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 via lacteals enter the systemic circulation. The apolipoproteins of chylomicrons include apoB-48, apoE, and apoC-II. Chylomicrons carry dietary fatty acids to various tissues, where they will be used for energy or storage. 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 to triacylglycerols and can be 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 get stored as triacylglycerols. After the partial removal of the triacylglycerol, VLDL remnants (IDL) are formed. Further removal of triacylglycerol produces LDL, the end-product of VLDL metabolism. LDL contains predominantly cholesteryl esters and apoB-100. LDL carries cholesterol to various tissues such as the adrenal gland, 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 the cells, free cholesterol regulates HMG-CoA reductase, which is 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 the level of intracellular cholesterol to prevent excess cholesterol deposition. Macrophages also take up the modified lipoproteins to become foam cells. However, in macrophages, the uptake is through the scavenger receptors, which, unlike the LDL receptor, are not under feedback control by cellular cholesterol. The LDL not taken up by the cells and tissues returns to the liver via LDL receptors present on the membranes of hepatocytes. In the liver, cholesterol may be converted to bile acids or neutral sterols or re-esterified and stored in the liver. 

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

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 the large buoyant LDL (lbLDL). The elevated levels of sdLDL have been considered as a risk factor for ASCVD. Also, increased susceptibility of sdLDL to oxidation could be due to its lipid composition and a decrease in antioxidant moieties. sdLDL has been shown to contain less sialic acid content compared to 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 lead 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 via LDL, measuring serum LDL levels could be useful to predict the risk for ASCVD. As per the NIH-sponsored National Cholesterol Education Program (NCEP) Adult Treatment Panel III (ATPIII) guidelines, serum levels of both total cholesterol (TC) and LDL-cholesterol (LDL-C) are useful to predict the risk for ASCVD. The therapy is also targeted to lower LDL cholesterol below a target value ( less than 100 mg/L).[8][9][10][11][12]

Ultracentrifugation can be used for the separation of lipoproteins based on equilibrium and rate methods. When plasma or serum is ultracentrifuged in native non-protein solute density (1.006 g/ml), TG-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 floating of LDL, or more commonly, the total cholesterol and HDL- cholesterol obtained in the infranatant is assayed to calculate the LDL-cholesterol. This method is tedious and time-consuming for high-throughput assays required in routine clinical practice. Alternately, agarose gel electrophoresis with subsequent enzymatic staining using cholesterol esterase and cholesterol oxidase could give a precise detection of lipoproteins. Although this method is quantitative, it is semi-automated and requires experience.

A commonly used and most cost-effective method is the Friedewald calculation, where LDL-C can be estimated from the measurements of TC, triglycerides, and HDL-C in a fasting sample. The Friedewald equation: LDL-cholesterol = TC – 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 of a fasting sample and unreliability as TGs exceed 400 mg/dl. Hence, several direct homogenous methods have evolved to measure cholesterol, and the advantage is the non-requirement of a fasting sample. They are reliable to TG 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 the number of particles, does not require a fasting sample, and is available on most automated platforms. However, it is more expensive than the measurement of LDL-cholesterol and reports apoB in VLDL and IDL.

Clinical Significance

We have previously detailed the approach to hypercholesterolemia in this series and will be brief.

FH is a monogenic disorder with a very high LDL-C, such as 200 mg/dl in heterozygous FH and 600 mg/dl in the homozygous FH, and promotes premature ASCVD. In these patients, the excess cholesterol from LDL 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). The oxidized LDL can bind to SRA, CD36, and TLR-4 and elicit several inflammatory pathways.

The preferred drug for these patients is the use of statins, which are 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 (cholesterol absorption inhibitor) and/or bile acid sequestrants, along with statins, could be used. Recently research has shown that the use of PCSK9 inhibitors (monoclonal antibodies) along with statins could lower LDL-C up to 60% in these patients. Hence, LDL-C can be lowered by both lifestyle modifications and medication.