Biochemistry, Lipoprotein Metabolism

Fundamentals

Lipoproteins are complex molecules that involve several different components. They contain a central core made of triglycerides and cholesterol esters.[1] Fatty acids released from triglycerides can be used for energy storage or production, and cholesterol is critical for steroid synthesis, cellular membrane formation, and bile acid production. Surrounding this core is a mix of phospholipids, free cholesterol, and apolipoproteins (apo). The apolipoproteins are particularly important, as they play a role in classifying lipoproteins into 5 main classes: chylomicrons, very-low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL). They provide structure and function in lipid metabolism. The differentiation between these classes depends on the size of the molecule, its lipid content, and the type of apolipoprotein it features. HDL, colloquially known as “good cholesterol,” participates in reverse cholesterol transport, while LDL, colloquially known as “bad cholesterol,” promotes atherosclerosis.

Issues of Concern

An impairment in lipoprotein metabolism could lead to catastrophic implications in an affected individual. A pathologic increase in LDL, for example, is a known risk factor in cardiovascular disease as it leads to premature atherosclerotic changes of vessels. Disorders of lipoproteins have both genetic and environmental underpinnings. In Western countries specifically, lifestyle has been identified as an insidious precipitant of these disorders. Studies, campaigns, and public health interventions are underway to encourage positive lifestyle changes to prevent lipoprotein disorders from becoming prevalent.

Molecular Level

Cholesterol synthesis begins with acetyl-CoA, which is derived from amino acids, fats, and carbohydrates. Thereafter, a series of enzymatic reactions occurs, divided into 4 main steps, each with unique products. The process starts with mevalonate. Second, it transforms into activated isoprenes. The third step is where squalene is produced, and the 4th and last step is where cholesterol forms. 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) catalyzes the formation of mevalonate, a critical enzyme reaction in cholesterol biosynthesis and the rate-limiting step. HMG-CoA reductase is also the target of statins to lower high LDL-cholesterol.

Acetyl-CoA can also be converted to triglycerides, which ultimately form lipoproteins. Each lipoprotein has a unique composition with different apolipoproteins on its outer surface. Chylomicrons, which are of intestinal origin, are very large and carry dietary lipids. They are associated with the following apolipoproteins: AI, AII, AIV, B48, CI, CII, CIII, and E. VLDL are particles that carry endogenous triglycerides and some cholesterol from the liver. It is associated with B100, CI, CII, CIII, and E apolipoproteins. IDL carries cholesterol esters and triglycerides and carries apolipoproteins B100, CIII, and E. LDL carries cholesterol esters and is associated with B100. HDL carries cholesterol esters and is associated with apolipoproteins AI, AII, CI, CII, CIII, D, and E.

Function

Lipids are 1 of the 4 main biological molecules of the human body, along with carbohydrates, proteins, and nucleic acids. Lipids are essential components of life on a cellular level. They are involved in multiple processes, such as storing energy, serving as chemical messengers, forming cell membranes, and transporting fat-soluble vitamins such as vitamin E. For lipids to carry out these roles in the cell, they must travel to their destination cells after being absorbed in the gastrointestinal tract. Without lipoproteins, this transport would not be possible, as the hydrophilic environment of blood is not compatible with the hydrophobic nature of lipids such as cholesterol. Therefore, lipoproteins play an integral role in the human body's ability to utilize lipids, and the metabolism of these lipoproteins directly affects lipid levels in the serum and subsequent lipid processes within cells.

Mechanism

Lipid metabolism can be divided into 2 main pathways: exogenous and endogenous. Lipids can be derived from exogenous sources, such as the diet, or from endogenous sources, such as liver synthesis. Lipids found in the diet are packaged as chylomicrons in the small intestine and carried as triglycerides in the molecule's hydrophobic core. In the intestinal cell, free fatty acids react with glycerol to produce triglycerides. Cholesterol is also esterified by acyl-CoA: cholesterol acyltransferase (ACAT). ACAT’s role in lipid metabolism first became established in animal models with ACAT deficiency. These animals were resistant to diet-induced hypercholesterolemia due to their inability to esterify cholesterol and reduced cholesterol absorption.[2] However, ACAT inhibitors were not shown to treat atherosclerosis and may have potentially harmful effects on disease progression.[3] Chylomicrons travel through the lymphatic system to the subclavian vein and ultimately travel throughout the body, delivering triglycerides where necessary.

The Neiman-Pick C1-like-1 protein (NPC1L1) is a human sterol transport protein expressed at the enterocyte/gut lumen and hepatobiliary interface. This protein has a sterol-sensing domain that facilitates the internalization of free cholesterol into the enterocyte. When extracellular cholesterol is high, cholesterol is incorporated into the cell membrane and sensed by NPC1L1, which internalizes it via interaction with the AP2-clathrin complex. The lipid-lowering medication ezetimibe prevents this interaction, thereby reducing plasma LDL-cholesterol levels.[4]

Triglycerides get packaged into the chylomicron through their interaction with the apoB48, the backbone apolipoprotein. ApoC-II and E are acquired from HDL as the chylomicron circulates. A cholesteryl ester transfer protein (CETP) promotes the transfer of cholesteryl esters from HDL to apoB-containing lipoproteins, including VLDL, VLDL remnant, IDL, and LDL in exchange for triglycerides. As a result, HDL cholesterol decreases, and cholesterol content in VLDL increases.[5] Subsequently, it is returned to the liver and endocytosed by hepatocytes via scavenger receptors. HDL can then be excreted or recycled by the Golgi.[6] Apo-B-48 allows lipid binding to the chylomicron from circulation before lipoprotein lipase (LPL) hydrolyzes the core triglycerides and releases fatty acids. ApoC-II is a cofactor for LPL, making chylomicrons smaller with each reaction. As the chylomicron becomes metabolized, the chylomicron remnant forms and is ultimately cleared by hepatic chylomicron remnant receptors. The apoE on the chylomicron remnant acts as a high-affinity ligand to signal the hepatic chylomicron remnant receptor. The surface constituents from the chylomicron remnant get transferred to form HDL. When LPL is deficient, termed chylomicronemia, patients can have severely elevated triglyceride levels exceeding 2000mg/dl, potentially leading to pancreatitis.[7]

In the endogenous pathway, lipid metabolism begins with VLDL synthesis, which produces a triglyceride-rich core containing cholesterol esters. On the surface of VLDL are apo B-100, C-II, and E. Microsomal triglyceride transfer protein (MTP), an intracellular lipid transfer protein in the endoplasmic reticulum, is critical for VLDL lipidation. If the MTP is not functional, VLDL does not get secreted into the circulation. In nascent VLDL, the triglyceride core is metabolized in the muscle and adipose tissue, releasing fatty acids through its interaction with LPL, which is activated by apoCII. LPL catalyzes the hydrolysis of triglycerides into fatty acids for absorption. Insulin increases the regulation of apoCII. Once the VLDL core is reduced, a remnant particle called IDL is formed. This particle is depleted of triglycerides by a process similar to that of chylomicron remnant clearance.[8] The IDL picks up cholesterol esters from HDL by CETP. Ultimately, IDL and HDL cholesterol form LDL through hepatic lipase-mediated interaction. LDL can transport cholesterol to tissues and is eventually recycled back to the liver, where apoB100 mediates endocytosis of LDL by binding to the apoB-100 receptor or LDL receptor on tissues and hepatic cells. A rare gain-of-function mutation in the serine protease PCSK9 can cause functional LDL receptor deficiency by targeting the receptor for lysosomal degradation, leading to a severe increase in LDL-C levels and premature ASCVD.

Ultimately, LDL can be recycled in the liver cells’ Golgi apparatus to make more lipoproteins, or it can be excreted through bile. The “empty” HDL can enter the circulation to pick up excess cholesterol from the tissues. ABCA1 mediates this transfer, and mutations in the ABCA1 gene are linked to low serum HDL cholesterol and familial HDL deficiency, Tangier disease. HDL can also acquire cholesterol from cells via SR-B1 or passive diffusion and directly transport cholesterol to the liver by interacting with hepatic SR-B1 or indirectly by transferring cholesterol to VLDL or LDL, facilitated by CETP. Lecithin cholesterol acyltransferase (LCAT), which is activated by apoA1 on HDL, mediates the esterification of cholesterol in HDL. Other ABC transporters may carry implications in cholesterol metabolism. Sitosterolemia is a rare genetic disorder characterized by elevated plasma levels of plant sterols, including sitosterol, and premature atherosclerosis. Mutations in the ABC half-transporters ABCG5 and ABCG8 can increase intestinal uptake of plant sterols because they fail to efflux them into the lumen. Overexpression of ABCG5 and ABCG8 increases biliary cholesterol secretion, suggesting that they may regulate it.[9]

Testing

Screening for dyslipidemia is through a routine test performed during health maintenance visits, most often by primary care physicians. Routine testing generally includes total cholesterol, triglycerides, LDL-C, and HDL-C, with desirable values for adults being below 200 mg/dL, under 150 mg/dL, less than 100 mg/dL, and greater than 60 mg/dL, respectively. Plasma or serum measurements of lipoprotein levels are useful. Plasma is typically collected in ethylenediaminetetraacetic acid (EDTA), which allows rapid cooling to prevent lipid peroxidation and inhibit bacterial enzymes. Plasma cholesterol and triglyceride values are about 3% lower than in serum. At least 2 lipid assessments, ideally 2 weeks apart, are performed before a diagnosis of dyslipidemia is made. The recommendation is that lipid screening takes place in males aged 25 to 30 years and females aged 30 to 35 years for patients with high cardiovascular risk. Patients with lower cardiovascular risk should have lipid screening at 35 years in males and 45 years in females. Screenings are generally repeated every 5 years. Patients typically fast for 10 to 12 hours before testing, avoid alcohol the evening before sampling, and wait for 2 to 3 weeks after a major illness, surgery, or trauma.[10] However, there is significant traction for screening with a non-fasting sample, with confirmation, if needed, with a fasting sample.

Pathophysiology

LDL-cholesterol is a leading cause of atherosclerotic cardiovascular disease (ASCVD); this involves many processes that ultimately result in ASCVD. LDL accumulates in circulating macrophages following modifications, such as oxidation. Oxidized LDL acts as a chemoattractant for monocytes, which then differentiate into macrophages. These macrophages become trapped in the vessel wall, likely due to abnormal endothelial leukocyte adhesion molecule-1, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1.[11] The LDL particle promotes an inflammatory response, leading to increased cytokine and antibody levels. The modified LDL in macrophages, now called foam cells, has the potential to rupture, releasing oxidized LDL, enzymes, and free radicals that can further damage the vessel wall.[12] Additionally, the modified LDL impairs nitric oxide release due to dysfunctional endothelial function. This process reduces the vessel’s ability to vasodilate appropriately. Lastly, oxidized LDL causes increased platelet aggregation and thromboxane release. The net result is that platelet activity and aggregation become enhanced.[13]

Triglyceride-rich HDL also carries implications for ASCVD, as it reduces macrophage efflux capacity, promotes monocyte infiltration into the arterial wall, and increases expression of pro-inflammatory genes. Apolipoprotein C-III, which inhibits LPL, may also promote atherosclerosis by resulting in elevated triglyceride-rich lipoproteins. Studies have found that lower C-III is associated with lower triglyceride levels and reduced risk for ASCVD.[14] IDL may also be a predictive measurement of ASCVD, and in patients with normal total cholesterol but an elevated IDL/HDL ratio, there is a significantly increased risk for ASCVD.[15]

Apo B-100 is directly related to the LDL particle number. When ApoB-100 lipoproteins are elevated, the risk of atherosclerosis increases, even in the absence of other risk factors. Some propose that atherogenesis is due to the subendothelial retention of apo B-100 by proteoglycans in the extracellular matrix.[16]

Clinical Significance

The clinical definition of dyslipidemia is total cholesterol, LDL, or triglyceride levels above the 90th percentile or HDL levels below the 10percentile for the general population. Dyslipidemia, especially elevated LDL-C, is strongly associated with ASCVD, and the prevalence is in the 75 to 85% compared to 40 to 48% of age-matched controls.[1] In some cases, dyslipidemia is familial, particularly when the disease's onset occurs earlier in age. However, lifestyle, mainly obesity and a high saturated fat diet, significantly increases dyslipidemia rates. The monogenic disorder familial hypercholesterolemia is primarily due to mutations in the LDL receptor gene, leading to LDL receptor deficiency and elevated LDL-C.

ASCVD is among the leading causes of death in the United States. Hyperlipidemia is a significant risk factor for the development of ASCVD, and medications that target lipoproteins and their constituents are critical in disease management and prevention. Recently, the American Heart Association/American College of Cardiology published new guidelines for managing cholesterol levels. As primary prevention of ASCVD, a healthy lifestyle, including weight loss, diet, smoking cessation, and aerobic activity, is emphasized. Among the lipid-lowering medications, statins are by far the most commonly prescribed and utilized. High-intensity statins are indicated for persons aged 20 to 75 years with LDL cholesterol over 190 mg/dL. Type 2 diabetics, who are 40 to 75 years old, should begin moderate-intensity statins unless they have additional risk factors.[17] 

Statins have an indirect effect on lipoprotein metabolism. The mechanism of action involves competitive inhibition of HMG-CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis in the liver. As the liver senses a decrease in cholesterol production, it attempts to compensate by increasing the number of LDL receptors on its cells, leading to increased uptake of LDL and VLDL into the liver, which then metabolizes them into cholesterol and other molecules. Statins effectively reduce circulating LDL and VLDL levels, thereby decreasing cholesterol deposition in tissues.[18] Other medications include ezetimibe and PCSK9 inhibitors, in addition to statins, to lower LDL-C by more than 50%, especially in high-risk patients. Newer drugs targeting cholesterol and lipoprotein metabolism are critical for managing or preventing ASCVD.