Lipid basics
Lipids are fatty substances that are required for the maintenance of normal bodily function. Cholesterol and triglycerides are the major lipids that circulate in blood plasma and are transported in globules known as lipoproteins. Cholesterol is an important component of cell membranes and is required for the synthesis of steroid hormones and bile acids. As the daily requirement for cholesterol cannot be met from dietary intake, the majority (80%) is derived from biosynthesis in the liver and peripheral tissues. Triglycerides are the storage form of long chain fatty acids, derived from the diet or synthesised in the liver, which are an important source of energy and structural fatty acids required for formation of phospholipids, an essential component of cell membranes. Both the liver and the gut package cholesterol, triglycerides and fat-soluble vitamins into lipoproteins for delivery to other tissues.
Lipoprotein fractions
All lipoproteins have a common basic structure (figure 1) but they vary greatly in their size, density and composition (figure 2 and table 1).
| Key: HDL = high-density-lipoprotein cholesterol; IDL = intermediate-density lipoprotein; LDL = low-density lipoprotein; Lp(a) = Lipoprotein(a); VLDL = very-low-density lipoprotein |
Table 1. The composition of the various lipoproteins and their transport pathways
| Composition | Name | Transport pathway |
|---|---|---|
| Cholesterol-rich | HDL (high-density lipoprotein) | Reverse |
| LDL (low-density lipoprotein) | Endogenous | |
| Intermediate | IDL (intermediate-density lipoprotein) | Endogenous |
| Chylomicron remnants | Exogenous | |
| Triglyceride-rich | VLDL (very-low-density lipoprotein) | Endogenous |
| Chylomicrons | Exogenous | |
| Key: HDL = high-density lipoprotein; IDL = intermediate-density lipoprotein; LDL = low-density lipoprotein; VLDL = very-low-density lipoprotein | ||
Lipid fractions are mixtures of lipoproteins of similar size and density which can be separated by the ultracentrifuge and are named accordingly as:
- high-density lipoproteins (HDL)
- low-density lipoproteins (LDL)
- very-low-density lipoproteins (VLDL).
As VLDL are metabolised into LDL (see below), short-lived intermediate-density lipoprotein (IDL) are also seen.
Chylomicrons, which appear after meals, are the largest and lowest-density lipoproteins and rapidly float to the top of stored plasma without ultracentrifugation. Chylomicrons are rapidly metabolised into smaller VLDL sized particles (chylomicron remnants).
Apolipoproteins
In addition to their lipid components, lipoproteins contain specific protein components known as apolipoproteins which provide a structural framework by binding lipids to form lipoproteins, allowing the transport of lipids through the lymphatic and circulatory systems. Apolipoproteins have a number of other important functions; they serve as enzyme cofactors, receptor ligands, and lipid transfer carriers that regulate the metabolism of lipoproteins and their uptake in tissues. More detail on specific apolipoproteins and examples of their beneficial role e.g. apolipoprotein E (apoE) in hepatic clearance of chylomicrons can be found in the drop-down box below.
Apolipoproteins
- ApoE is required for hepatic clearance of chylomicron and VLDL remnants (IDL) via the LDL receptor (LDLR) and LDLR-like receptor protein 1 (LRP1)
- Apolipoproteins B100 (apoB100) and B48 (apoB48) are two proteins produced from the same gene, due to editing of messenger ribonucleic acid (mRNA) in the gut
- ApoB48 is exclusive to chylomicrons and chylomicron remnants, which carry dietary (exogenous) lipids, and is secreted by the intestine. ApoB48 contains 48% of the sequence of apoB100, and lacks the ligand-binding domain recognised by the LDLR, so that hepatic removal of chylomicron remnants is dependent upon ApoE
- ApoB100 is the bulk carrier of endogenously produced lipids and is secreted by the liver as the major apolipoprotein component of VLDL and LDL, one molecule per lipoprotein, which stays with the particle until it is removed from the circulation by the LDLR. ApoB100 remains with VLDL as it undergoes lipolysis to IDL and LDL whereas exchangeable apolipoproteins (e.g. apoE and apolipoprotein CII) can transfer between particles. As each LDL particle contains one molecule of apoB100, apoB concentration is a measure of LDL particle numbers
- The large triglyceride-rich VLDL and chylomicrons also contain multiple copies of small exchangeable apolipoproteins (AI, AII, AIV, CI, CII, CIII and E), which enter the HDL fraction as triglyceride and are removed by triglyceride-metabolising enzymes lipoprotein lipase (LPL) and hepatic lipase
- Apolipoprotein A1 (apoA1) is secreted by both the gut and the liver and is the major apolipoprotein component of HDL, which has an important role in the reverse transport of cholesterol back to the liver from peripheral tissues.
Plasma lipid pathways
The three major plasma lipid pathways are co-ordinated by the liver to ensure that balance (homeostasis) of the major lipid classes is maintained, avoiding the twin perils of deficiency and overload. They are:
- exogenous
- endogenous
- reverse.
Intestinal (exogenous) pathway
| Key: CM = chylomicron; FFA = free fatty acids; HDL = high-density-lipoprotein cholesterol; LDLR = low-density-lipoprotein receptor; LPL = lipoprotein lipase; LRP = LDL receptor-related protein; RLP = remnant lipoprotein |
Figure 3 shows the intestinal (exogenous) pathway. Cholesterol and triglycerides derived from dietary lipids are absorbed in the gut and incorporated into chylomicrons, which are regulated by the Niemann-Pick C1-like 1 (NPC1L1) transporter and microsomal triglyceride transfer protein (MTP). These very large triglyceride-rich lipoproteins (TRLs) are secreted into the lymph and bypass the liver, entering the plasma post-prandially via the thoracic duct. Chylomicrons deliver the fat to adipose tissue via lipoprotein lipase (LPL) which allows it to be taken up rapidly in the form of fatty acids. Once small enough, the resulting chylomicron remnant is removed from plasma by the liver via apoE (see drop down box) binding to the remnant receptor (LDL receptor-related protein [LRP]) or to the LDL receptor (LDLR). Leftover surface material and exchangeable apolipoproteins (AI, AII, AIV, CI, CII, CIII and E) may enter the HDL pool as ‘nascent HDL’. Between meals (post-absorptive phase), as insulin levels fall, fatty acids are released from adipose tissue by lipolysis (hormone-sensitive lipase and adipose tissue triglyceride lipase) and enter the circulation where they are rapidly bound to albumin.
Hepatic (endogenous) pathway
| Key: ApoB = apolipoprotein B; FFA = free fatty acids; HDL = high-density lipoprotein cholesterol; HTGL = hepatic triglyceride lipase; IDL = intermediate-density lipoprotein cholesterol; LDLR = low-density lipoprotein receptor; LRP = LDL receptor-related protein; VLDL = very-low-density lipoprotein cholesterol |
Fatty acids arriving at the liver bound to albumin or those newly synthesised (de novo lipogenesis), are re-esterified to form triglyceride and, together with cholesterol, are loaded onto apoB to form VLDL. These large triglyceride-rich lipoproteins enter the plasma between meals and deliver the fat to adipose tissue and muscle via LPL, which allows it to be taken up in the form of fatty acids (see figure 4). Once small enough, the resulting IDL is either taken up directly by the liver (compare with chylomicron remnant) or converted to LDL by hepatic lipase. The resulting LDL may then be taken up by peripheral tissues via the LDLR to meet local cholesterol needs. Any surplus LDL particles are finally removed by the liver via the LDLR.
Reverse cholesterol pathway
| Key: ABCA1 = ATP-binding cassette, sub-family A; ApoB = apolipoprotein B; FFA = free fatty acids; HDL = high-density lipoprotein cholesterol; HTGL = hepatic triglyceride lipase; IDL = intermediate-density lipoprotein cholesterol; LDLR = low-density lipoprotein receptor; LPL = lipoprotein lipase; LRP = LDL receptor-related protein; SR-B1 = scavenger receptor class B member 1; VLDL = very-low-density lipoprotein cholesterol |
HDL inhibits the development of atheroma and coronary artery disease by transporting excess tissue cholesterol to the liver where it is converted into bile acids and excreted (figure 5). Lower levels of HDL have been correlated with an increased risk of atherosclerosis, the primary cause of cardiovascular disease. The principal HDL pathway, termed ‘reverse cholesterol transport’, is a major contributor to lipid homeostasis. Cholesteryl ester transfer protein (CETP) is responsible for the exchange of cholesteryl esters from HDL for triglyceride in more atherogenic cholesterol fractions, including LDL and VLDL. Some individuals with loss of function in the gene encoding CETP appeared to be at lower cardiovascular risk; therefore, CETP had become a target for pharmaceutical inhibition (see module 4 for more on CETP inhibitors as a drug class).
Nascent HDL, in the form of flattened discs, is generated from LPL-mediated lipolysis of TRLs (including VLDL and chylomicrons, or TRLs secreted directly from the gut or liver) and enters the plasma where it picks up additional exchangeable apolipoproteins.
Free cholesterol is removed from peripheral tissues (including cholesterol-laden macrophages in the arterial wall) via ATP-binding cassette A1 (ABCA1) activated by apoA1, rapidly esterified by the action of lecithin cholesterol acyl transferase (LCAT) and funnelled into the core of the new HDL particle converting it to the mature spherical form. Core lipid exchanges between HDL and TRLs occur in the circulation, catalysed by CETP. This allows cholesterol to be offloaded from HDL into VLDL and LDL, which are destined for hepatic uptake, permitting further cholesterol uptake from tissues. Finally, the cholesterol-enriched HDL particle returns cholesterol to the liver via the scavenger receptor B1 (SR-B1) for excretion in bile. The cholesterol-depleted HDL particles can then return to the circulation to undertake more reverse cholesterol transport. The resulting biliary cholesterol is partly reabsorbed via NCP1L1 and may be re-secreted via ATP-binding cassette subfamily-G transporters 5 and 8 (ABCG5 and ABCG8, which function as a heterodimeric complex [ABCG5/G8]), including direct secretion from the gut (trans-intestinal cholesterol excretion), which may also be an important route for cholesterol elimination.
Cholesterol homeostasis
The rate of cholesterol formation by the liver and absorption by the small intestine is highly responsive to the cellular level of cholesterol. This feedback regulation is controlled primarily by changes in the amount and activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase). This enzyme catalyses formation of mevalonate, the committed step in cholesterol biosynthesis. (For more detail on this process of cholesterol homeostasis, see drop-down box).
| Key: acetyl CoA = acetyl coenzume A; ApoB = apolipoprotein B; HDL = high-density lipoprotein cholesterol; HMG-CoA = 3-hydroxy-3-methylglutaryl-coenzyme A; IDL = intermediate-density lipoprotein cholesterol; LDLR = low-density lipoprotein receptor; LRP = LDL receptor-related protein; SR-B1 = scavenger receptor class B member 1; VLDL = very-low-density lipoprotein cholesterol |
Cholesterol homeostasis
The concentration of free cholesterol determines the fluidity and function of cell membranes and regulates overall cholesterol homeostasis (see figure 6). The concentration of free cholesterol is ‘sensed’ by membrane-bound transcription factors known as sterol regulatory element-binding proteins (SREBPs). When hepatic cholesterol content is reduced by export in lipoproteins or conversion to bile acids, membrane cholesterol concentration falls and SREBP-2 activates the enzymes of cholesterol synthesis, including HMG-CoA reductase which is the rate-limiting step in the pathway. SREBP-2 also activates the synthesis of LDLRs, thus accelerating the uptake of cholesterol in LDL to help restore membrane cholesterol concentration. In addition, SREBP-2 activates synthesis and secretion of proprotein convertase subtilisin/kexin type-9 (PCSK9), a counterbalancing factor which ‘applies the brakes’ by binding LDLRs and directing them to destruction in the lysosome, thereby preventing their recycling to the cell membrane.
Conversely, when hepatic cholesterol is increased by receptor-mediated uptake of cholesterol in lipoproteins or return of cholesterol to the liver by HDL particles, membrane cholesterol increases, preventing activation of SREBP-2 and leading to LDLR downregulation and inactivation of cholesterol synthesis.
Lipoproteins and atherogenesis
LDL
The majority of circulating cholesterol is carried in LDL, which is the lipoprotein most closely associated with the development of atherosclerosis.
Table 2. Steps in atherogenesis
| 1. Infiltration of low-density lipoprotein cholesterol (LDL) into artery wall |
| 2. Entrapment of LDL in wall |
| 3. Modification of LDL |
| 4. Uptake of modified LDL by macrophages |
| 5. Foam cell formation |
| 6. Fatty streak formation |
| 7. Conversion of fatty streaks to fibrous plaques |
The steps in atherogenesis are summarised in table 2.
Under normal circumstances, LDL may pass from the plasma into the subendothelial space and return to the liver to be removed from the circulation. At this point, it has performed its transport function without being taken up by macrophages and indeed, is unable to stimulate foam cell formation in vitro. However, if retention of the LDL in the endothelial space is increased due to endothelial injury (e.g. with smoking, hyperglycaemia, hypertension) or if removal of LDL from the circulation is delayed, it can become damaged by oxidation or modified in other ways (e.g. glycation).
Oxidised or otherwise modified LDL is retained in the subendothelial space and taken up by monocyte-derived macrophages via the scavenger receptor leading to the formation of foam cells and the development of arterial sub-endothelial fatty streaks – the precursor of atheroma. Small, dense LDL particles, typically found in association with prolonged post-prandial hypertrigylceridaemia and low HDL cholesterol, appear more susceptible to oxidation which may make them more atherogenic. Retention of oxidised lipoproteins in the subendothelial space generates an inflammatory reaction – the ‘response-to-retention’ hypothesis of atherosclerosis.
Partially metabolised remnants of TRLs (remnant lipoproteins) that appear post-prandially are able to induce foam cell formation without modification. These are considered the most highly atherogenic of all. Other atherogenic lipoproteins readily retained in the subendothelial space include glycated LDL and lipoprotein(a) (Lp[a]). However, HDLs are able to penetrate deep into the subendothelial space and are able to remove oxidised lipid from macrophages and prevent foam cell formation, in addition to having a protective effect on the endothelium. Reduction of HDL particle numbers or functional activity is therefore pro-atherogenic.
Figure 7 shows the progression of atherosclerosis. For more information on the process of atherosclerotic plaque development, please visit module 3 of our angina e-learning programme.
ApoE
Additionally, certain ApoE genotypes also play a role in atherosclerosis of which it has three common isoforms (E3, E4, E2). Of the six common genotypes, E3/E3 is the most prevalent. E2 exhibits defective binding to LDLR (<2% of E3), thus predisposing to remnant (type III) hyperlipidaemia (familial dysbetalipoproteinaemia), which is usually associated with the E2/E2 genotype.
Lp(a)
Lp(a) is a highly atherogenic and pro-thrombogenic lipoprotein formed by covalent bonding between apoB of the LDL particle and apolipoprotein(a) (apo[a]), an apparently vestigial plasminogen-like protein which increases its retention in the artery wall (see figure 8). Genetic variants of the apo(a) protein, which are smaller in size due to fewer numbers of repeats of the kringle IV-type 2 domain, generate greater numbers of Lp(a) particles, concentrations of which show 100-fold variation between individuals. As these genetic variants are co-dominantly inherited, family members may also be at risk.1
| Key: HDL = high-density lipoprotein cholesterol; IDL = intermediate-density lipoprotein cholesterol; LDL = low-density lipoprotein cholesterol; Lp(a) = lipoprotein(a); RLP = remnant lipoprotein; VLDL = very-low-density lipoprotein cholesterol |
Routine lipid profile measurements
The routine lipid profile is based on measurements of:
- total cholesterol (TC)
- HDL cholesterol (HDL-C)
- triglycerides
These can be measured easily on automated laboratory analysers and some point-of-care systems.
Measuring TC provides limited information about risk because the number includes both atherogenic (LDL, IDL and VLDL) and anti-atherogenic (HDL) fractions. HDL-C is essential for calculation of the TC/HDL-C ratio, as is required for cardiovascular risk assessment.
Triglyceride, mainly carried in VLDL, is not considered directly atherogenic but is a risk modifier, a component of the ‘metabolic syndrome’, a sentinel marker of secondary hyperlipidaemias and a risk factor for pancreatitis when severely elevated (>20 mmol/L). In addition, a 12-hour fasting measurement of triglyceride is required for the calculation of LDL-C. LDL is usually considered the most important class of atherogenic lipoproteins. Measurement of LDL-C is complex, so it is usually calculated using the Friedewald equation as follows:
LDL-C = TC – (HDL-C + TG/2.2) [in mmol/L]
However, this equation assumes a constant ratio (1:2.2 or 0.45) of cholesterol to triglyceride in VLDL. Additionally, it requires a fasting sample to ensure the absence of post-prandial lipoproteins, including chylomicrons and chylomicron remnants, and is valid only if the fasting triglyceride level is less than 4.5 mmol/L. As the ratio is altered by statin treatment, the equation significantly underestimates LDL-C in treated patients; however, newer equations (e.g. Sampson) give more accurate results.
|
Both LDL-C and non-HDL-C measurements are used for targeting treatment, but the latter is more reliable in patients already receiving high-intensity statin therapy, since the standard LDL-C calculation is increasingly inaccurate at lower concentrations (see table 3). |
Non-HDL-C is a simple alternative to LDL-C, which is easier to calculate and is preferred when TGs are elevated or the patient has not been fasting when sampled, giving the concentration of total atherogenic lipoproteins. It is calculated as follows:
Non-HDL-C = TC – HDL-C [in mmol/L]
Understanding clinical results
A patient with low LDL-C and high non-HDL-C levels is an example of a patient with an increased risk who may slip through the net because we only look at LDL-C. These patients are also likely to have a high LDL particle number, as well as high apoB levels.
Apolipoproteins are measured by immunoassay and are therefore more expensive tests. ApoB is an alternative measure of atherogenic lipoproteins. The ratio of apoB/apoA1, which has been used in epidemiological studies such as INTERHEART (Effect of Potentially Modifiable Risk Factors Associated with Myocardial Infarction),2 provides similar information to the TC/HDL ratio.
In patients with mixed hyperlipidaemia, the ratio of non-HDL-C (total atherogenic cholesterol) to apoB is a measure of the average size of atherogenic particles and is characteristically very high (>5 mmol/L) in remnant (type III) hyperlipidaemia (familial dysbetalipoproteinaemia).3
Lp(a) is also measured by immunoassay and careful method selection is required to ensure equal recognition of particles containing different apo(a) isoforms, but the measurement is stable throughout life and a single measurement is usually sufficient to assess Lp(a)-associated cardiovascular risk.1
Therefore, there is a need for a lipid parameter that better reflects the amount of cholesterol within all atherogenic particles. This is of particular importance when triglyceride levels are high, which is quite common among some people such as those with abdominal obesity and/or metabolic syndrome; they tend to have elevated triglycerides, low HDL-C and relatively normal calculated LDL-C levels. Despite this, they harbour highly atherogenic lipoproteins such as TRL remnants and IDL as well as small, dense LDL particles.
|
NICE recommends non-HDL-C as the preferred marker to monitor lipid-lowering therapy.4 |
Non-HDL-C has been shown to be a better marker of risk in both primary and secondary prevention studies. In a meta-analysis of data combined from 68 studies, non-HDL-C was the best predictor among all cholesterol measures, both for coronary artery disease events and for strokes.
Background reading
- Duan Y, Gong K, Xu S et al. Regulation of cholesterol homeostasis in health and diseases: from mechanisms to targeted therapeutics. Signal Transduct Target Ther 2022;7:265. https://doi.org/10.1038/s41392-022-01125-5
- Yusuf S, Hawken S, Ounpuu S, on behalf of the INTERHEART Study Investigators. Effect of potentially modifiable risk factors associated with myocardial infarction in 52 countries (the INTERHEART study): case-control study. Lancet 2004;364:937–52. https://doi.org/10.1016/S0140-6736(04)17018-9
- Tabas I, Williams KJ, Borén J. Subendothelial lipoprotein retention as the initiating process in atherosclerosis: update and therapeutic implications. Circulation 2007;116:1832–44. https://doi.org/10.1161/CIRCULATIONAHA.106.676890
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References
- Preiss D, Neely D. Biochemistry laboratories should routinely report non-HDL-cholesterol. Ann Clin Biochem 2015;52:629–31. https://doi.org/10.1177/0004563215594818
- Boot CS, Middling E, Allen J, Neely RDG. Evaluation of the non-HDL cholesterol to apolipoprotein B ratio as a screening test for dysbetalipoproteinemia. Clin Chem 2019;65:313–20. https://doi.org/10.1373/clinchem.2018.292425
- Cegla J, France M, Marcovina SM et al. Lp(a): when and how to measure it. Ann Clin Biochem 2021;58:16–21. https://doi.org/10.1177/0004563220968473
- National Institute for Health and Care Excellence. Cardiovascular disease: risk assessment and reduction, including lipid modification. NICE guideline [NG238]. London: NICE, December 2023. https://www.nice.org.uk/guidance/ng238 (accessed 12 March 2024)
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