Lipids and Lipoproteins

Classes of lipids:

• Fat-soluble vitamins
• Cholesterol
• Simple lipids: esters of fatty acids with alcohols
  • Triglycerides: fatty acids with glycerol
  • Fatty acids
  • Glycerol
• Complex or conjugated lipids 
  • Phospholipids and glycolipids: esters of fatty acids with alcohols and molecules with other groups (e.g.)

Lipid chemistry

Lipids are organic substances that are insoluble in water, soluble in organic solvents

Roles of lipids (fats): a rich source of energy & efficient way for the body to store calories; integral components of cell membranes and structure

Fatty acids: chains of carbon-hydrogen bonds terminating in carboxyl groups that are utilized for energy

Triglycerides: 3 fatty acid molecules attached to 1 molecule of glycerol by ester bonds; used to store energy

Key players

• Insulin is important in regulating fatty acid synthesis and storage; after meals it stimulates: Lipogenesis, Glycolysis, Pyruvate dehydrogenase to produce acetyl-CoA (stimulating the TCA cycle and increasing citrate levels)
• During fasting and starvation, insulin levels decrease
• Glucagon activates gluconeogenesis (liver) and causes lipid mobilization from fat stores (lipolysis)
  • Results in ↑ free fatty acids (energy) and ↑ glycerol (glucose backbone) in the blood
• Cortisol exerts a chronic effect on lipolysis and causes insulin resistance
• Cushing’s syndrome: excess of cortisol results in hyperglycemia, muscle wastage, and redistribution of fat to cheeks and upper back

Lipoproteins

• Lipids are insoluble (hydrophobic) in water and in order to be transported in the body, they must be made soluble
• Lipids are incorporated into lipid-protein complexes called lipoproteins for transportation throughout the body
• Lipoproteins distribute cholesterol esters and triacylglycerol or TAG (esters of glycerol and fatty acids, synonymously called triglycerides)
• Abnormalities of lipoprotein metabolism are key in atherosclerosis, which is the build-up of plaque on arterial walls
• Cause of coronary heart disease, stroke, and peripheral vascular disease
• Atherosclerosis related cardiovascular disease (CVD) is the number one cause of death in the world

Lipoproteins: used in the diagnosis of dyslipidemia, related to coronary heart disease (CHD)

Chylomicrons = transport exogenous triglycerides to the liver

VLDLs = transport endogenous TAGs to cells for energy and storage as fat

LDLs = transport cholesterol into the cells and liver after delivering the main cargo of TAGs

HDL = transports cholesterol out of cells (i.e. heart)

Fatty acids

Fatty acids that contain no carbon-carbon double bonds are termed saturated fatty acids 

- no bents, very rigid -

Those that contain double bonds are unsaturated fatty acids

Saturated fat (animal fat e.g. cheese, butter) raises total cholesterol by raising LDL

Unsaturated fat (olive oil) increases your HDL

- Most unsaturated are in the cis conformation -

Trans fat: hydrogenated fatty acids

Very bad for you; raises total cholesterol by ↑ LDL, and lowers HDL

Esterification of fatty acids

Esterification of fatty acids with glycerol forms a triacylglycerol (TAG)

Most fatty acids (FA) are in the esterified form

All fatty acid esters (TAG) are transported by lipoproteins

Free fatty acids circulate bound to albumin

When energy is needed TAGs can be degraded to glycerol and free fatty acids

Triglycerides (TAG): storage and transport form of fat (fatty acids)

Fatty acid oxidation

Fatty acids are a major source of energy in the body

β-oxidation is done in the mitochondrion

β-oxidation results in acetyl-coenzyme A (acetyl-CoA), and reduced forms of the nucleotides FADH2 and NADH

Normally, the acetyl-CoA is used in the TCA cycle

During fasting, a low carbohydrate diet, and starvation, acetyl-CoA is used in ketogenesis

Ketogenesis

During fasting and starvation, insulin levels decrease

Glucagon levels increase and stimulate lipolysis for gluconeogenesis

Ketogenesis: ketone bodies are made from fat-derived acetyl-CoA in the liver as an energy source for the body

Occurs during low blood glucose levels in healthy people (e.g. fasting, low carbohydrate diet, and starvation)

It also occurs when insulin levels are extremely low as seen in type 1 diabetes; no insulin results in elevated lipolysis - more glucagon

If the amount of acetyl-CoA made from fat overwhelms the TCA cycle then ketogenesis occurs; during ketogenesis, 1 of 3 ketone bodies are made: acetoacetate, β-hydroxybutyrate, and acetone

Oxidation of ketone bodies: most tissues, even the brain, can use ketones as an energy source

Almost all ketogenesis occurs in the liver, but the liver cannot use ketones as an energy source and exports them into the blood

Biosynthesis and storage of fatty acids

• Lipogenesis occurs during excess energy intake in the form of carbohydrates
• Insulin promotes lipogenesis and inhibits lipolysis
• Excess carbohydrates are converted to fatty acids in the liver via the TCA cycle > esterified into TAGs > transported by VLDL to adipocytes
• The liver cannot store TAGs

Phospholipids

Phospholipids: contain 2 fatty acids, alcohol (glycerol), and a phosphate group

Phospholipids are the major lipids in biological membranes

Types of head groups: choline, inositol, serine, ethanolamine, all of which are hydrophilic in nature

Sphingomyelin is found primarily in cell membranes in the myelin sheath (nervous system)

Lecithin is found in bile acids and cell membranes

Cholesterol

Cholesterol consists of 4 sterol rings

The major site of cholesterol synthesis is in the liver

Cholesterol synthesis requires a source of carbons (acetyl–CoA)

HMG-CoA Reductase (Hydroxymethyl glutaryl CoA) – the major enzyme that regulates endogenous cholesterol synthesis 

the major target of statin drugs - HMG-CoA Reductase 

Precursor to all steroid hormones - Cholesterol

Excess cholesterol is excreted by the liver in bile acids (emulsification of dietary fats)

The majority of cholesterol is transported by LDL

Regulation of cholesterol synthesis

High free cholesterol causes:

• A reduction in HMG-CoA reductase (limiting synthesis)
• Downregulation (# of receptors) of LDL receptors (limits the uptake of cholesterol)
• Increase in the transfer of cholesterol from cell to apolipoproteins A (i.e. HDL)
• Increased production of bile acids (increases excretion of cholesterol)

Lipoprotein structure

Lipoproteins contain lipids and protein

Lipids include triacylglycerol and cholesteryl esters surrounded by polar phospholipids and protein

The apolipoprotein is the protein portion of a lipoprotein 

The inner portion is triacylglycerol and cholesterol esters, and is hydrophobic

The outer portion consists of phospholipids and free cholesterol together with hydrophilic apolipoproteins (protein)

These lipid-protein complexes vary in their content of lipid and protein

All lipoproteins: spheres composed of lipids & proteins; deliver fuel to peripheral cells

Chylomicrons: largest & least dense

produced by the intestines and deliver dietary lipids to hepatic & peripheral cells - chylomicrons

Chylomicrons appear in serum after a fatty meal giving it a milky appearance (do not appear in fasting specimens)

Very-Low-Density Lipoproteins (VLDL): Produced by the liver; carries endogenous triglycerides

Intermediate-Density Lipoproteins (IDL): VLDL remnants; transient

Low-Density Lipoproteins (LDL): major transporter of cholesterol (heart) 

LDL is formed from VLDL

Significantly smaller than VLDLs; atherogenic

High-Density Lipoproteins (HDL): Smallest & most dense; synthesized by the liver

Removes excess cholesterol from peripheral cells (heart)

Pathway of lipoprotein metabolism

Chylomicrons transport exogenous (dietary) TAGs from the intestines to the liver

VLDL transports endogenous TAGs from the liver to peripheral adipose tissue

IDL (VLDL remnant) forms after VLDL delivers TAGs to cells [atherogenic]

LDL (forms from IDL after removing remaining TAG content): transports cholesterol to cells (e.g. heart) [atherogenic]

HDL transports cholesterol out of cells (e.g. heart) and to the liver [anti-atherogenic]

Lipoprotein size is directly proportional to TAG content

Density is inversely proportional to TAG content

Apolipoproteins

Apolipoprotein: the protein portion of the lipoprotein found on the surface

Apolipoproteins A (AI and AII) are present in HDL and are a marker for HDL

Apolipoprotein B exists in two forms: apoB100 and apoB48

apoB100 controls metabolism for LDL, VLDL

apoB48 is present in chylomicrons

Apolipoprotein E: present in all lipoprotein classes

Apolipoprotein (a) is a component of lipoprotein (a) (Lp(a))

Elevated Lp(a) is a risk factor for cardiovascular disease

Apolipoproteins predict the risk of CVD better than lipid testing (total cholesterol, LDL, and HDL), but old research guidelines are based on the lipid panel

Lipid and lipoprotein population distributions

Women have, on average, higher HDL cholesterol levels but lower total cholesterol & triglyceride levels than men

After menopause, there is no difference in total cholesterol; total & LDL cholesterol & triglyceride levels all increase with age, in both men & women

Total & LDL cholesterol & triglycerides are much lower in young children than in adults

At puberty, the HDL cholesterol levels in young men drop to 20% to adult male levels but does not change in women

Atherosclerosis

Atherosclerosis is a type of arteriosclerosis, the deposition of plaque (build-up of fats and cholesterol) on the arterial wall

Plaque can narrow an artery enough to slow or block blood flow

The development of atherosclerotic plaque is termed atherogenesis; this is termed cardiovascular disease (CVD) or coronary heart disease (CHD)

Inflammation is fundamental to atherogenesis

After initial damage of the endothelium, inflammatory chemokines call in monocytes, which convert to macrophages

Macrophages scavenge remnants and LDL that have penetrated the arterial wall and become overloaded with lipids

Dying foam cells (overloaded macrophages) turn into fatty streaks (plaque), changing the structure of the cell wall

Lipid disorders (dyslipidemias)

Arteriosclerosis: the single leading cause of death & disability in the U.S.

Cholesterol is deposited in artery walls, plaque (fatty streaks) forms when foam cells (macrophages) die

Hyperlipoproteinemia: diseases associated with elevated lipoprotein levels; includes hypercholesterolemia, hypertriglyceridemia, & combined hyperlipidemia

Lipid disorders (dyslipidemias)

Hypercholesterolemia: Most common lipid abnormality most closely linked to heart disease due to health behavior (elevated levels of LDL or apo-B100)

high cholesterol = high LDL

Familial hypercholesterolemia (FH): genetic abnormality predisposing people to elevated cholesterol levels

Hypertriglyceridemia: common among alcoholics; increases risk for pancreatitis; also seen in renal failure and diabetes

HyperTAG - Alcoholics, pancreatitis

Combined Hyperlipoproteinemia (CH): Elevated levels of serum total cholesterol & triglycerides - Increased risk for coronary heart disease (CHD)

Combined - Total cholesterol and TAG

Familial dysbetalipoproteinemia: increased apo B synthesis results in ↑ VLDL and LDL

Lp(a) Elevation: increased risk of CHD & cerebrovascular disease

Hypolipoproteinemia: low levels of lipoproteins 

hypoalphalipoproteinemia & hypobetalipoproteinemia; Alpha and beta denote regions in which HDL migrates on agarose electrophoresis

Familial hypobetalipoproteinemia results in low levels of LDL or apo B in the blood

Tangier disease or familial hypoalphalipoproteinemia: Genetic, linked to increased risk of premature CHD

Secondary dyslipidemias

Diabetics have an increased risk of CVD due to combined hyperlipidemia

Diabetic dyslipidemia: decreased levels of HDL and increased levels of TAG (VLDL), LDL, and total cholesterol

⬆ VLDL (TAGs), LDL; ⬇ HDL = unhealthy

Alcohol abuse causes increased VLDL (triglycerides) blood levels but also increases HDL levels along with LDL and total cholesterol

alcoholism = all high

All chronic renal disease patients present with secondary dyslipidemia (nephrotic syndrome); mimics the laboratory results of diabetic dyslipidemia

Non-HDL cholesterol

Reflects total cholesterol minus HDL-C:

LDL, VLDL, IDL, Lp(a)

Elevated non-HDL-C associated with increased risk of CVD

Lipids, lipoproteins, and triglycerides are important indicators of cardiovascular disease (CVD) or coronary heart disease (CHD)

National Cholesterol Education Programs (NCEP) recommends that adults over 20 have a fasting “lipid” or “lipoprotein” panel performed at least once every 5 years

Lipid panel reference ranges (borderline)

Total Cholesterol: 220 ± 20 mg/dL

HDL Cholesterol:  50 ± 10 mg/dL - high is desirable -

LDL Cholesterol: 130 ± 30 mg/dL

TAGs: 175 ± 25 mg/dL

Cardiovascular risk factors

• Male sex, risk equalizes in postmenopausal women
• Age
• Smoking
• High plasma total cholesterol (high LDL-cholesterol)
• Low plasma HDL-cholesterol
• Diabetes Mellitus: CVD is the main cause of death in diabetics

Lipid and lipoprotein analysis

• Total cholesterol measurement: hexane extraction, followed by reaction with Liebermann-Burchard color reagent
• Enzymatic method (cholesterol oxidase) has replaced the colorimetric method in measuring cholesterol

Cholesteryl esterase: Cholesteryl ester + H2O > Cholesterol + FAs

Cholesterol oxidase: Cholesterol + O2 > Cholestenone + H2O2

Peroxidase: H2O2 + Dye > Color

• Triglyceride measurement: enzymatic method involves the liberation of glycerol by lipase
• Glycerol contamination from the stoppers of evacuation tubes or ingestion of glycerol coated medication can cause falsely elevated results

Bacterial lipase: TAG + H2O > FAs + Glycerol

Glycerolkinase: Glycerol + ATP > Glycerophosphate + ADP

Glycerophosphate oxidase: Glycerophosphate + O2 > Dihydroxyacetone + H2O2

Peroxidase: H2O2 + Dye > Color

Lipoprotein methods:

Electrophoresis – separate based on size and charge

Ultracentrifugation reference method separates based on density or rates of flotation

HDL direct or homogenous assay methods: measure HDL without pretreatment or separation

HDL indirect assay methods: pretreat with an antibody to apo-B to remove and precipitate LDL, VLDL, and chylomicrons and analyze supernatant for HDL

remove other lipoproteins to test for HDL indirectly

LDL Methods

Beta-quantification: most common; combines ultracentrifugation & chemical precipitation

Friedewald calculation: bypasses centrifugation; commonly used in routine labs

Compact analyzers: mobile point-of-care testing systems

can measure cholesterol, triglycerides, HDL cholesterol, & glucose from a finger stick sample

Lipid panel testing

Lipid panel includes: total cholesterol, LDL, HDL, triglycerides

10-12 hour fast is required; non-fasting will cause increased triglycerides

Serum is the specimen of choice for lipid panel

Triglycerides, HDL, and total cholesterol are measured on an analyzer

LDL is commonly calculated: the Friedewald calculation involves direct measurement of fasting total cholesterol, triglycerides, and HDL cholesterol

Friedewald equation for LDL

VLDL cholesterol is estimated by dividing TAG by 5

Total cholesterol = LDL + HDL + VLDL

LDL = total cholesterol – (HDL + VLDL)

Friedewald calculation of LDL cholesterol is only considered valid in patients with fasting triglyceride concentrations less than 400 mg/dL