1. Lipid metabolism II Phospholipids and glycolipids Eicosanoids. Synthesis and metabolism of cholesterol and bille acids Biochemistry I Lecture 9 20 11 ( E.T .)

2. Glycero ph os ph olipid s C H 2 C H O C O C H 2 O C O O P O O O X Phosphatidylcholine – PC Phosphatidylethanolamine – PE Phosphatidylserine – PS Phosphatidylinositol – PI Cardiolipin - CL

4. Synthesis of triacylglycerols and glycerophospholipids – common aspects P i PI, cardiolipin P C H 2 O C O R C H O C O R C H 2 O R R C O S C oA H S C oA C H 2 O C O R C H O C O R C H 2 O C O R PC,PE,PS triacylglycerol hydrolase diacylglycerol phosphatidate Addition of the head group C H 2 O C O C H O C O R C H 2 O H

6. Diacylglycerol accepts CDP-activated choline or ethanolamine. Activation of choline in two steps: 1 Synthesis of phosphatidyl choline, phosphatidyl ethanolamine, and phosphatidyl serine Choline + ATP  choline phosphate + ADP Choline phosphate + CTP  CDP-choline + PP i CDP-choline plays a part formally similar to that of UDP-glucose in the synthesis of glycogen. activated choline CH 3 CH 3 OH OH O N N O NH 2 O – CH 2 O – O O CH 3 –N–CH 2 –CH 2 –O–P–O–P–O– + O – O + CH 3 –N–CH 2 –CH 2 –O–P–O – CH 3 CH 3

7. 1,2-diacylglycerol + CDP-choline CMP + phosphatidyl choline (PC) The biosynthesis of phosphatidyl ethanolamine (PE) is similar. N -Methylation of PE (in the liver, the donor of methyl group is S -adenosylmethionine) Synthesis of phosphatidylcholine O – CH 2 O P–O– R–CO–O–CH 2 R–CO–O–CH CH 2 –O – CH 2 –N–CH 3 CH 3 CH 3 + O – O P–O–CH 2 –CH 2 –NH 2 R–CO–O–CH 2 R–CO–O–CH CH 2 –O

8. Phosphatidyl ethanolamine + serine  phosphatidyl serine + ethanolamine Phosphatidyl serine (PS) is not, in animals, formed directly in this way, but as exchange of serine for the ethanolamine of PE: Phosphatidyl serine can be also decarboxylated to form PE . CH 2 –CH–NH 2 O – O P–O– R–CO–O–CH 2 R–CO–O–CH CH 2 –O COOH O – O P–O–CH 2 –CH 2 –NH 2 R–CO–O–CH 2 R–CO–O–CH CH 2 –O Serine Ethanolamine CO 2

9. Phosphatidic acid is activated in a reaction with CTP to CDP-diacylglycerol : CDP-diacylglycerol = activated phosphatidate 2 Synthesis of phosphatidyl inositol Phosphatidic acid + CTP  CDP-diacylglycerol + PP i OH OH O N N O NH 2 O – CH 2 O – O O P–O–P–O– R–CO–O–CH 2 R–CO–O–CH CH 2 –O

10. CDP-diacylglycerol + inositol  CMP + phosphatidyl inositol (PI) CDP-Diacylglycerol then reacts with free inositol to give phosphatidyl inositol (PI) Further phosphorylations of PI generate phosphatidyl inositol bis phosphate (PIP 2 ) which is an intermediate of the phosphatidyl inositol cycle generating important intracellular messengers IP 3 and diacylglycerol.

11. Phosphatidyl inositol phosphates (PIP, PIP 2 , PIP 3 ) are minor components of plasma membranes, and their turnover is stimulated by certain hormones. A specific phospholipase C, under hormonal control, hydrolyses phosphatidyl 4,5-bisphosphate (PIP 2 ) to diacylglycerol and inositol 1,4,5-trisphosphate (IP 3 ), both are second messengers IP 3 Inositol 1,4,5-trisphosphate R–CO–O–CH R–CO–O–CH 2 CH 2 -O-PO O - O- R–CO–O–CH R–CO–O–CH 2 CH 2 -OH O-PO 3 2 - 2- O 3 PO OH OH OH 2- O 3 P-O O-PO 3 2 - 2- O 3 PO OH OH OH PIP 2 PI 3,4-bisphosphate DG

12. Plasmalogens m odified glycerophospholipids ( alkoxylipids or ether glycerophospholipids ) . Plasmalogens represent about 20 % of glycerophospholipids. O – O P–– CH 2 – O–CH=CH–R R–CO–O–CH CH 2 –O choline (in myocard) ethanolamine (in myelin) serine Alkenyl Ether bond

13. PAF (platelet activating factor) PAF induces aggregation of blood platelets and vasodilation and exhibits further biological effects, e.g. it is a major mediator in inflammation, allergic reaction and anaphylactic shock. Acyl reduced to alkyl Acetyl in place of the fatty acyl O – O P–O–CH 2 –CH 2 –N–CH 3 CH 2 – O–CH 2 –CH 2 –R CH 3 –CO –O–CH CH 2 –O CH 3 CH 3 +

14. Transacylation reactions Exchange of acyls on the C-2 in phosholipids: diacylglycerols: oleic acid on the C-2 phospholipids: polyunsaturated FA (often arachidonic acid) on the C-2

16. Anchoring of proteins to membrane The linkage between the COOH-terminus of a protein and phosphatidylinositol fixed in the membrane lipidic bilayer exist in several ectoenzymes (alkaline phosphatase, acetylcholinesterase, some antigens).

17. Lung surfactant The major component is dipalmitoylphosphatidylcholine . It contributes to a reduction in the surface tension within the alveoli (air spaces) of the lung, preventing their collapse in expiration. Less pressure is needed to re-inflate lung alveoli when surfactant is present. The respiratory distress syndrome (RDS) of premature infants is caused, at least in part, by a deficiency in the synthesis of lung surfactant.

18. Catabolism of glycerophospholipids Enzymes catalysing hydrolysis of glycerophosholipids are called phospholipases . Phospholipases are present in cell membranes or in lysosomes. Different types (A 1 , A 2 , C, D) hydrolyse the substrates at specific ester bonds: O – O P–O–X (head group) R–CO–O–CH 2 R–CO–O–CH CH 2 –O C A 1 A 2 D (only in brain and plants)

19. Sphingo phospho lipids Binding of phosphate Binding of choline Binding of FA sphingosine Components of membranes, signal transduction, myelin sheat 1 2 3 4 H O N H 2 O H

20. S ph ingosin e has 18 C ( 16 from palmit ate , 2 from serin e) serin e (3C) palmitoyl-CoA (16C) amino group binds FA Prim. alcohol group binds phosphate or sugar

22. C H 3 ( C H 2 ) 1 4 C O S - C oA + C H C H 2 O H C O O - N H 3 + C H 3 ( C H 2 ) 1 4 C O C H C H 2 O H N H 3 + oxosfinganine Biosynthesis of sphingosine 1. Palmitic acid serine 2 C O + C oA - S H

23. C H 3 ( C H 2 ) 1 2 C H 2 C H 2 + C C H C H 2 O H O N H 3 C H 3 ( C H 2 ) 1 2 C H 2 C H 2 + C H C H C H 2 O H O H N H 3 C H 3 ( C H 2 ) 1 2 C H C H C H C H C H 2 O H O H N H 3 + NADPH + H + NADP FAD FADH 2 oxosfinganine sfinganine sfingosine 2. 3. 4.

24. S ph ingomyelin es FA – lignocer ic 24:0 a nd nervon ic 24:1(15) fatty acid sphingosine phosphate choline

25. 1.Attachment of fatty acid by amide bond = ceramid 2. Reaction with CDP-choline: Phosphocholine is attached to CH 2 OH = sphingomyelin Biosynthesis of sphingomyeline 18 1 2 3 4 H O N H 2 O H

26. Cerebrosid e ( monogly c osylceramid e) gala c tosylceramid e ceramide + UDP-galactose galactose O-glycosidic bond Glycosphingolipids oligoglycosylceramides, acidic sulphoglycosylceramides, and sialoglycosylceramides ( gangliosides) . N O O H H O O O H O H O H H O ceramide

27. Glycolipids can be sulfated Sulfosphingolipids are formed by transfer of sulphate from 3´-phosphoadenosine-5´-phosphosulfate ( PAPS).

28. Saccharidic components of glycolipids - examples: Cerebroside Ceramide– (1 ← 1β) Glc Oligoglycosylceramide Ceramide– (1 ← 1β) Glc (4 ← 1β) Gal Sulphoglycosphingolipid Ceramide– (1 ← 1β) Glc-3´-sulphate Gangliosides G M3 (monosialo ganglioside type III) Ceramide– (1 ← 1β) Glc- (4 ← 1β) Gal (3 ← 2α) NeuAc G M2 Ceramide– (1 ← 1β) Glc- (4 ← 1β) Gal- (4 ← 1β) GlcNAc (3 ← 2α) NeuAc G M1 Ceramide– (1 ← 1β) Glc- (4 ← 1β) Gal- (4 ← 1β) GlcNAc- (3 ← 1β) Gal (3 ← 2α) NeuAc

29. Sialic acid is attached to oligosaccharide chain + NeuAc -CMP Ganglioside s Ceramide– (1 ← 1β) Glc- (4 ← 1β) Gal- (4 ← 1β) GlcNAc (3←2α) NeuAc

30. Degradation of sphingolipids in lysosomes In lysosomes, a number of specific enzymes catalyse hydrolysis of ester and glycosidic linkages of sphingolipids. Sphingomyelins loose phosphocholine to give ceramide . Glycolipids due to the action of various specific glycosidases get rid of the saccharidic component to give ceramide . Ceramide is hydrolysed (ceramidase) to fatty acid and sphingosine . Sphingosine is decomposed in the pathway that looks nearly like the reversal of its biosynthesis from palmitoyl-CoA and serine. After phosphorylation, sphingosine is broken down to phosphoethanolamine (decarboxylated serine) and palmitaldehyde , that is oxidized to palmitate.

31. Phosphocholine FATTY ACID CERAMIDE ( N- Acylsphingosine) SPHINGOSINE Sphingosine-1-P Phosphoethanolamine Palmitaldehyde PALMITIC ACID Ceramide Glc Gal GalNAc Gal NeuNAc Ceramide Gal Ceramide Glc Ceramide Gal–O-SO 3 – Ceramide–P–-choline SPHINGOMYELIN CEREBROSIDE SULPHATIDE GANGLIOSIDE G M1 ATP NAD + Degradation of sphingolipids

32. In general, the turnover of sphingolipids is very slow, particularly in brain. Sphingolipidosis Inherited defects in production of the enzymes that catabolize sphingolipids . They result in accumulation of their substrates in lysosomes, leading to lysosomal damage and to disruption of the cell as new lysosomes continue to be formed and their large number interferes with other cellular functions. In the sphingolipidosis mainly the cells of the central nervous system (including brain and retina) are affected.

33. Eicosanoids

34. Eicosanoids Local hormons Synthesis of eicosanoids: PG, TX (prostanoidy) – cyclooxygenase pathway LT (Leukotriens) – lipoxygenase pathway The main types of eicosanoids: prostaglandins (PG) tromboxans (TX) leukotriens (LT) They are synthesized from polyunsaturetd fatty acids with 20 carbons

35. signal molecule (adrenalin, trombin, bradykinin, angiotensin II) ph os ph olipas e A 2 receptor 1. The release of C 20 fatty acids from membr ane phospholids arachidon ic ac. EP E ei c osatrien oic ac. cytopla sm Biosynthesis of eicosanoids

36. Inhibito rs of ph os ph olipas e A 2 Membr ane ph os ph olipid s PUFA ph os ph olipas e A 2 c orti c oid s lipo c ortin Steroid al antiphlogistics (hydro c ortiso ne , prednison e ) stimul ate the synthesis of protein lipo c ortin which inhib its ph os ph olipas e A 2 and block the release of PUFA and eicosanoids formation 

38. Involvement of prostanoids in physiological processes - examples TXA 2 (tromboxane A 2 ) It is produced in platelts, it stimulates vasoconstriction and and platelet agregation Duration of action  30-60 s PGI 2 (prostacycline A 2 ) It is antagonist of TXA 2, , it is produced by vascular endothelium, it inhibits platelet coagulation and has vasodilatation effects, half-life 3 min. Their equilibrated effects takes part in platelet coagulation and vasomotor and smooth muscle tone.

39. PGE 2 is produced by mucose cells of the stomach and inhibits HCl secretion PGE 2 and PGF 2  are synthesized in endometrium and induce uterine contractions. Their concentration in amniotic fluid during pregnancy is low , it significantly increases during delivery. Together with oxytocin is involved in the induction of labor. It r educes the risk of peptic ulcer They can be used to induce abortion by inttravenous or intravaginal application

40. Examples of some biological effects of prostanoids platelet aggregation, vasoconstriction blood platelets thromboxane TXA 2 vasodilation, inhibition of platelet aggregation endothelial cells, smooth muscle cells of blood vessels prostacyclin PGI 2 vasoconstriction increase of body temp. nearly all cell types prostaglandin F PGF 2α inflammatory reaction, vasodilation, inhibition of HCl secretion nearly all cell types prostaglandin E PGE 2 The most remarkable effect Synthesized in Structural group prostanoid

41. Synthesis of prostanoids The enzyme cyclooxygenase (COX) has two enzyme activities: cyclooxygenase peroxidase

42. Synthesis of prostanoids (cycloxygenase pathway) Arachidonic acid PGG 2 (two double bonds) PGH 2 - p recursor of all prostanoids of the 2-series cyklooxygenase (cyklooxygenase activity) cyklooxygenase (peroxidase activity) 2O 2

43. PGE synthase Prostaglandin H 2 Thromboxane TXA 2 Prostacyclin PGI 2 Prostaglandin PGE 2 Prostaglandin PGF 2 α PGE 9-keto reductase TXA synthase PGI synthase

44. The enzyme equipment of various tissues is different E.g., in the lung and the spleen, the enzyme equipment enables biosynthesis of all eicosanoid types. In blood platelets , only thromboxan synthase is present. The endothelial cells of blood vessels synthesize only prostacyclins. The catabolism of prostanoids is very rapid - Enzyme catalyzed ( t 1/2 ~ 0,1-10 min) - non-enzymic hydrolysis ( t 1/2 sec-min)

47. I nhibition of cyclooxygenase suppresses the effects of prostanoids … it has the positive effects ( the anti-inflammatory effect, relief of pain, mitigation of fever. …) … .o n the contrary, there may be some undesirable effects of blocked prostanoid production, e.g. decline in blood platelet aggregation, decreased protection of endothelial cells and of gastric mucosa. Therefore d rugs are being developed which w ould act as selective inhibitors of COX-2 without the adverse gastrointestinal and anti-platelet side effects of non-specific inhibitors of COX.

48. COX-2 inhibitors They are proposed to act as potent anti-inflammatory agents by inhibiting COX-2 activity, without the gastrointestinal (stomach ulcer) and antiplatelet side effects associated with NSAIDs Examples: celecoxib, rofecoxib However further studies indicated that specific COX-2 inhibitors may have a negative effect on cardiovascular function. Coxibs were withdrawn from the market by its manufacturer because of negative patients study Nimesulid (AULIN, COXTRAL), meloxikam (ANTREND,LORMED,MELOBAX) – are still used, they inhibit more COX-2 than COX-1 and must be used with caution

49. Acetylsalicyl ic acid (Aspirin) salicylic acid acetylsalicylic acid It covalently acetylates the active site of cyclo-oxygenase, causing its irreversible inhibition anti-thrombotic action (prevention) ~ 50 mg analgetic, anti-pyretic actions ~ 500 mg

50. Low-doses of aspirin (ASA, 81-325 mg daily) has been shown to be effective in prevention of acute myocardial infarction. Aspirin blocks the production of TXA 2. by inhibition of COX The principal effect of TXA 2 is the stimulation of platelets aggregation. It may initiate the formation of trombus at sites of vascular injury or in the vicinity of ruptured atherosclerotic plaque. Such thrombi may cause sudden total occlusion of vascular lumen. By aspirin treatment the effect the effects of thromboxane are attenuated. Protective effect of acetylsalicylic acid

51. all of them have three conjugated double bonds ( trienes ), the position of which may be different and the configuration either trans or cis.. Lipoxygenase pathway Synthesis of leukotrienes Precursor of all leukotrienes of the 4-series 5-Lipoxygenase COO – OOH COO – O COO – Arachidonate 5-HydroperoxyETE Leukotriene LTA 4 O 2

52. Leukotrienes are produced primarily in leuko cytes and mast cells The classes of LTs are designated by letters A – E, the subscript denotes the total number of double bonds. COO – O LTA 4 LTB 4 OH S Cys  Gly LTD 4 Slow-reacting substance of anaphylaxis (SRS-A) LTB 4 12-Lipoxygenase GSH Glu

53. Eicosanoids bronchoconstriction, vasodilation various cell types lipoxin LXA 4 bronchoconstriction, vasoconstriction leukocytes, mast cells leukotriene LTD 4 The most remarkable effect Synthesized in Structural group Example

54. Cholesterol HO 5-cholesten-3  -ol Essential component of membranes Source for synthesis of bile acids, steroids and vitamin D3 1 2 10 5 11 14 6 7 12 18 17 19 20 21

56. 3-hydroxy-3-methylglutarylCoA (HMG-CoA) acetylCoA acetoacetylCoA ER 1. phase of cholesterol synthesis - synthesis of 3-HMG-CoA Compare with the synthesis of keton bodies in mitochondrial matrix

57. 3-HMG-CoA Mevalonic acid 2. phase - formation of mevalonate 3-HMG-CoA reductase + Double reduction of carboxylic group to primary alcohol group

62. Regulation of HMG-CoA reductase by covalent modification Forms of the enzyme phosphorylated dephosphorylated inactive active Glucagon, intracelular sterols (cholesterol, bill acids), glucocorticoids Insulin, thyroidal hormons Activation: kinase –AMP dependent phosphatase

63. Inhibition of HMG-CoA-reductase by drugs The statin drugs are reversible competitive inhibitors of HMG-CoA-reductase in liver. The synthesis of cholesterol in liver is decreased by their action. Statins – various structures part of their structure resembles to HMGCoA. Simvastatin (Zocor), Lovastatin (Mevacor), Pravastatin (Mevalotin), Pravastatin (Pravachol), Simvastatin (Lipovas), Fluvastatin (Lescol),…

65. mevalonyldiphosphate mevalonate Isopentenyl diphosphate Dimethylallyl diphosphate 5C 5C CO 2 H 2 O 3.phase of cholesterol synthesis: formation of five carbon units O H - O O C - C H 2 - C - C H 2 - C H 2 O H C H 3 - O O C - C H 2 - C - C H 2 - C H 2 O P P O H C H 3 2ATP 2ADP C H 2 =C - C H 2 C H 2 O P P C H 3 ATP ADP + P i C H 3 C H 3 - C =C H C H 2 O P P

66. Dimethylallyldiphosphate isopentenyldiphosphate + PP i prenyltransferase geranyldiphosphate

67. Dimethylallyldiphosphate + isopentenyldiphosphate  geranyldiphosphate 5 C 5 C 10 C 10 C 5 C 15 C farnesyl diphosphate 15 C 15 C 15 C 15 C + + 30 C squalene synthesis of dolichol and ubiquinon + Prenylation of proteins Prenylation of proteins geranyl diphosphate

68. Synthesis of oligosaccharide chains of glycoproteins Respiratory chain Dolichol diphosphate ubiquinon

70. Squalene is linear molecul that can fold into a structure that closely resembles the steroid structure cholesterol squalen

72. Esterification of cholesterol Higher hydrophobicity Cholesterol + acylCoA ACAT Most often linoleic and linolenic acid acyl-CoA-cholesterol acyltransferase Located in ER

73. Transport of cholesterol in blood in form of lipoproteins From liver transported in form of VLDL Most of VLDL is converted to LDL after the utilization of main part of TG contained in them LDL transfers cholesterol into the periferal tissues Reverse transport of cholesterol to the liver - HDL 25-40% - esterified cholesterol

74. Cholesterol in blood Recommended value < 5 mmol / l When the total cholesterol level exceeds 5 mmol /l further investigation of lipid metabolism is necessary, especially the finding of the cholesterol distribution in the lipoprotein fractions LDL-cholesterol = „bad“ cholesterol HDL-cholesterol = „good“ cholesterol A high proportion of serum total cholesterol incorporated in HDL is considered as a sign of the satisfactory ability of an organism to eliminate undesirable excess cholesterol. On the contrary, an increased concentration of LDL-cholesterol represents the high coronary risk involved in hypercholesterolaemia. See Biochemistry II – 4.semestr

76. Cholesterol balance per 24 h FOOD BIOSYNTHESIS 80-500 mg 800 – 1000 mg Steroid hormons, sebum and ear-wax, secluded enterocytes 200 mg Bile acids (primary) 500 mg Cholesterol (bile) 800 mg Cholesterol pool 1000-1500 mg/day is excreted

78. S tructurally related to cholesterol; only the side chain on C-17 is changed  -sitosterol C onsumption of phytosterols reduces the resorption of cholesterol. Recommended intake for people with increased level of cholesterol - 2g/day Phytosterols - sterols of plant origin Plant oils (corn, rapeseed, soya, sunflower, walnut) contain up to 0.9 % phytosterols. Average intake of phytosterols in Czech republic - about 240 mg per day,

79. How do phytosterols function? They penetrate into the mixed micelles that are in contact with intestine mucosa , they compete with cholesterol in resor p tion into the enterocytes.

80. Synthesis of bile acids 7- α -hydroxylase NADPH, O 2 cytP450 Located in ER (monooxygenase reaction) LIVER 7 rate-limiting step in the synthesis NADP + , H 2 O

81. In subsequent steps, the double bond in the B ring is reduced and additional hydroxylation may occur. Two different sets of compounds are produced. One set has  -hydroxylgroups at position 3,7, and 12, the second only at positions 3 and 7. Three carbons from the side chain are removed by an oxidation reaction. Primary bile acids chenodeoxycholate cholate LIVER 24 C pK A  6 pK A  6 12

82. Conjugation with glycine and taurine (ER) LIVER BILE INTESTINE deconjugation and partial reduction chenodeoxycholate cholate lithocholate deoxycholate feces enterohepatal circulation bacterias ABC-transporter

83. Conjugated bile acids taurocholic glycocholic Conjugation increase pK a values , increases detergent efficiency pK A  2 pK A  4 H O O H C O N H S O 3 - O H O H

84. lithocholate deoxycholate Secondary bile acids – do not have OH on C-7 Less soluble, excreted by feces

85. Enterohepatal circulation of bile acids Synthesis 0,2-0,6 g/den recyclation > 95% Feces 0,2-0,6 g/den Digestion of lipids Reabsorption 12-32 g/den Efficiency > 95% Including secondary bile acids Cholesterol  bile acids Gallbladder Intestine Liver

86. 7-dehydrocholesterol cholecalciferol - vitamin D 3 (calciol) Skin cholesterol Liver vit. D 3 – absorption with animal food (fish oils, eggs) Plants Intake and synthesis of vitamins D ergosterol ergocalciferol-vitamin D 2 (ercalciol) vit.D 2 – absorption with plant food (also supplementations) D 3 Transport in blood into the liver D 2  

87. Liver calciol - vitamin D 3 25-hydroxylase 1  -hydroxylase 1,25-dihydroxycholecalciferol (calcitriol) Kidney Active form – regulation of calcium level Synthesis of calciols HO O H O H calcidiol HO O H