5 triP
- 27. Eicosanoids • Are derived from Arachadonic Acid 20:4(Δ5,8,11,14 ) • NSAIDs (Aspirin and ibuprofin) block production of Prostaglandiins and thromboxanes • Prostaglandins - C8-C12 bond generates 5 membered ring. Stimulate adenylyl cyclase • Thromboxanes - C8 -C12 bond + Oxygen in heterocyclic ring • Leukotrienes involved in asthma and other processes
- 28. Steroid Hormones • low solubility in water • transported by proteins, • can pass through membranes
- 29. Vitamin A and D • Isoprene is a common precursor for sterols, Vitamin D and Vitamin A – CH2=C(CH3)-CH=CH2 • D vitamins derived from Sterols
- 31. Vitamin E and K - Redox Cofactors • Vitamin E and other tocopherols are antioxidants • Vitamin K is an isoprenoid blood Clotting cofactor – Warfarin is a vitamin K derivative (named after the Wisconsin Alumni Research Foundation WARF) – rat poison that kills by inducing hemorrhage, internal bleeding – Can be used medically to inhibit clotting • Dolichols – Serve as membrane anchors for the assembly of oligosaccharides by glycosyl transferases
- 33. 10.3 Summary • Minor lipids are essential cofactors • PI bisP => inositol trisP + DAG • Eicosanoid hormones from arachidonic acid • Steroid Hormones • Fat Soluble Vitamins • Fat soluble quinones - electron transport • Dolichols - membrane anchors for "solid phase" oligosaccharide synthesis
- 34. 10.4 Working with Lipids • Lipid Extraction • Adsorption Chromatography • Gas Liquid Chromatography • Specific Hydrolysis • Mass Spectrometry
- 35. Lipid Extraction • Lipid Definition: Any of a group of organic compounds, including the fats, oils, waxes, sterols, and triglycerides, that are insoluble in water but soluble in nonpolar organic solvents, are oily to the touch, and together with carbohydrates and proteins constitute the principal structural material of living cells. - First step is an organic solvent extraction to separate the lipids (by definition) from everything else - Commonly Chloroform Methanol Water Mixtures
- 36. Adsorption Chromatograph y • Silica gel is insoluble in and more polar than CHCl3 • Polar lipids adsorb from the solvent, bind to the column • Elution with successively more polar solvents eg. Acetone then MeOH
- 37. Gas Liquid Chromatography • Good for analysis of volatile components • Fatty acids analysed as Methyl esters after acid or base hydrolysis in MeOH • Adsorption from gas phase to the liquid (instead of solid from liquid) slows the passage through the column
- 38. Specific Hydrolysis • Acid or base hydrolysis is non-specific • Enzymatic hydrolysis requires specific interactions • Lipid identity can be deduced from Chromatograms taken before and after hydrolysis
- 39. Mass Spectrometry • Chromatography fractions or continuous flow can be monitored by MS • Accurate masses of intact lipids, specific hydrolysis products, and internal fragments can be used to deduce structure
- 40. 10.4 Summary • Extraction and Chromatography • Phospholipase hydrolysis • ID by chromatagraphy, specific hydrolysis and/or Mass. Spec.
Editor's Notes
- #14: FIGURE 10-9 (part 2) Glycerophospholipids. The common glycerophospholipids are diacylglycerols linked to head-group alcohols through a phosphodiester bond. Phosphatidic acid, a phosphomonoester, is the parent compound. Each derivative is named for the head-group alcohol (X), with the prefix "phosphatidyl-." In cardiolipin, two phosphatidic acids share a single glycerol (R 1 and R 2 are fatty acyl groups).
- #31: FIGURE 10-21 Vitamin A 1 and its precursor and derivatives. (a) β -Carotene is the precursor of vitamin A 1 . Isoprene structural units are set off by dashed red lines (see p. 359). Cleavage of β -carotene yields two molecules of vitamin A1 (retinol) (b). Oxidation at C-15 converts retinol to the aldehyde, retinal (c), and further oxidation produces retinoic acid (d), a hormone that regulates gene expression. Retinal combines with the protein opsin to form rhodopsin (not shown), a visual pigment widespread in nature. In the dark, retinal of rhodopsin is in the 11- cis form (c). When a rhodopsin molecule is excited by visible light, the 11- cis -retinal undergoes a series of photochemical reactions that convert it to all- trans -retinal (e), forcing a change in the shape of the entire rhodopsin molecule. This transformation in the rod cell of the vertebrate retina sends an electrical signal to the brain that is the basis of visual transduction, a topic we address in more detail in Chapter 12.
- #33: FIGURE 10-22 Some other biologically active isoprenoid compounds or derivatives. Units derived from isoprene are set off by dashed red lines. In most mammalian tissues, ubiquinone (also called coenzyme Q) has 10 isoprene units. Dolichols of animals have 17 to 21 isoprene units (85 to 105 carbon atoms), bacterial dolichols have 11, and those of plants and fungi have 14 to 24.