Biochemistry, Low Density Lipoprotein

Fundamentals

The density of the lipoproteins is directly proportional to the protein content. Chylomicrons mobilize dietary lipids from the intestine to other tissues. They are the largest in size among the lipoproteins, are the least dense, and comprise about 80% of triacylglycerols. They are assembled in the enterocytes and then enter the systemic circulation via lacteals. The apolipoproteins of chylomicrons include apoB-48, apoE, and apoC-II. Chylomicrons carry dietary fatty acids to various tissues, where they are used for energy or stored. The remnants of chylomicrons containing cholesterol, apoE, and apoB-48, are cleared by the liver via remnant receptors.[5][6][7]

When there are excess fatty acids and cholesterol, they are converted to triacylglycerols and cholesteryl esters, respectively, in the liver and packaged with apolipoproteins into VLDL. Excess carbohydrates can also be converted into triacylglycerols and transported as VLDL. VLDL also contains apoB-100, apoC-I, apoC-II, apoC-III, and apoE. VLDL is transported from the liver to various tissues via capillaries. In the tissues, lipoprotein lipase (LPL), activated by apoC-II, catalyzes the release of free fatty acids from the triacylglycerols present in the VLDL, like with chylomicrons. ApoC-III inhibits LPL. The free fatty acids are taken up by the adipose tissues, where they are stored as triacylglycerols. After the partial removal of the triacylglycerol, VLDL remnants (IDL) are formed. Further removal of triacylglycerol yields LDL, the end product of VLDL metabolism. LDL contains predominantly cholesteryl esters and apoB-100. LDL carries cholesterol to various tissues, including the adrenal glands, gonads, muscle, and adipose tissue. All these tissues have LDL receptors on their plasma membranes that recognize apoB-100. The LDL particle is taken up by receptor-mediated endocytosis, as classically described by Goldstein and Brown. 

In cells, free cholesterol regulates HMG-CoA reductase, the rate-limiting step in cholesterol biosynthesis. Also, excess cholesterol is esterified and stored in the cell. The expression of the LDL receptor is finely regulated by intracellular cholesterol levels to prevent excessive cholesterol deposition. Macrophages also take up modified lipoproteins, becoming foam cells. However, in macrophages, uptake occurs via scavenger receptors, which, unlike the LDL receptor, are not under feedback control by cellular cholesterol. LDL not taken up by cells and tissues returns to the liver via LDL receptors on hepatocyte membranes. In the liver, cholesterol may be converted to bile acids or neutral sterols or re-esterified and stored. 

LDL particles are cleared from the serum through interaction with the LDL receptor. The outer membrane of LDL contains a phospholipid monolayer, unesterified cholesterol, and Apo-B protein, whereas the inner core contains a highly nonpolar core enriched in cholesterol esters. The cholesterol is transported via blood and endocytosed in the target tissues via LDL receptor-mediated endocytosis. When LDL binds to its receptor in plasma membranes, small invaginations called caveolae, composed of caveolin and receptor proteins, form and are endocytosed via clathrin-coated pits and vesicles. Once endocytosed, the LDL receptor recycles to coated pits, while cholesterol is transported to lysosomes, where hydrolases degrade the apo-B protein into amino acids and cleave cholesterol esters into cholesterol and fatty acids. Cholesterol is either incorporated into the cell membrane or reesterified and stored as lipid droplets by ACAT. When mutations are present in the LDL receptor, normal uptake of LDL by the liver is impaired, leading to familial hypercholesterolemia with markedly elevated LDL cholesterol levels, an autosomal-dominant disorder. 

Numerous studies have shown a positive link between LDL-cholesterol and the composition of the circulating LDL and the risk for atherosclerotic cardiovascular diseases (ASCVD). The large buoyant LDL (lbLDL) comprises LDL1 (large) and LDL II (intermediate), whereas the small dense LDL (sdLDL) comprises LDL III (small) and LDL IV (very small). The half-life of small dense (sdLDL) is greater than that of large buoyant LDL (lbLDL). The elevated levels of sdLDL have been considered a risk factor for ASCVD. Also, the increased susceptibility of sdLDL to oxidation could be due to its lipid composition and decreased antioxidant moieties. sLDL has been shown to have less sialic acid content than buoyant LDL. Desialylation of sdLDL may result in a higher affinity to proteoglycans present in the arterial wall. Hence, LDL trapped by proteoglycans in the sub-endothelial space leads to the formation of atherosclerotic plaque. ApoB lipoprotein present in sdLDL is more prone to glycation compared to the one present in lbLDL.

Testing

Since most of the cholesterol in serum is transported by LDL, measuring serum LDL levels could be useful for predicting ASCVD risk. According to the NIH-sponsored National Cholesterol Education Program (NCEP) Adult Treatment Panel III (ATPIII) guidelines, serum levels of both total cholesterol and LDL-cholesterol (LDL-C) are useful for predicting the risk of ASCVD. The therapy is also targeted to lower LDL cholesterol to a target value (less than 100 mg/L).[8][9][10][11][12]

Ultracentrifugation can be used to separate lipoproteins using equilibrium and rate methods. When plasma or serum is ultracentrifuged in native non-protein solute density (1.006 g/ml), triacylglycerol-rich VLDL and chylomicrons can be floated, which can be recovered by aspiration. The density of the bottom fraction can be adjusted to 1.063 g/ml with potassium bromide, and ultracentrifugation leads to the flotation of LDL, or, more commonly, the total cholesterol and HDL-cholesterol obtained in the infranatant are assayed to calculate LDL-cholesterol. This method is tedious and time-consuming for high-throughput assays required in routine clinical practice. Alternatively, agarose gel electrophoresis followed by enzymatic staining with cholesterol esterase and cholesterol oxidase could provide precise detection of lipoproteins. Although this method is quantitative, it is semi-automated and requires experience.

A commonly used and cost-effective method is the Friedewald calculation, which estimates LDL-C from measurements of total cholesterol, triglycerides, and HDL-C in a fasting sample. The Friedewald equation: LDL-cholesterol = total cholesterol – HDL-C – triglycerides (VLDL)/5, since the triglyceride to cholesterol ratio in VLDL in normal fasting persons is 5/1. Although widely used, this method has several limitations, including the requirement for a fasting sample and unreliability when triacylglycerols exceed 400 mg/dL. Hence, several direct homogeneous methods have evolved to measure cholesterol, with the advantage of not requiring a fasting sample. They are reliable for triacylglycerol measurements up to 1000 mg/dL. Homogeneous assays were developed in the third-generation methods, where the LDL-C can be determined directly with improved precision and accuracy, and are available on automated platforms. An additional measure of LDL is assaying its protein, apoB. This, unlike cholesterol, provides a measure of particle number, does not require a fasting sample, and is available on most automated platforms. However, it is more expensive than LDL-cholesterol measurement and reports apoB in VLDL and IDL.

Clinical Significance

We have previously detailed the approach to hypercholesterolemia in this series, and we keep this discussion brief. Familial hypercholesterolemia is a monogenic disorder with a very high LDL-C, such as 200 mg/dl in heterozygous familial hypercholesterolemia and 600 mg/dl in homozygous familial hypercholesterolemia, and promotes premature ASCVD. In these patients, excess LDL cholesterol permeates the intima and accumulates as fatty streak lesions, an early step in atherosclerosis. It is demonstrated that native LDL does not cause lipid accumulation; whereas, the modified forms of LDL, such as oxidized, acetylated, and glycated, possess pro-atherogenic properties. While it remains yet to be proven, the best hypothesis is that the oxidatively modified LDL promotes fatty streak formation (foam cells). Oxidized LDL can bind to SRA, CD36, and TLR-4, thereby activating several inflammatory pathways. The preferred drug for these patients is statins, which have been shown to decrease LDL-C by 22% to 50% and reduce ASCVD events and mortality. If adequate control is not achieved, additional drugs, such as ezetimibe (a cholesterol absorption inhibitor) and/or bile acid sequestrants, along with statins, could be added. Recently, research has shown that the use of PCSK9 inhibitors (monoclonal antibodies) in combination with statins can lower LDL-C by up to 60% in these patients. Hence, LDL-C can be lowered by both lifestyle modifications and medication.