The Usual Suspects Cholesterol and Triglyceride Lipid Structure

The Usual Suspects: Cholesterol and Triglyceride

Lipid Structure Cholesterol: Membranes Bile Acids Steroid Hormones Protein modification H O Fatty Acids: Fuel, Prostanoids + Glycerol Triglycerides: FA for Fuel, Prostanoids Protein modification COOH COO H O Phospholipid: Lecithin COO H O COO + N OPOO Membranes 2 nd Messengers

Structure of a Typical Lipoprotein Free cholesterol (surface and core) Phospholipid (amphipath at surface only) Apolipoprotein (amphipath at surface only) Triglyceride (core only) Cholesteryl ester (core only)

Lipoprotein classes and sub-Classes 0. 95 Chylomicron VLDL Density (g/ml) VLDL Remnants 1. 006 IDL Chylomicron Remnants 1. 02 LDL 1. 06 HDL 2 Directly atherogenic (found in plaque) Lp(a) 1. 10 HDL 3 pre-β 2 HDL 1. 20 pre-β 1 HDL 5 10 20 40 60 Particle Size (nm) 80 1000

Substrates for Triacylglycerol Synthesis Plasma NEFA Glucose Triglycerides Acyl-Co. A Synthetase Glc-6 -Pase Co. A Acyl-Co. A Multiple steps Glucose-6 -P CPT I Pyruvate kinase Mitochondria Acyl-Carnitine Fatty Acid Synthase CPT II Malonyl-Co. A PEP Acyl-Co. A PEPCK = PEP carboxylase Citrate Acetyl-Co. A ATP Citrate Lyase Acetyl-Co. A CO 2 HMG-Co. A Synthase Ketone Bodies PEP = phosphoenolpyruvate Acyl-Co. A Carboxylase Krebs Cycle Pyruvate Acyl-Co. A Hepatocyte PEPCK Citrate Beta-oxidation CPT = Carnitine palmitoyl transferase

Structures of Fatty Acids O C HO 16: 0 (palmitic) cis-18: 1 -6 (oleic) O C HO trans-18: 1 -6 (elaidic O C HO (alpha 18: 3 -3 linolenic) O C HO 18: 2 -6 (linoleic) 20: 5 -3 (EPA)

Exogenous (dietary) lipid metabolism Muscle and adipose tissue Lipoprotein lipase Fatty acids Lipoprotein Lipase Chylomicron Remnant Bloodstream Plasma chylomicron I N T E S T I N E Apo C-II enhances and apo C-III inhibits LPL activity Apo B and apo E are ligands for LDL receptor LDL (apo B, E) receptor clears Chylomicron Remnants LDL receptor Lymphatic chylomicron Xenical blocks diatary fat digestion Hepatocyte Liver

Endogenous (hepatic) lipid metabolism Muscle and adipose tissue Lipoprotein lipase Fatty acids Lipoprotein Lipase And hepatic lipase LDL Apo C-II enhances and apo C-III inhibits LPL activity Bloodstream IDL Apo B and apo E are ligands for LDL receptor LDL (apo B, E) receptor clears VLDL, IDL & LDL receptor VLDL Hepatocyte Liver

Clinical Hypertriglyceridaemia Condition Features Secondary Relatively common (obesity, diabetes, renal impairment, liver disease, drugs) Polygenic Accounts for the majority of cases Familial HTG TG predominates. CVD risk varies Predisposes to massive HTG Familial Combined H/L Overproduction of apo B lipoproteins TG and TC vary with age and weight Massive HTG Lipoprotein Lipase deficiency or saturation. Risk of pancreatitis

Therapy for Hypertriglyceridaemia Intervention Features Diet, Exercise Relatively responsive Alcohol restriction Often sufficient in heavy intake Manage 2 o causes Fibrates Statins Fish oils (eg 6 gm/d) Niacin Future Diabetes, renal Effective in high TG, low HDL Mild TG and HDL benefit Benefits TG rather than HDL Effective, but increases glu, urate. DGAT 2 Inhibitors? Bile acid resins Contraindicated. Increase TG

NORMAL CHOLESTEROL ABSORPTION 1, 300 mg/day 400 mg/day Oil phase

NORMAL CHOLESTEROL ABSORPTION 1, 300 mg/day 400 mg/day Oil phase Plant sterols compete with cholesterol here

NORMAL CHOLESTEROL ABSORPTION 1, 300 mg/day 400 mg/day Oil phase 17, 400 mg/day 850 mg/day Ezetimibe competes with cholesterol here

NORMAL CHOLESTEROL ABSORPTION 1, 300 mg/day 400 mg/day Oil phase 17, 400 mg/day 850 mg/day

Intracellular cholesterol sensing by SREBPs (Sterol Regulatory Element Binding Proteins) SCAP or SREBP activating protein SCAP SREBP WD Reg ER b. HLH Cytosol Nucleus SRE b. HLH Lumen Sterols Golgi Apparatus WD Reg b. HLH ZN++ S 1 P Serine protease S 2 P Metalloproteinase Membrane fluidity reflects intracellular cholesterol. Low levels allow cleavage to active form which binds nuclear receptor to control gene expression. • SREBP-2 controls cholesterol synthesis and sterol metabolism • SREBP-1 c is the major isoform in liver and is a key regulator of fatty acid & triglyceride synthesis Other nuclear receptors: FXR, LXR.

LDL Receptor activity reflects intracellular cholesterol homeostasis Cholesterol delivery via LDL-R alters intracellular membrane cholesterol and SREBP, which a) Reduces synthesis via HMGCo. A Reductase b) Reduces LDL-R synthesis c) Increases storage as ester d) Reduces counter-regulatory PCSK 9 *[SREBP] = sterol regulatory element-binding protein. 1. Goldstein JL, et al. Arterioscler Thromb Vasc Biol. 2009; 29: 431 -438. .

The Role of HDL in Reverse Cholesterol Transport Bile ABCG 1 &SR-B 1 Spheroidal HDL Liver LCAT UC LDL Receptor ABCA 1 Hepatic lipase, endothelial lipase SR-BI UC Pre-β HDL PL&UC SR-A CETP tion a xid O Macrophage VLDL/LDL ABCA 1, ATP-binding cassette protein A 1; CETP, cholesterol ester transfer protein; FC, free cholesterol; LCAT, lecithin: cholesterol acyltransferase; SR-A, scavenger receptor class A; SR-BI, scavenger receptor class B type I. Adapted with permission from Cuchel C et al. Arterioscler Thromb Vasc Biol. 2003; 23: 1710– 1712

Clinical Hypercholesterolaemia Condition Features Secondary Relatively uncommon, but potent (hypothyroidism, nephrotic syndrome, primary biliary cirrhosis) Polygenic Accounts for the majority of cases Familial Prevalent, Accelerates CVD. Hyperchol’aemia Due to defects in genes related to LDL-R Familial Combined H/L Overproduction of apo B lipoproteins TG and TC vary with age and weight Increased HDL ? OK if LDL not raised?

Therapy for Hypercholesterolaemia Intervention Features Diet Manage 2 o causes Plant sterols, avoid sat & trans FA Rarer, but potent: (Thyroid, liver, renal. ) First line for LDL reduction 2 nd line. Neutral for TG & HDL Statins Ezetimibe Niacin Bile acid resins Future Improves LDL, TG, HDL & Lp(a) Colesevalam better tolerated PCSK 9 Inhibitors, MTP inhibitors? Apo B antisense oligonucleotides

Triglyceride and Cholesterol: Why are they linked? • Most lipoproteins have TG and/or CE in their core • Hepatic Triglyceride rich lipoproteins are precursors of cholesterol-rich LDL • Cholesterol-ester transfer protein allows all triglyceride -rich lipoproteins to modify the composition of cholesterol-rich HDL and LDL. As a result, hypertriglyceridaemia is associated with reduced HDL cholersterol as well as “small dense LDL”. • The major gene regulators for lipid metabolism affect both TG and Chol •

Key Regulators of Genes in Fatty Acid and Triglyceride Metabolism Bile Acids SHP = Short Heterodimer Partner SHP SREBP-1 c LXR Acetyl Co. A Carboxylase Fatty Acid Synthase Spot 14 NEFA PPAR FXR Acyl Co. A Unsaturated Saturated HNF-4α L-FABP RXR Fatty acid metabolism Transport Oxidation Fatty Acid Binding Protein Ketogenesis Adapted from Pegorier JP et al. J Nutr 2004; 134: 2444 S-9 S Apolipoproteins Pyruvate kinase Glucose-6 -phosphalase Transferin Bile Acids

Key Regulators of Genes in Lipid Metabolism Sterol Regulatory Element Binding Protein (SREBP 2) Peroxisome proliferated activator receptors PPARs Synthesis Delivery Acquisition Cellular Cholesterol Homeostasis Excretion Hepatobiliary Farnesoid X Receptor (FXR) Intestine Liver X Receptor (LXR)

Link to mixed HL cases

Case MC Patient is a 43 year-old male with a strong family history of premature CVD who presents for initial evaluation. He has a 10 year history of dyslipidaemia and hypertension, for which he has received beta blockers in the past. More recently he has been on an ARB/diuretic combination. Three months prior to this visit a fasting lipid profile showed: Total Cholesterol: 5. 7 mmol/L HDL-C: 0. 7 mmol/L Triglyceride: 2. 8 mmol/L LDL-C: 3. 6 mmol/L He has managed to lose 3 kg and today results include: Total Cholesterol: 6. 9 mmol/L Triglyceride: 1. 8 mmol/L HDL -C: 0. 8 mmol/L LDL-C: 5. 2 mmol/L

Questions concerning Mr M. C. • 1) Is ethnicity an independent risk factor for CVD? Yes / No? • 2) In the absence of any symptoms or signs of hypothyroidism, would you perform thyroid function tests? Yes / No? • 3) His brother’s lipids include LDL = 5. 4 mmol/l, TG = 1. 9 mmol/l, HDL = 0. 9 mmol/l. What is the most likely cause of MC’s lipid abnormality? • • • A) B) C) D) E) Dyslipidaemia secondary to Insulin resistance and the Metabolic Syndrome Polygenc dyslipidamia Familial Combined Hyperlipidaemia Familial Hypercholesterolaemia Lipids aren’t really an issue in this patient

Patient is a 43 year-old male with a strong family history of premature CVD who presents for initial evaluation. He has a 10 year history of dyslipidaemia. Hypertension, for which he has received beta blockers in the past. More recently he has been on an ARB/diuretic combination. Three months prior to this visit a fasting lipid profile showed: TC: 5. 7 Triglyceride: 2. 8 mmol/L HDL-C: 0. 7 mmol/L LDL-C: 3. 6 mmol/L He has managed to lose 3 kg and today results include: TC: 6. 9 Triglyceride: 1. 8 mmol/L HDL-C: 0. 8 mmol/L LDL-C: 5. 2 mmol/L • 1) • 2) Is ethnicity an independent risk factor for CVD? Yes / No? In the absence of any symptoms or signs of hypothyroidism, would you perform thyroid function tests? Yes / No? His brother’s lipids include LDL = 5. 4 mmol/l, TG = 1. 9 mmol/l, HDL = 0. 9 mmol/l. What is the most likely cause of MC’s lipid abnormality? • 3) • • • A) B) C) D) E) Dyslipidaemia secondary to Insulin resistance and the Metabolic Syndrome Polygenc dyslipidamia Familial Combined Hyperlipidaemia Familial Hypercholesterolaemia Lipids aren’t really an issue in this patient

Is ethnicity an independent risk factor for CVD? Yes / No? • YES: • NO:

Is ethnicity an independent risk factor for CVD? Case for a qualified “Yes”. Same risk factors, different pattern INTERHEART, Karthikeyan et al 2009, INTERHEART, Joshi et al 2007

2) In the absence of any symptoms or signs of hypothyroidism, would you perform thyroid function tests? Yes / No? • Yes: • No:

2) In the absence of any symptoms or signs of hypothyroidism, would you perform thyroid function tests? The case for “Yes”

What is the most likely cause of MC’s lipid abnormality? • A) Dyslipidaemia secondary to Insulin resistance and the Metabolic Syndrome • B) Polygenc dyslipidamia • C) Familial Combined Hyperlipidaemia • D) Familial Hypercholesterolaemia • E) Lipids aren’t really an issue in this patient

What is the most likely cause of MC’s lipid abnormality? The case for “C”, maybe “A” or “B” Condition Features Secondary Relatively uncommon, but potent (hypothyroidism, nephrotic syndrome, primary biliary cirrhosis) Polygenic Accounts for the majority of cases Familial Prevalent, Accelerates CVD. Hyperchol’aemia Due to defects in genes related to LDL-R Familial Combined H/L Overproduction of apo B lipoproteins TG and TC vary with age and weight Increased HDL ? OK if LDL not raised?

Case MC (continued) Mr MC started statin, therapy, Atorvastatin 20 mg/ day, but unfortunately he had and inferior AMI still 4 months later. His discuharge medication include: Atorvastatin 20 mg, Metoprolol 20 mg, Aspirin 100 mg, and his previous ARB/diuretic. Follow-up 2 months later reveals: 2 kg weight loss, BP 118 / 78, Fasting tests: • Glu 5. 3 mmol/l, TC 4. 4 mmol/l TG 4. 2 mmol/l, • HDL 0. 7 mmol/l, LDL 1. 8 mmol/l

Case MC: Further questions: • Should you stop his beta blocker? Yes / No? • Do you trust the LDL-C result? Yes / No? • Is it practical to try to manage Mr M. C’s lipid profile to target levels? Yes / No? • What is the next lipid-lowering drug that you would add to his therapy? a) Ezetimibe b) Niacin c) I would increase Atorvastatin to 80 mg but I wouldn’t give anything other than a statin d) Fenofibrate e) Fish Oil

Mr MC started statin, therapy, Atorvastatin 20 mg/ day, but unfortunately he had and inferior AMI still 4 months later. His discuharge medication include: Atorvastatin 20 mg, Metoprolol 20 mg, Aspirin 100 mg, and his previous ARB/diuretic. Follow-up 2 months later reveals: 2 kg weight loss, BP 118 / 78, Fasting tests: • Glu 5. 3 mmol/l, TC 4. 4 mmol/l TG 4. 2 mmol/l, • HDL 0. 7 mmol/l, LDL 1. 8 mmol/l • Should you stop his beta blocker? Yes / No? • Do you trust the LDL-C result? Yes / No? • Is it practical to try to manage Mr M. C’s lipid profile to target levels? Yes / No? • What is the next lipid-lowering drug that you would add to his therapy? a) Ezetimibe b) Niacin c) Increase Atorvastatin to 80 mg but don’t give anything other than a statin d) Fenofibrate e) Fish Oil

Should you stop his beta blocker? • Yes • No

Should you stop his beta blocker? The case for “no” Some β-blockers decrease HDL and increase triglycerides. 25 In spite of this, BHAT data showed that propranolol improves survival after MI. 26 Low-dose metoprolol CR/XL alone or in combination with a statin resulted in significant slowing of the progression of carotid artery’s intima-media thickness over a 3 year period. 27 M Gheorghiade et al Circulation. 2002; 106: 394 -398

Do you trust the LDL-C result? • Yes • No

Do you trust the LDL-C result? The case for “No” Discussion of the effect of Cholesterol ester transfer protein will Explain why LDL-C underestimates risk when TG is elevated

Is it practical to try to manage Mr M. C’s lipid profile to target levels? • Yes • No

Is it practical to try to manage Mr M. C’s lipid profile to target levels? The case for “Yes” Combination therapy is safe and effective, but yet to be supported by clinical endpoint data.

What is the next lipid-lowering drug that you would add to his therapy? a) Ezetimibe b) Niacin c) I would increase Atorvastatin to 80 mg but I wouldn’t give anything other than a statin d) Fenofibrate e) Fish Oil

What is the next lipid-lowering drug that you would add to his therapy? The case for “d” or “b”, possibly “c” or “e” anticipate “residual risk” module

Case GS Patient is a 47 year-old female who has been gaining weight for several years. She has a 10 year history of mildly elevated triglyceride. She received therapeutic lifestyle counseling but she has been largely non-compliant. Three months prior to this visit, a fasting lipid profile showed: Total Cholesterol: 5. 5 mmol/L Triglyceride: 2. 4 mmol/L HDL-C: 1. 0 mmol/L LDL-C: 3. 6 mmol/L Now she has symptoms of hyperglycaemia and repeat fasting glucose confirms Type 2 diabetes

Case GS Review of Symptoms: Thirst, polyuria, folliculitis, Weight unchanged (increased 2 kg, then lost when polyuria commenced BP 118/72 Pulse 72 Wt 85 kg Ht 175 cm Waist 93 cm BMI 27. 8: Physical examination unremarkable Current fasting lipid results surprise you: Total Cholesterol: 8. 5 mmol/L Triglyceride: 7. 4 mmol/L HDL-C: 1. 0 mmol/L LDL-C: unable to be calculated

Questions: How could you obtain an LDL-C result? a) Friedewald equation b) Abusive phonecall to lab c) “Direct method” involving detergents d) Ultracentifugation e) Subtract HDL-C from Total cholesterol Which class or classes of lipoproteins would you expect to be increased? a) Chylomicrons and LDL b) VLDL and LDL c) VLDL and HDL d) IDL and chylmicron “remnants” e) Why bother? It doesn’t matter Which combination of extra tests would be most useful? a) LDL size+HDL subfractions b) Lipid EPG+Apo. E phenotype c) LDL subfractions HDL size d) Lp(a)+ homocysteine e) Routine fasting lipids are the only lipid tests that are ever required

She has a 10 year history of mildly elevated triglyceride. She received therapeutic lifestyle counseling but she has been largely non-compliant. Three months prior to this visit, a fasting lipid profile showed: Total Cholesterol: 5. 5 mmol/L Triglyceride: 2. 4 mmol/L HDL-C: 1. 0 mmol/L LDL-C: 3. 6 mmol/L Now she has symptoms of hyperglycaemia and repeat fasting glucose confirms Type 2 diabetes. Symptoms: Thirst, polyuria, folliculitis, Weight increased 2 kg, then lost when polyuria commenced BP 118/72 Pulse 72 Wt 85 kg Ht 175 cm Waist 93 cm BMI 27. 8: Physical examination unremarkable Current fasting lipid results surprise you: TC: 8. 5 mmol/L TG: 7. 4 mmol/L HDL-C: 1. 0 mmol/L LDL-C: unable to be calculated • How could you obtain an LDL-C result? • Which class or classes of lipoproteins would you expect to be increased? • Which combination of extra tests would be most useful?

How could you obtain an LDL-C result? a) Friedewald equation b) Abusive phonecall to lab c) “Direct method” involving detergents d) Ultracentifugation e) Subtract HDL-C from Total cholesterol

How could you obtain an LDL-C result? The case for “d”, but “c” is misleading Lab Tests Online: “Direct LDL-C is ordered whenever calculation of LDL cholesterol will not be accurate because the person's triglyceridesare significantly elevated. It may be ordered by a doctor when prior test results have indicated high triglycerides. In some laboratories, this direct LDL test will automatically be performed when the triglyceride levels are too high to calculate LDL-C. This saves the doctor time by not needing to order another test, saves the patient time by not needing to have a second blood sample drawn, and speeds up the time to provide the test result. ” Ultracentrifuge gives absolute result. Detergent methods assume LDL

Which class or classes of lipoproteins would you expect to be increased? a) Chylomicrons and LDL b) VLDL and LDL c) VLDL and HDL d) IDL and chylmicron “remnants” e) Why bother? It doesn’t matter

Which class or classes of lipoproteins would you expect to be increased? The case for “b”(orange) or “d” (green)

Which combination of extra tests would be most useful? • a) LDL size+HDL subfractions • b) Lipid EPG+Apo. E genotype/phenotype • c) LDL subfractions HDL size • d) Lp(a)+ homocysteine • e) Routine fasting lipids are the only lipid tests that are ever required

Which combination of extra tests would be most useful? The case for “b” CM β preβ α Apo. E isoforms

Case GS (continued) The patient subsequently complied with diet and started on Simvastatin 40 mg daily and other treatment, which she tolerates without difficulty. Current Medications: 1. Metformin 850 mg bid 2. Enalapril 10 mg q day 3. ASA 81 mg q day 4. Simvastatin 40 mg q day Subsequent results include: LEPG – Broad beta pattern present Apo E Genotype: Apo E 2: E 2

Questions This implies the accumulation of which lipoprotein class(es)? a) VLDL + LDL b) IDL and Chylomicron “remnants” Which lipid-lowering drug is the ideal treatment for this situation? a) Simvastatin b) Nicotinic Acid c) Questran d) Fibrate e) Fish oil f) Ezetimibe Which lipid-lowering therapy is strongly CONTRAindicated? Simvastatin b) Nicotinic Acid c) Questran d) Fibrate e) Fish oil f) Ezetimibe a) Would you stop his statin therapy? Yes / No Do you agree with the use of low-dose aspirin in this patient? Yes / No

The patient subsequently complied with diet and started on Simvastatin 40 mg daily and other treatment, which she tolerates without difficulty. Current Medications: 1. Metformin 850 mg bid 2. Enalapril 10 mg q day 3. ASA 81 mg q day 4. Simvastatin 40 mg q day Subsequent results include: LEPG – Broad beta pattern present Apo E Genotype: Apo E 2: E 2 This implies the accumulation of which lipoprotein class(es)? Which lipid-lowering drug is the ideal treatment for this situation? Which lipid-lowering therapy is strongly CONTRAindicated? Would you stop his statin therapy? Do you agree with the use of low-dose aspirin in this patient? The patient asks you about his risk of Alzheimers’ Disease. Is it increased?

This implies the accumulation of which lipoprotein class(es)? a) VLDL + LDL b) IDL and Chylomicron “remnants”

This implies the accumulation of which lipoprotein class(es)? The case for “b”

Which lipid-lowering drug is the ideal treatment for this situation? a) Simvastatin b) Nicotinic Acid c) Questran d) Fibrate e) Fish oil f) Ezetimibe

Which lipid-lowering drug is the ideal treatment for this situation? The case for “d”

Which lipid-lowering therapy is strongly CONTRAindicated? a) Simvastatin b) Nicotinic Acid c) Questran d) Fibrate e) Fish oil f) Ezetimibe

Which lipid-lowering therapy is strongly CONTRAindicated? The case for “c” Hepatocyte Heterodimerization with RXR Acetyl Co. A SREPB-1 c Bile Duct FA, TG MDRP 2/ 3 Phospholipids FXR VLDL (TG levels)

Do you agree with the aspirin dose? Comment on the role of aspirin in this patient. • Yes • No

Do you agree with the aspirin dose? Comment on the role of aspirin in this patient. Evidence and opinion tending towards “no”?