FATTY ACD METABOLISM 100.pptx Biochemistry

1) The acyl group is to the hydroxyl group of carnitine to form acyl carnitine. This reaction is catalyzed by carnitine acyl transferase I

2) Acyl carnitine is then shuttled across the inner mitochondrial membrane by a carnitine translocase.

3) The acyl group is transferred back to CoA on the matrix side of the membrane. This reaction, which is catalyzed by carnitine acyl transferase II.
Finally, the translo case returns carnitine to the cytosolic side in exchange for an incoming acyl carnitine
n= Number of carbon atoms present in fatty acid
Number of acetyl CoA produced = n/2
Number of cycles for fatty acids = (n/2 -1)
Number of reduced coenzyme = (n/2-1) (FADH2 + NADH)
For example if 16C (palmitic acid) undergoes beta oxidation
No. of acetyl CoA produced = 8 {1 Acetyl CoA = 12 ATP in TCA}
8×12 = 96 ATPs
No. of cycles for palmitic acid = 7
No. of reduced coenzymes produced= 7 (FADH2 + NADH)
7(2+3) = 35 ATPs
Total no. of ATPs produced= 96+35= 131 ATPs
No. of ATPs utilized during activation= 2 ATPs
Net gain = 131 - 2= 129 (ATPs According to old energetics concept)

1. OXIDATION OF FATTY ACIDS & CLINICAL RELEVANCE V ICTORIA UNIVERSITY BPHARM STUDENTS PRESENTED BY KAWALYA STEVEN

2. Fatty acids can be obtained from- A fatty acid contains a long hydrocarbon chain and a terminal carboxyl ate grou p. The hydrocarbon chain may be saturated ( with n o double bond) or may be unsaturated ( c ontaining double bond). FATTY ACIDS Diet Adipolysis De novo synthesis 2

3. 1) Fatty acids are building blocks of phospholipids and glycolipids . 2) Many protei ns are modified by the covalent attachment of fatty acids , which target them to membrane loca tions 3) Fatty acids are fuel molecules . They ar e stored as triacylglycerol s . Fatty acids mobili zed from triacylglycerol s are oxidized to meet the energy needs of a cell or or ganism . 4) Fatty acid der ivatives serve as hormones and intracellular messengers e .g. steroi ds, sex hormones and pro staglandins. FUNCTIONS OF FATTY ACIDS 3

4. . Triglycerides are a highly concentrated stores of energy because they are reduced and anhydrous . . The yield from the complete oxid ation of fatty acids is a bout 9 kcal g-1 (38 kJ g-1 ) . Triacylglycerols are nonpola r, and are stored in a nea rly anhydrous form, whereas much mor e polar proteins and carbohydrates are more highly TRIGLYCERIDES 4

5. . A gram of nearly anhydrous f at stores more than six times as much energy as a gram of hydrated gly cogen , which is likely the reason that triacylglycero ls rather than glycogen were s elected i n evolution as the major energy reservoir . . The glycogen and glucose st ores provide enough energy to sustain biologica l function for about 24 hours , wher eas the Triacylglycerol stores allow s urvival for several wee ks. TRIGLYCERIDES V / S GLYC OGEN 5

6. o Free fatty acids and monoacylglycero ls obtained by digestion of dietary triglycerides are abs orbed by intestinal epithelial cells . o Triacylglycero ls are resynthesized and packaged with other lipids and apoprotein B -48 to form chylomi crons, which are then released into the lymph system . PROVISION OF DIETA R Y FATTY ACIDS Most lipids are ingested in the form of triacylglycerols , that must be degraded to fatty acids for A bsorption , across the intestinal epithelium . 6

7. The triacylglycerols are degraded to fatty acids and glyc erol by hormone sensitive lipase . The released fatty are transported to the energy-requiring tissues . PROVISION OF FATTY ACIDS FROM ADIPOSE TISSUE 7

8. Free fatty acids — also called unesterified ( UFA ) or nonesterif ied ( NEFA ) fatty acids — are fatty acids that are in the unesteri fied state. In plasma , longe r-chain FFA are combined with albumin , and in th e cell they are attached to a fatty acid-binding protein. Shorter - chain fatty acids ar e more water-soluble and exi st as the un-ionized acid or as a fatty acid anion . By these means, free fatty acids are made accessible as a fuel i n other tissues. TRANSPORTATI ON OF FREE FATTY AC IDS 8

9. Types of fatty acid oxidation 1.Major fatty acid oxidation Beta Oxidation Beta oxidation proper Beta oxidation of odd fatty a cid chains 2.Minor fatty acid oxidation Alpha oxidation Omega oxidation Peroxisomal beta oxidation

10. Fatty acids can be oxidiz ed by- 1) Beta oxidation - Major mechanism , o ccurs in the mitochondria matrix . 2- C units are released as acetyl Co A p er cycle . 2) Alpha oxidation - Predominantly takes place in brain and liver, one carbon is lost in the form of CO2 per cycle . 3) Omega oxidation - Min or mechanism, but becomes important in conditions of impaired beta oxidation 4) Peroxisomal oxidation - Mainly for the trimmi ng of very long chain fatty acid s. TYPES OF FATTY ACID OXIDATION 9

11. Overview of b eta oxidation Beta oxidation proper occurs in mitochondrial matrix and involves 4 steps. A saturated acyl Co A is degraded by a recurring sequence of f our reactions: 1 ) Oxidation by F lavin adenine dinucleotide (FAD ) 2) Hydration 3) Oxidation by NAD + 4) Thiolysis by Co ASH BETA OXIDATION PROPER 10

12. The fatty acyl chain is shortened by two carbon atoms as a r esult of these reactions , FADH2 , NADH , and acetyl Co A are generated . Because oxidation i s on the β carbon and the chain is broken between the α (2)- and β (3)- carbon atoms — hence the name – β oxidation . BETA OXIDATION 11

13. Fatty acids must first be converted to an active intermediate before they can be catabolized. This is the only step in the complete degradation of a fatty acid that requi res energy from ATP. The activation of a fatty acid is accomplished in two steps- ACTIVATION OF FAT TY ACIDS 12

14. . Carnitine (ß- hydroxy -Υ- trimeth yl ammonium buty rate), ( CH 3 ) 3 N + — CH 2 — CH ( OH )— CH 2 — COO – , is widely distributed and is particularly abundant in muscle . Carnit ine i s obtained f rom foods , par ticularly animal-based foods, and via endogenous s ynthesis. TRANSPORT OF FATTY ACID IN TO MITOCHONDRI AL MATRIX Fatty acid s are activated on the outer mitochondrial membrane , whereas they are oxidized in the m itochondrial matrix. Activated long-chain fatty acids are transported across the membrane by conjugating them to carnitine , a zwitterionic alcohol 13

15. 1 ) The acyl group is to the hydroxyl group of carnitine to form acyl car nitine . This reaction is catalyzed by carnitine acyl transfe rase I 2 ) Acyl carnitine is then shuttled across the inner mitochondrial membrane by a carnitine t ra nslocase . 3 ) The acyl grou p is transferred back to CoA on the matrix side of the membrane . This reacti on , which is catalyzed by carnitin e acyl transferase II . Finally , the translo c ase returns carnitine to the cytosolic side in exchange for a n incoming acyl carnitine ROLE OF CARNITINE 14

16. ROLE OF CARNITINE 15

17. STEPS OF BET A OXIDATION Step-1 Dehydrogenation- The first step is the removal of two hydrogen a toms from the 2(α)- and 3(β)- carbon atoms , catalyzed by acyl- CoA dehydrogenase and requiring FAD . This results in the formation of Δ 2 - trans - enoyl -CoA and FADH 16

18. Electrons from the FADH2 prosthetic group of t he reduced acyl CoA dehydrogenase are transferre d to electron- transferring flavoprot ein (ETF). ETF donates electro ns to ETF: ubiquinone reductase , an iron - s ulfur protein. Ubiquinone is thereby reduced to ub iquinol, which delivers its high - potential electrons to the second proton-pumping site of the respiratory chain. STEPS OF BETA OXIDATION 17

19. STEPS OF BETA OXIDATION Step-2- Hydration Water is added to saturate the double bond and form 3 -hydroxyacyl-CoA , catalyzed by Δ 2 -enoyl-CoA hydratase . 18

20. Step-3- dehydrogena tion- The 3-hydroxy derivative undergoes further dehydrogenation on the 3-carbon catalyzed by L (+)- 3-hydroxyacyl- CoA dehydrogenase , to form the cor responding 3-ketoacyl-CoA compound . In this case, NAD + is the coe nzyme involved. STEPS OF BETA OXIDATION 19

21. Step -4- Thioly sis 3-ketoacyl-CoA is split at the 2,3- position by thiolase (3- ketoacyl-CoA- thiolase ) , F or ming acetyl-CoA and a new acyl-CoA tw o carbons shorter than the original acyl-Co A molecule . 20 STEPS OF BET A OXIDATION

22. STEPS OF BETA OXIDATION The acyl-CoA formed in the cleavage reaction re - enters the oxidative p athway at reaction 2 . Since acetyl- CoA can be oxidized to CO 2 and water v ia the citric acid cycle , the complete oxidation of fat ty acids is achieved 21

23. BETA OXIDATION The overall reaction can be repre sented as follows- 22

24. The degradation of palmitoyl C oA ( C16-acyl Co A ) requires seven reaction cycl es. In the seventh cycle, the C 4- ketoacy l CoA is thio lyzed to two molecules of acetyl CoA . BET A OXIDATION- ENERG Y YIELD 106 (129 As per old concept) ATP are produced by the complete oxidation of one mol of Palmitic acid . Energy yield by the complete oxidation of one mol of Palmitic ac id- 23

25. FORMULA FOR CALCULATING ENERGETICS FOR EVEN CHAIN FATTY ACID n= Number of carbon atoms present in fatty acid Number of acetyl CoA produced = n/2 Number of cycles for fatty acids = (n/2 -1) Number of reduced coenzyme = (n/2-1) (FADH2 + NADH) For example if 16C (palmitic acid) undergoes beta oxidation No. of acetyl CoA produced = 8 {1 Acetyl CoA = 12 ATP in TCA} 8×12 = 96 ATPs No. of cycles for palmitic acid = 7 No. of reduced coenzymes produced= 7 (FADH2 + NADH) 7(2+3) = 35 ATPs Total no. of ATPs produced= 96+35= 131 ATPs No. of ATPs utilized during activation= 2 ATPs Net gain = 131 - 2= 129 (ATPs According to old energetics concept)

26. BET A OXIDATION- ENERG Y YIELD Therefore 2 ATP consumed during activat ion of palmitate to Palmitoyl CoA Net Energy output - 108 - 2 = 106 ATP Total = 108 ATPs 2 ATP equivalents ( ATP AM P + P p i PPi 2 Pi 2.5 ATPs per NADH = 17.5 1. 5 ATPs per FADH 2 = 10.5 10 ATPs per acetyl - C oA = 80 24

27. Regulation of beta-oxidation Increased availability of FFA increases the rate of beta oxidation Glucagon increases FFA and Insulin decreases FFA CAT-I is inhibited by Malonyl CoA ( substrate for fatty acid synthesis). Thus during denovo synthesis of fatty acid beta oxidation is inhibited

28. WITH IMPAIRED BETA OXIDATION DISORDERS ASSOCIATED 25

29. deficiencies of carnitine or carnitine palmitoyl transferase or carnitine translocate Causes: Deficiency of carnitine Inherited CPT-I deficiency affects only the liver. CPT-II deficiency affects primarily skeletal muscle and, when severe, the liver. Symptoms Include: Muscle cramps are precipitated by fasting, exercise and high fat diet . Severe muscle weakness related to importance of fatty acids as long term energy source and death . Hypoglycemia and hypo-ketosis Diet containing MCFAs (milk fat and coconut oil) is recommended since they do not require carnitine shuttle to enter mitochondria.

30. 2 ) Dicarboxylic aciduria is characterized b y- i ) Excretion of C 6 –C 10 -di carboxylic acids and ii ) Nonketotic hypoglycemia which is caused by lack of mitochondrial , medium chain A cyl- CoA dehydrogenases. DISORDERS ASS OCIATED WITH IMPAIRED BETA OXIDATION 26

31. 3 . Jamaican vomiting sickness Caused by eating unripe A ckee fruit which contains unusual toxic amino acids hypoglycin A and B It inhibits the medium and short-chain enzyme acyl CoA dehydrogenase . Beta-oxidation is blocked leading to serious complications. Symptoms : Severe hypoglycemia vomiting Convulsions Coma Ackee fruit DISORDERS ASSOCIATED WITH IMPAIRED BETA OXIDATION

32. 4) Acute fatty liver of pregnancy Manifests in the second half of pregnancy, usually close to term , but may als o develop in the postpartum period. The patient developed sympt oms of hepatic dysfunction at 36 weeks of gestation . Short history of illness, hypoglycemia , liver failure , renal failure, and coagulo pathy are observed. Diagnosis is made based on an incidental finding of abnormal liver enzy me levels . Affected patients may become jaundiced or develop encephalopathy from liver f ailure, usually reflected by an elevated ammo nia level . Profound hyp oglycemia is common. DISORDERS ASS OCIATED WITH IMPAIRED BETA OXIDATION 27

33. Medium chain acyl- coa dehydrogenase deficiency (MCAD deficiency) Most common inborn error of fatty acid oxidation . Being found in 1:14,000 births worldwide. Decreased ability to oxidize fatty acids with six to ten carbons. MCFA accumulates in tissue and also excreted in urine . Symptoms: Hypoglycemia Sleepiness Vomiting Fat accumulation in liver

34. Fatty acids with an odd number of carbon atoms are o xidized by the pathway of β- oxidation , produ cing acetyl - CoA , until a three - carbon ( propio nyl -CoA) residue remains . This compound converted to Succinyl - CoA , a constituent of the TCA cycle BETA OXIDATION OF ODD CHAIN FATT Y ACIDS The propionyl residue from an odd-chain fatty acid is the only part of a fatty acid that is glucogenic. Acetyl CoA cannot be converted into p yruvate or Oxaloacetate in animals . 28

35. BETA OXIDATI ON OF UNSATURATED FATTY ACIDS In the oxidation of unsaturated fa tty acids, most of the reactions are the same as those for saturated fatty acids , only two additiona l enzymes an isomerase and a reductase are needed to degrade a wide range of unsa turated fatty acids . Energy yield is le ss by the oxidation of unsaturated fatty acids since they are less reduced . Per double bonds 2 ATP are less formed , since the first step o f dehydrogenation to introduce double b ond is not required. 29

36. Palmitoleoyl CoA undergoes a series of chain degradation losing 2 carbons from (-CH 2 ) 7 which are carried out by the same enzymes as in the oxidation of saturated fatty acids. The cis - Δ 3-enoyl CoA formed in the third round is not a substrate for acyl CoA dehydrogenase. An isomerase converts this double bond into a t rans- Δ 2 double bond . The subsequent reactions are those of the saturated fatty acid oxidation pathway, in which The cis - Δ 3- enoyl CoA is a BETA OXIDATION OF UNSATURATED FATTY ACIDS

37. 31 BETA OXIDATI ON OF POLY UNSATURATED FATTY ACIDS

38. A different set of enzymes is required for the oxidation of Linoleic acid , a C 18 polyunsatura ted fatty acid with cis -Δ 9 and cis -Δ12 double b onds . The cis - Δ 3 double bond for med after three rounds of β oxidation is converted into a trans - Δ 2 dou ble bond by isomerase . The acyl CoA produced by another round of β oxid ation contains a cis - Δ 4 double bond. Dehydrogenation of this species by acyl CoA dehydrogenase yields a 2,4-dienoyl intermediate, which is not a substrate for the next enzyme in the β -oxidation pathway reductase , an enzyme that uses NADPH to reduce the 2,4 - dienoyl intermediate to tran s -D 3-enoyl CoA. Cis - Δ 3- Enoyl CoA isomerase then converts trans- Δ 3- enoyl CoA into the, trans- Δ 2 form, a customary intermediate in the beta-oxidation pathway BETA OXIDATION OF POLY UNSATURATED FATTY ACIDS 32

39. 1) α- Oxidation - Oxidation occ urs at C-2 instead of C-3 , as in β oxidation 2) ω- Oxidation – Oxidation occurs at the methyl end of the fatty acid molec ule . 3) Peroxisomal fatty acid oxidat ion- Occurs for the chain shortening of very long chain f atty acids . MINOR PATHWAY S OF FATTY ACID OXIDATION 33

40. Alpha ( α ) - oxidation Defined as the oxidation of fatty acid (methyl group at beta carbon) with the removal of one carbon unit adjacent to the α carbon from the carboxylic end in the form of CO2 Alpha oxidation occurs in those fatty acids that have a methyl group (CH3) at the beta-carbon, which blocks beta oxidation . Substrate : - Phytanic acid, which is a lipid present in milk or derived from phytol present in chlorophyll and a constituent in animal fat and meat peroxisomes is the cellular site of brain and liver No production of ATP

41. Involves decarboxyla tion process for the removal of single carbon atom at one time . with the resultant production of an odd chain fatty acid that can be subsequently oxidized by beta oxidation for energy production. It is strictly an aerobi c process. No prior activation of th e fatty acid is required. The process involve s hydroxylation of the alpha carbon with a specific , α- hydroxyla se that requires Fe ++ and vitamin C / FH 4 as c ofactors. 34 ALPHA- - OXIDATION -

42. α- Oxidation is most suited for t he oxidation of phytanic acid Normally it is metabol ized by an initial α- hydroxylation foll owed by dehydrogenation and decarboxylation. Beta oxidation can not occur initially becaus e of the presence of 3- methyl groups, but it can proceed after decarboxylation. The whole reaction produces three molecules of propionyl C o A , three molec ules of Acetyl C o A, and one molecule of iso butyryl co A . BIOLOGICAL SIGNIFICANCE O F ALPHA OXIDATI ON 35

43. Steps of alpha oxidation Activation of phytanic acid Hydroxylation Removal of formyl CoA (CO2) Oxidation of Pristanal Beta-oxidation of pristanic acid

44. Alpha Oxidation Phytanic acid ATP AMP+ ppi Phytanoyl CoA α KG + O2 Succinate +CO2 2-hydroxy phytanoyl CoA Formyl CoA CO2 Pristanal NADP NADPH Pristanic acid Phytanoyl CoA synthetase Phytanoyl CoA Hydroxylase Lyase Aldehyde dehydrogenase

45. Pristanic acid undergoes beta oxidation Pristanic acid Activation Beta oxidation proper 2- methyl propionyl CoA + 3Acetyl CoA + 3 Propionyl CoA

46. Phytanic acid is oxidized by Phytanic acid α oxidase (α- hydroxylase enzyme ) T o yield CO 2 and odd chain fa tty acid Pristanic acid that can be subsequently oxidized by beta oxidation . 36

47. 2) The hydroxy fatty acids produced as intermediates of this pathway like Cerebro nic acid can be used for the synthesis of immunological C erebrosides and sulfatides ( precursor in white matter formation) 3) Odd chain fatty acids produced upon decarboxylation in this pathway, can be used for the synthes is of sphingolipids in myelin sheath, gangliosides in nerves and can also undergo beta oxidation to form propion yl C o A and Acety l C o A. The number of acetyl co A depend upon the chain length. Propionyl C o A is converted to Succi nyl C o A to gain entry in to TCA cycle for further oxidation. BIOLOGICAL SIGNIFICANCE O F ALPHA OXIDATI ON 37

48. Refsum's disease (RD )- is a neurocutaneous syndrome that is characterized biochemically by the accumulation of phytan ic acid in plasma and tissues . Patients with Refsum d isease are unable to degrade phytanic acid because of a deficient activity of Phytanic acid oxida se enzyme catalyzin g the first step of phytanic acid alpha - oxidatio n . CLINICAL SI GNIFICANCE OF ALPHA OXI DATION 38

49. Adult Refsum’s Disease Biochemical defect Defect in enzyme Phytanoyl CoA H ydroxylase (Phytanic acid oxidase) Autosomal recessive disorder in PHYH gene Phytanic acid accumulates in brain and other nervous tissues lab Findings Plasma Level of phytanic acid > 200µmol/L Normal< 3oµmol/L

50. Molecular Toxicology of Refsum’s Disease PA is directly toxic to ciliary ganglion cells and induces calcium – driven apoptosis in purkinji cells Recent studies has found that PA has a Rotenone like action in inhibiting complex –I and producing reactive oxygen species ROS. This is the reason why neuronal cells and retina rich in mitochondria are prime tissue affected in Refsum’s disease

51. Refsum’s Disease Clinical manifestations Severe neurological symptoms such as ., Polyneuropathy, Retinitis pigmentosa (associated with night blindness) Nerve deafness Cerebellar ataxia Patients should avoid intake of diet such as green vegetables and milk.

52. Omega( ω) Oxidation Cellular site : Endoplasmic reticulum oxidation occurs at ( ω- omega) carbon — the carbon most distant from the carboxyl group. Substrates : Medium and long chain fatty acid Importance : It is a minor pathway but becomes active when beta oxidation is defective . The product formed are di -carboxylic acid

53. Involves hydroxylation and occur s in the endoplasmic reticulum of many t issues. Hydroxylation takes place on the methyl carbon at the other end of the molecule from t he carboxyl group or on the carbon next to th e methyl end . It uses the “ m ixed function oxidase” type of reaction requiring Cytochrome P4 50, O2 and NADPH , as well as the necessary enzymes . Hydroxy fatty acids can be further oxidized to a dicarboxylic acid via sequential re actions of Alcohol dehydr ogenase and aldehyde dehydrogenases . The process occurs primar ily with medium chain fatty acids . OMEGA OXIDATION OF FATTY ACIDS 39

54. OMEGA OXIDATION O F FATTY ACIDS Dicarboxylic acids so formed can undergo beta oxidation to produce shorter chain dicarboxylic acids such as ; Adipic A cids ( C6 ) and succinic acid (C4). 40

55. The microso mal ( endop lasmic reticulum, ER) pathway of fatty acid ω- oxidation represents a minor pathway of overall fatty acid oxidation . However , in certain pathophysiologic al states, such as diabetes, chronic alcohol consumption , and starvatio n, the ω-oxidation pathway may prov ide an effective means for the elimination o f toxic levels of free fatty acids. SIGNIFICAN CE OF OMEGA OXIDATION 41

56. In peroxisomes , a flavoprotein d ehydrogenase transfers electrons to O 2 to yield H 2 O 2 instead of capturing the high-energy electrons as FADH 2 , as occurs in mitochondrial beta oxida tion . Catalase is needed to conver t the hydrogen peroxide produced in the initial reaction into water and oxygen . Subsequent steps are identical with th eir mitochondrial count erparts , They are carr ied out by different isoform of the enzymes. PERO XISOMAL OXIDATION OF VER Y LONG CHAIN FATTY ACI DS 42

57. The specificity of the peroxiso mal enzymes is for longer chain fatty acids . Thus per oxiso mal enzymes function to shorten the chain length of relatively long chain fatty acids to a point at which beta oxidation can be completed in mitochondria. PEROXISOMAL OXIDATION OF VERY LONG CHAIN FATTY ACI DS 43

58. Peroxisomal reactions include ; ch ain shortening of very long chain fatty acids and dicarboxylic acid s conversion of cholesterol to bile acids and formatio n of ether lipids. The congenital abse nce of functional peroxisomes , an inherited defect , c auses Zellweger syndrome. SIGNIFICANCE OF PEROXISOMAL OXIDATION 44

59. Infantile Refsum’s Disease Biochemical defect It is a disorder observed in zellweger syndrome . Congenital peroxisomal biogenesis and assembly disorder Lab findings Phytanic acid in the serum is More than 30 µmol/L and less than 200µmol/L VLCFA and LCFA in serum is increased

60. Zellweger syndrome A.k.a cerebro-hepatorenal syndrome i s a rare, congenital disorder ( pre sent at birth ) Biochemical defect Defect in the gene for peroxisome biogenesis and assembly   Characterized by a reduction or absence of Peroxisomes in the cells of the liver, kidneys, and brain . VLCFA and LCFA are not oxidized and accumulates in tissue , particularly in brain ,liver and kidney. Lab findings Increased level of Phytanic acid in the serum is More than 30 µmol/L and less than 200µmol/L Increased level of VLCFA and LCFA in serum

61. The most common features of Zellweger syndrome include Vision D isturbances Prenatal growth failure Lack of muscle tone , unusual fa cial characteristics M ental retardation Seizures An inabil ity to suck and / or swa llow. ZELLWEGER SYNDROME 45

62. The abnormally h igh levels of VLCFA ( Very long chain fatty a cids), are most diagnostic. There is no cure for Zellweg er syndrome, nor is there a standard course o f treatment . Most treatments are s ymptomatic and supportive . Most infants do not survive past the first 6 months , and usually succumb to respiratory distress , gas trointestinal bleeding, or liver failure. ZELLWEGER SYNDROME 46

63. THANKS FOR LISTENING