Chapter 14 Lipids, lipoproteins and cardiovascular disease
The major lipids present in the plasma are fatty acids, triglycerides, cholesterol and phospholipids. Other lipid-soluble substances, present in much smaller amounts but of considerable physiological importance, include steroid hormones and fat-soluble vitamins; these are discussed in Chapters 8 and 20, respectively.
Elevated plasma concentrations of lipids, particularly cholesterol, are causally related to the pathogenesis of atherosclerosis, the process responsible for the majority of cardiovascular disease (coronary, cerebrovascular and peripheral vascular disease). Cardiovascular disease is the commonest cause of death in the UK: about one-quarter of all deaths are due to coronary heart disease (CHD). Many of these are in people under the age of 60. Effective management of hypercholesterolaemia and other risk factors is of proven benefit in reducing cardiovascular disease mortality.
Triglycerides are more correctly called ‘triacylglycerols’, but this term is not in general use in clinical medicine, and the more colloquial term is used in this book to avoid confusion. They consist of glycerol esterified with three long-chain fatty acids, such as stearic (18 carbon atoms) or palmitic (16 carbon atoms) acids. Triglyceride is present in dietary fat, and can be synthesized in the liver and adipose tissue to provide a source of stored energy; this can be mobilized when required, for example during starvation. Although the majority of fatty acids in the body are saturated, certain unsaturated fatty acids are important as precursors of prostaglandins and in the esterification of cholesterol. Triglycerides containing both saturated and unsaturated fatty acids are important components of cell membranes.
Cholesterol is also important in membrane structure and is the precursor of steroid hormones and bile acids. Cholesterol is present in dietary fat, and can be synthesized in the liver by a mechanism that is under close metabolic regulation. Cholesterol can be excreted in the bile either per se, or after metabolism to bile acids.
Because they are not water soluble, lipids are transported in the plasma in association with proteins. Albumin is the principal carrier of free fatty acids (FFAs); the other lipids circulate in complexes known as lipoproteins. These consist of a non-polar core of triglyceride and cholesteryl esters surrounded by a surface layer of phospholipids, cholesterol and proteins known as apolipoproteins (Fig. 14.1). The latter are important both structurally and in the metabolism of lipoproteins (Fig. 14.2).
Figure 14.1 Diagram showing the composition of a lipoprotein particle. A segment has been removed to reveal the non-polar core of cholesteryl ester and triglyceride surrounded by phospholipids and apolipoprotein.
Lipoproteins are classified on the basis of their densities as demonstrated by their ultracentrifugal separation. Density increases from chylomicrons (CM, of lowest density) through lipoproteins of very low density (VLDL), intermediate density (IDL) and low density (LDL), to high density lipoproteins (HDL). HDL can be separated, on the basis of density, into two metabolically distinct subtypes: HDL2 (density 1.064–1.125) and HDL3 (density 1.126–1.21). Distinct subtypes of LDL (LDL-I, II and III, in increasing order of density) are also recognized. IDL are normally present in the bloodstream in only small amounts but can accumulate in pathological disturbances of lipoprotein metabolism. This classification is illustrated in Figure 14.3 and the approximate lipid and apolipoprotein content in Figure 14.4. However, it is important to appreciate that the composition of the circulating lipoproteins is not static. They are in a dynamic state with continuous exchange of components between the various types. Their principal functions are summarized in Figure 14.3 and discussed in greater detail in the next section.
Figure 14.4 Composition of lipoproteins; although the composition in each class is similar, the particles are heterogeneous, so the percentages given are approximate. Figures shown for HDL are for HDL3; HDL2 contains less protein and more lipid. Only the principal apolipoproteins are shown.
Lipoprotein(a), or Lp(a), is an atypical lipoprotein of unknown function. It is larger and more dense than LDL but has a similar composition, except that it contains in addition one molecule of apo(a) for every molecule of apo B-100. Apo(a) shows considerable homology with plasminogen. The concentration of Lp(a) in the plasma varies considerably between individuals, ranging from 0 to 1000 mg/L. An elevated concentration of Lp(a) appears to be an independent risk factor for CHD. Conventional drug treatments that lower LDL have little effect on Lp(a) concentration.
Chylomicrons (Fig. 14.5) are formed from dietary fat (principally triglycerides, but also cholesterol) in enterocytes; they enter the lymphatics and reach the systemic circulation via the thoracic duct. Chylomicrons are the major transport form of exogenous (dietary) fat. Triglycerides constitute about 90% of the lipid. Triglycerides are removed from chylomicrons by the action of the enzyme lipoprotein lipase (LPL), located on the luminal surface of the capillary endothelium of adipose tissue, skeletal and cardiac muscle and lactating breast, with the result that free fatty acids are delivered to these tissues to be used either as energy substrates or, after re-esterification to triglyceride, for energy storage. LPL is activated by apo C-II.
Figure 14.5 Chylomicrons transport dietary triglycerides to tissue where they are removed by the action of lipoprotein lipase. The resulting remnant particles are removed from the bloodstream by the liver. They bind to remnant receptors (which recognize apo E) and LDL receptors (not shown) on hepatic cells, are internalized and catabolized. Apolipoproteins A and B-48 are synthesized in intestinal cells; apo C and apo E are acquired, together with cholesteryl esters (CE), from HDL. Apolipoprotein C-II activates lipoprotein lipase. As triglycerides (TRIG) are removed from chylomicrons, apo A, apo C, cholesterol (CHOL) and phospholipids are released from their surfaces and transferred to HDL where the cholesterol is esterified. Cholesteryl esters are transferred back to the remnant particles in exchange for triglycerides by cholesteryl ester transport protein (CETP).
Apo A and apo B-48 are synthesized in the gut and are present in newly formed chylomicrons; apo C-II and apo E are transferred to chylomicrons from HDL. As triglycerides are removed from chylomicrons by the action of LPL, these become smaller; cholesterol, phospholipids, apo A and apo C-II are released from the surface of the particles and taken up by HDL. Esterified cholesterol is transferred to the chylomicron remnants from HDL, in exchange for triglyceride, by cholesteryl ester transfer protein. The chylomicron remnants, depleted of triglyceride and enriched in cholesteryl esters, are cleared rapidly from the circulation by hepatic parenchymal cells. This hepatic uptake depends on the recognition of apo E by hepatic remnant receptors (also known as LDL-related receptor protein) and LDL receptors (see below).
Although their major function is the transport of dietary triglyceride, chylomicrons also transport dietary cholesterol and fat-soluble vitamins to the liver. Under normal circumstances, chylomicrons cannot be detected in plasma in the fasting state (>12 h after a meal).
VLDLs (Fig. 14.6) are formed from triglycerides synthesized in the liver either de novo or by re-esterification of free fatty acids. VLDL also contain some cholesterol, apo B, apo C and apo E; the apo E and some of the apo C is transferred from circulating HDL.
Figure 14.6 VLDLs are synthesized in the liver and transport endogenous triglyceride from the liver to other tissues where they are removed by the action of lipoprotein lipase. At the same time, cholesterol, phospholipids and apo C and apo E are released and transferred to HDL. By this process, VLDL are converted to IDL. Cholesterol is esterified in HDL and cholesteryl esters are transferred to IDL by cholesteryl ester transfer protein. Some IDL is removed by the liver, but most has more triglyceride removed by hepatic triglyceride lipase and is thereby converted into LDL. Thus the triglyceride-rich VLDL are precursors of LDL, which comprise mainly cholesteryl esters and apo B-100.
VLDL are the principal transport form of endogenous triglycerides and initially share a similar fate to chylomicrons, triglycerides being removed by the action of LPL. As the VLDL particles become smaller, phospholipids, free cholesterol and apolipoproteins are released from their surfaces and taken up by HDL, thus converting the VLDL to denser particles, IDL. Cholesterol that has been transferred to HDL is esterified and the cholesteryl ester is transferred back to IDL by cholesteryl ester transfer protein in exchange for triglyceride. More triglycerides are removed by hepatic triglyceride lipase, located on hepatic endothelial cells, and IDL are thereby converted to LDL, composed mainly of cholesteryl esters, apo B-100 and phospholipid. Some IDL are taken up by the liver via LDL receptors. These receptors, also known as B, E receptors, are capable of binding apo B-100 and apo E (but not apo B-48). Under normal circumstances, there are very few IDL in the circulation because of their rapid removal or conversion to LDL.
LDLs are the principal carriers of cholesterol, mainly in the form of cholesteryl esters. LDL are formed from VLDL via IDL (Fig. 14.6). LDL can pass through the junctions between capillary endothelial cells and attach to LDL receptors on cell membranes that recognize apo B-100. This is followed by internalization and lysosomal degradation with release of free cholesterol (Fig. 14.7). Cholesterol can also be synthesized in these tissues, but the rate-limiting enzyme, HMG-CoA reductase (hydroxymethylglutaryl CoA reductase), is inhibited by cholesterol, with the result that, in the average adult, cholesterol synthesis in peripheral cells probably does not occur. Free cholesterol also stimulates its own esterification to cholesteryl ester by stimulating the enzyme acyl CoA: cholesterol acyl transferase (ACAT).
Figure 14.7 LDL uptake and catabolism. LDL are derived from VLDL, via IDL. They are removed by the liver and other tissues by a receptor-dependent process involving the recognition of apo B-100 by the LDL receptor. The LDL particles are hydrolysed by lysosomal enzymes, releasing free cholesterol which (i) inhibits HMG-CoA reductase, the rate-limiting step in cholesterol synthesis, (ii) inhibits LDL receptor synthesis and (iii) stimulates cholesterol esterification by augmenting the activity of the enzyme acyl CoA: cholesterol acyl transferase (ACAT).
LDL receptors are saturable and subject to down-regulation by an increase in intracellular cholesterol. Macrophages derived from circulating monocytes can take up LDL via scavenger receptors. This process occurs at normal LDL concentrations but is enhanced when LDL concentrations are increased and by modification (e.g. oxidation) of LDL. Uptake of LDL by macrophages in the arterial wall is an important event in the pathogenesis of atherosclerosis. When macrophages become overloaded with cholesteryl esters, they are converted to foam cells, the classic components of atheromatous plaques.
In human neonates, plasma LDL concentrations are much lower than in adults and cellular cholesterol uptake is probably all receptor mediated and controlled. LDL concentrations increase during childhood and reach adult levels after puberty.
HDLs (Fig. 14.8) are synthesized primarily in the liver and, to a lesser extent, in small intestinal cells, as a precursor (‘nascent HDL’) comprising phospholipid, cholesterol, apo E and apo A. Uptake of cholesterol is stimulated by ATP-binding cassette protein A1 (ABCA1). Nascent HDL is disc shaped; in the circulation, it acquires apo C and apo A from other lipoproteins and from extrahepatic tissues, and in doing so assumes a spherical conformation. The free cholesterol is esterified by the enzyme lecithin-cholesterol acyltransferase (LCAT), which is present in nascent HDL and activated by its cofactor, apo A-I. This increases the density of the HDL particles, which are thus converted from HDL3 to HDL2.
Figure 14.8 HDL metabolism and reverse cholesterol transport. Nascent HDL acquires free cholesterol from extrahepatic cells, chylomicrons and VLDL, and is thereby converted to HDL3. The cholesterol is esterified by the enzyme LCAT and cholesteryl esters are transferred to remnant lipoproteins by CETP in exchange for triglyceride. Remnant particles are removed from the circulation by the liver, whence the cholesterol is excreted in bile both per se and as bile acids. Much HDL is recycled, although some is probably taken up by the liver and steroidogenic tissues. Apoprotein transfers have been omitted for clarity.
Cholesteryl esters are transferred from HDL2 to remnant particles in exchange for triglycerides, this process being mediated by cholesteryl ester transfer protein. Cholesteryl esters are taken up by the liver in chylomicron remnants and IDL and excreted in bile, partly after metabolism to bile acids.
The triglyceride-enriched HDL2 are converted back to HDL3 by the removal of triglycerides by the enzyme hepatic triglyceride lipase, located on the hepatic capillary endothelium. Some HDL2 is probably removed from the circulation by the liver and steroidogenic tissues, through receptors that recognize apo A-I (scavenger receptor type B1).
Thus HDL has two important functions: it is a source of apoproteins for chylomicrons and VLDL, and it mediates reverse cholesterol transport, taking up cholesterol from senescent cells and other lipoproteins and transferring it to remnant particles, which are taken up by the liver. Cholesterol is excreted by the liver in bile, both as free and esterified cholesterol and through metabolism to bile acids.
• HDL acquire cholesterol from peripheral cells and other lipoproteins and this is esterified by LCAT. Cholesteryl esters are transferred to remnant particles, which are taken up by the liver, whence the cholesterol is excreted.
At birth, the plasma cholesterol concentration is very low (total cholesterol less than 2.6 mmol/L, LDL cholesterol less than 1.0 mmol/L). There is a rapid increase in concentration in the first year of life; the mean value in childhood is approximately 4.2 mmol/L. In affluent societies particularly, concentrations rise further in early adulthood. Elevated plasma cholesterol concentrations are a major risk factor for CHD. The relationship between cholesterol concentration and CHD mortality is curvilinear (Fig. 14.9). The curve becomes increasingly steep as cholesterol concentration increases: CHD mortality doubles between concentrations of 5.2 and 6.5 mmol/L, and quadruples between 5.2 and 7.8 mmol/ L. Approximately two-thirds of adults in the UK have a plasma cholesterol concentration >5.2 mmol/L and one-quarter have >6.5 mmol/L. While at a concentration <5.2 mmol/L, the curve becomes shallow, it does not become flat. In individuals with other risk factors, for example cigarette smoking (see below), the curve is moved upwards and is steeper.
Figure 14.9 Mortality from coronary heart disease (CHD) and plasma cholesterol concentration. Mortality is expressed as risk relative to that associated with a concentration of 5.2 mmol/L. With additional risk factors (e.g. cigarette smoking) the curve is shifted upwards and is steeper.
Because of the gradation of CHD risk even within the range of cholesterol concentrations found in the bulk of the adult population, it is inappropriate to define a reference range for plasma cholesterol concentration. Rather, it is preferable to consider an individual person’s concentration in terms of what is ideal or desirable for that individual: this will depend on many factors, including the presence or absence of other CHD risk factors.
While there is an undoubted association between plasma cholesterol concentration (and, in particular, LDL cholesterol) and an increased risk of CHD, there is an inverse correlation between HDL cholesterol and CHD risk. Many physiological factors influence LDL and HDL cholesterol, some of which are indicated in Figure 14.10.
Figure 14.10 Some physiological and external influences on plasma lipoproteins. P/S is the ratio of polyunsaturated to saturated fats in the diet. Several drugs can also affect plasma lipoprotein concentrations (see text). aIn susceptible individuals. N, no significant effect.
Hypertriglyceridaemia is also a risk factor for CHD (probably more so in women than in men), albeit a less important one. Hypertriglyceridaemia due to small, dense, triglyceride-rich LDL particles (LDL-III), which are particularly associated with type 2 diabetes mellitus, is of particular significance, because these particles appear to be more atherogenic than other LDL subtypes. Plasma triglyceride concentrations >10 mmol/L carry an increasing risk of pancreatitis.
where all quantities are expressed in mmol/L. This formula is invalid if the triglyceride concentration exceeds 4.5 mmol/L. ‘Non-HDL cholesterol’ has been proposed as an alternative, as this takes account of potentially atherogenic triglyceride-rich particles as well, but has not been widely adopted.
Separation of lipoproteins by ultracentrifugation is not a convenient technique for routine use and is primarily a research tool. Formerly, separation of lipoproteins by electrophoresis was widely used, but this only provides qualitative information and is now obsolete. Genotyping, or phenotyping, of apo E is required to confirm the diagnosis of remnant hyperlipidaemia (see p. 250). Assays for lipoprotein lipase and apo C-II are required for the diagnosis of the cause of fasting chylomicronaemia. Assays for other apoproteins are available and may prove useful in the future (e.g. apo B in place of LDL or non-HDL cholesterol, apo A-I in place of HDL), but so far there is insufficient evidence to support their use in diagnosis and management.
The appearance of the plasma in the laboratory may provide the first clue that a patient has hyperlipidaemia. In health, in the fasting state, plasma is clear. Following a meal, it often becomes opalescent owing to the light-scattering properties of chylomicrons and VLDL. At triglyceride concentrations above about 4 mmol/L, the plasma becomes increasingly turbid; with severe hypertriglyceridaemia, it appears milky (lipaemic). If plasma is left undisturbed, chylomicrons float to the surface, leaving a clear infranatant layer; VLDL remain in suspension. LDL do not scatter light and, even at high plasma cholesterol concentrations, the plasma remains clear.
Blood for lipid studies should be drawn after an overnight fast, when chylomicrons, being derived from dietary fat, should normally have been cleared; a pathological disturbance may thus be inferred if they are present. The patient should have kept to his or her own normal diet for two weeks before the blood is taken. Alcohol should not have been taken on the evening before blood sampling. Alcohol is a common cause of hypertriglyceridaemia even in patients who have otherwise fasted. When lipid studies are done on a patient who has had a myocardial infarction or stroke, blood should either be taken within 24 h or after an interval of three months, because the metabolism of lipoproteins is disturbed during the convalescent period and analytical results may be misleading.
There is conclusive evidence from clinical trials that lowering plasma cholesterol concentration reduces mortality from CHD and decreases overall mortality. This has been demonstrated both in the context of secondary prevention (treatment of individuals with pre-existing disease) and primary prevention (treatment of individuals in whom there is no evidence of disease). Lowering cholesterol also reduces the risk of stroke. Lowering cholesterol is not associated with an increase in mortality from other diseases (e.g. cancer), with the exception that there is some evidence that it may increase the risk of haemorrhagic stroke, albeit to an extent that is far outweighed by the potential benefits.
It is therefore clear that lipid measurements should be made in all patients known to have vascular disease, and in those at increased risk (see p. 252). Thus plasma lipids should be measured in individuals with the following: