Lipids module 1: lipid metabolism and its role in atherosclerosis

Lipid basics
Lipids are fatty substances that are required for 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. Triglycerides are the storage form of fatty acids, derived from the
diet or synthesised in the liver, which are an important source of energy and 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

Figure 1. The components of a lipoprotein

All lipoproteins have a common basic structure (figure 1) but they vary greatly in their size,
density and composition (figure 2 and table 1).
 Figure 2. The lipoprotein particle family

Table 1. The composition of the various lipoproteins and their transport pathways

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 lipoproteins
are also seen (IDL).
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 and have a number of other important
functions, including binding to receptors and activation of lipid transporters and metabolising
enzymes. More detail on specific apolipoproteins can be found in the drop down box.

Specific apolipoproteinsHide details

Apolipoprotein B100 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 LDL receptor. A
shorter form of this apolipoprotein, apolipoprotein B48, is secreted by the intestine as the major
structural component of chylomicrons which carry dietary (exogenous) lipids.
The large, triglyceride rich VLDL and chylomicrons also contain multiple copies of small
exchangeable apolipoproteins (apolipoproteins AI AII AIV, CI CII CIII and E) which enter the HDL
fraction as triglyceride is removed by triglyceride metabolising enzymes lipoprotein lipase (LPL).
Apolipoprotein A1 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 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:
endogenous
exogenous
reverse.
Intestinal (exogenous) pathway

Figure 3. The exogenous lipoprotein pathway

Figure 3 shows the intestinal (exogenous) pathway. Cholesterol and triglycerides derived from
dietary lipids are absorbed in the gut and incorporated into chlyomicrons, regulated by the
Niemann-Pick C1-Like 1 (NPC1L1) gene and microsomal triglyceride transfer protein (MTP). These
very large, triglyceride-rich lipoproteins 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 (LRP) or to the LDL receptor
(LDLR). Left over 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 and enter the circulation
bound to albumin.

Hepatic (endogenous) pathway

Figure 4. The endogenous lipoprotein pathway

Fatty acids, arriving at the liver bound to albumin or 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 lipoprotein lipase (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 (cf. Chylomicron remnant) or converted to LDL by hepatic lipase (HL). That LDL is
then taken up by peripheral tissues via the LDLR to meet local cholesterol needs. Any left over
LDL particles are finally removed by the liver via the LDLR.

Reverse cholesterol pathway

Figure 5. The reverse cholesterol pathway

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 (RCT) is
a major component of lipid homeostasis. Cholesteryl ester transfer protein (CETP) is responsible
for the exchange of cholesteryl esters from HDL to more atherogenic cholesterol fractions
including LDL and VLDL. Individuals lacking in the gene encoding CETP have been identified as
having lower cardiovascular risk and the enzyme has become a pharmaceutical target (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
triglyceride rich lipoproteins (TRLs,) including (VLDL and cChylomicrons), or triglyceride rich
lipoproteins (TRLs) or secreted directly from the gut or liver, and enters plasma and where it
picks up additional exchangeable apolipoproteins.
Free cholesterol is removed from deposits in peripheral tissues 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 for biliary excretion. The
cholesterol-depleted HDL particles can then return to the circulation to undertake more reverse
cholesterol transport.
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 feed back regulation is controlled primarily by
changes in the amount and activity of 3-hydroxy-3 methylglutaryl CoA reductase (HMGCoA
reductase). This enzyme catalyses formation of mevalonate, the committed step in cholesterol
biosynthesis. (For more detail on this process of cholesterol homeostasis, see dropdown box).
Figure 6. Cholesterol homeostasis

Cholesterol homeostasis Hide details

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 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-Co-A reductase which is the rate limiting step in the pathway. SREBP-2
also activates the synthesis of LDL-receptors, 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 LDL-receptors 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 LDL-receptor downregulation and inactivation of cholesterol synthesis.

Lipoproteins and atherogenesis

The majority of circulating cholesterol is carried in LDL which is the lipoprotein most closely
associated with the development of atherosclerosis.
Table 2.

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
functions 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.
Oxidised or otherwise modified LDL are retained in the subendothelial space and are 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 found in association with prolonged postprandial
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 triglyceride-rich lipoproteins (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). HDL are, however, 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.

Figure 7. The progression of atherosclerosis

Apolipoproteins
Apolipoproteins are proteins that bind lipids to form lipoproteins. They transport lipids through
the lymphatic and circulatory systems.They also serve as enzyme cofactors, receptor ligands,
and lipid transfer carriers that regulate the metabolism of lipoproteins and their uptake in
tissues. For examples of their beneficial role e.g. apoE in hepatic clearance of chylomicrons and
their thrombogenic potential e.g. lipoprotein(a), see dropdown box.

Routine lipid profile measurements

The routine lipid profile is based on measurements of:
total cholesterol (TC),
HDL
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, the anti-atherogenic fraction, HDL. HDL is essential for
calculation of the TC/HDL ratio as is required for cardiovascular risk assessment.
Triglycerides, mainly carried in VLDL, are not considered directly atherogenic but are 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, 12 hour fasting measurement of triglycerides is required for calculation of LDL. LDL is
usually considered the most important class of atherogenic lipoproteins. Measurement of LDL is
complex so it is usually calculated using the Friedewald equation:
LDL = TC (HDL + TG/2.2)
However this equation assumes a constant ratio (1:2.2 or 0.45) of cholesterol to triglyceride in
VLDL, and requires fasting to ensure absence of postprandial lipoproteins, including chylomicrons
and chylomicron remnants and is valid only if fasting triglyceride is less than 4.5 mmol/L. As the
ratio is altered by statin treatment, the equation significantly underestimates LDL in treated
patients.
Non-HDL is a simple alternative to LDL 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:
Non-HDL = TC HDL
Both LDL and non-HDL measurements are used for targeting treatment but the latter is more
reliable in patients treated receiving highly effective statin therapy, since the LDL calculation is
increasingly inaccurate at lower concentrations (see table 3).
dropdown box

ApolipoproteinsHide details
Apolipoproteins B100 and B48 are two proteins produced from the same gene, due to editing of mRNA in the gut.
ApoB48 is exclusive to chylomicrons and chylomicron remnants. ApoB48 contains 48% of the sequence of apoB100, and
lacks the ligand binding domain recognised by the LDL-receptor (LDLR), so that hepatic removal chylomicron remnants is
dependent on ApoE.
ApoE is required for hepatic clearance of chylomicron and VLDL remnants (IDL) via the LDL receptor (LDLR) and LDLR-
like receptor protein 1 (LRP1). ApoE has 3 common Isoforms (E3, E4, E2). Of the 6 common genotypes, E3/E3 is the most
prevalent. E2 exhibits defective binding to LDLR (<2% of E3) predisposing to remnant (Type III) hyperlipidaemia (familial
dysbetalipoproteinaemia), which is usually associated with the E2/E2 genotype.
ApoB100 remains with VLDL as it undergoes lipolysis to IDL and LDL whereas exchangeable apolipoproteins (e.g. ApoE
and apoC-II) can transfer between particles. As each LDL particle contains one molecule of apolipoprotein B100,
apolipoprotein B concentration is a measure of LDL particle numbers.
Lipoprotein(a) is a highly atherogenic and thrombogenic lipoprotein formed by covalent bonding between the
apolipoprotein B of the LDL particle and apolipoprotein(a), an apparently vestigial plasminogen-like protein which
increases its retention in the artery wall (see figure 8). Genetic variants of apo(a) which are smaller in size generate
greater numbers of liporotein(a) particles, concentrations of which show 100-fold variation between individuals.

Figure 8. Major atherogenic lipoproteins

Table 3. Normal values (Health Survey for England 2003)

Understanding clinical results

A patient with low LDL and high non-HDL is an example of a patient with increased risk who may
slip through the net because we only look at LDL. These patients are also likely to have high LDL
particle number (LDL-P) 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 and the ratio of ApoB/ApoA1, which has
been used in epidemiological studies such as INTERHEART,1 provides similar information to the
TC/HDL ratio. Lipoprotein(a) is also measured by immunoassay and careful method selection is
required to ensure equal recognition of particles containing different apo(a) isoforms.
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, for example among people with abdominal obesity and/or metabolic
syndrome.
Such people have elevated triglycerides, low HDL and relatively normal calculated LDL. Despite
this, they harbour highly atherogenic lipoproteins such as TRL remnants and IDL (intermediate
density lipoprotein) as well as small dense LDL particles.
Non-HDL has been shown to be a better marker of risk in both primary and secondary prevention
studies. In a recent analysis of data combined from 68 studies, non-HDL was the best predictor
among all cholesterol measures, both for CAD events and for strokes.

References
1. 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;364:93752.
http://dx.doi.org/10.1016/S0140-6736(04)17018-9
2. Durrington PN. Hyperlipidaemia: diagnosis and management (3rd Edition). Hodder Arnold
2007. http://dx.doi.org/10.1201/b13464
3. Garg A (ed). Dyslipidaemias: pathophysiology, evaluation and management. Humana Press
2015. http://dx.doi.org/10.1007/978-1-60761-424-1
4. Tabas I, Williams KJ, Born J. Subendothelial lipoprotein retention as the initiating process in
atherosclerosis: update and therapeutic implications. Circ 2007;116:183244.
http://dx.doi.org/10.1161/CIRCULATIONAHA.106.676890
5. Calandra S, Tarugi P, Speedy HE, Dean AF, Bertolini S, Shoulders CC. Mechanisms and genetic
determinants regulating sterol absorption, circulating LDL levels, and sterol elimination:
implications for classification and disease risk. J Lipid Res 2011;52:1885926.
http://dx.doi.org/10.1194/jlr.R017855