Cholesterol - Factors determining blood levels
A high blood (serum) cholesterol level is a major risk factor for atherosclerotic coronary heart disease. Consequently, there has been much interest in the causes of elevated serum cholesterol concentrations. Cholesterol is transported in the bloodstream by several specific carriers called lipoproteins. Each lipoprotein has its own characteristics, and each is responsive to a number of factors, among which are diet constituents such as cholesterol, certain fatty acids, and total energy. Other factors affecting lipoprotein metabolism include age, menopause, and genetics. Consideration of each of the factors regulating serum cholesterol concentrations first requires a description of the different lipoprotein species.
Lipoproteins are macromolecular complexes that consist of discrete particles and are composed of both lipids and proteins. The lipids include cholesterol, phospholipids, and triacylglycerols (TAG). A portion of serum cholesterol is esterified with a fatty acid; the remainder is unesterified. The protein components go by the name of apolipoproteins.
Four categories of lipoproteins that carry cholesterol in the serum are chylomicrons, very low-density lipoproteins (VLDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL). The characteristics and metabolism of each lipoprotein will be reviewed briefly.
Dietary cholesterol enters the intestine together with fat, which is predominantly TAG. The latter undergoes hydrolysis by pancreatic lipase and releases fatty acids and monoacylglycerols. In the intestine, these mix with bile acids, phospholipids, and cholesterol from the bile. The mixture of hydrolyzed lipids associates with phospholipids and bile acids to form mixed micelles. Fatty acids, monoacylglycerols, and cholesterol are taken up by the intestinal mucosa. In the mucosal cells, the fatty acids and monoacylglycerols are recombined by enzymatic action to form TAG, which are incorporated with the cholesterol into lipoprotein particles called chylomicrons. Most of the cholesterol in chylomicrons is esterified with a fatty acid. The major apolipoprotein of chylomicrons is apo B-48; other apolipoproteins – apo Cs, apo Es, and apo As – attach to the surface coat of chylomicrons and aid in metabolic processing. In the mucosal cells, microsomal lipid transfer protein (MTP) facilitates the transfer of TAG and cholesterol ester into chylomicron particles. The presence of MTP is required for the secretion of chylomicrons from mucosal cells.
Chylomicrons are secreted by intestinal mucosal cells into the lymphatic system; from here they pass through the thoracic duct into the systemic circulation. When chylomicrons enter the peripheral circulation they come into contact with an enzyme, lipoprotein lipase (LPL), which is located on the endothelial surface of capillaries. LPL is activated by apo C-II on chylomicrons; this process is modulated by apo C-III, an inhibitor of LPL activity. Nonetheless, most chylomicron TAG is hydrolyzed by LPL; a residual lipoprotein particle, named chylomicron remnant, is released into the circulation and is rapidly removed by the liver. Hepatic uptake of chylomicron remnants is believed to be mediated by binding of remnants with a glycoprotein on the surface of liver cells. Almost all newly absorbed cholesterol thus enters the liver in association with chylomicron remnants.
The liver also secretes a TAG-rich lipoprotein called VLDL. Fatty acids used in the synthesis of TAG in the liver are normally derived from circulating nonesterified fatty acids (NEFA); even so, the liver has the capacity to synthesize fatty acids when the diet contains mainly carbohydrate. MTP inserts TAG into newly forming VLDL particles. The surface coat of VLDL contains unesterified cholesterol, phospholipids, and apolipoproteins. The major apolipoprotein of VLDL is apo B-100. Other apolipoproteins, notably apo Cs and apo Es, are also present. As VLDL circulate they acquire cholesterol esters from HDL. Circulating VLDL particles lose TAG through interaction with LPL in the peripheral circulation; in this process, VLDL are transformed into VLDL remnants. The latter can have two fates: Hepatic uptake or conversion to LDL. Hepatic uptake of VLDL remnants may occur via two mechanisms: Interaction with glycoproteins or interaction with LDL receptors. Both glycoproteins and LDL receptors are located on the surface of liver cells.
Conversion of VLDL remnants into LDL appears to be largely the result of hydrolysis of remaining TAG by hepatic triacylglycerol lipase (HTGL). Normally, approximately two-thirds of cholesterol is carried by LDL, most of this LDL cholesterol existing in the form of esters. The only apolipoprotein in LDL is apo B-100. LDL is removed from the circulation largely by hepatic LDL receptors. The level of expression of LDL receptors is a major determinant of serum LDL cholesterol concentrations. The synthesis of LDL receptors is regulated in large part by the liver’s content of cholesterol. An increase in the hepatic cholesterol content suppresses LDL receptor synthesis and increases serum LDL cholesterol; conversely, a decrease in hepatic cholesterol stimulates receptor synthesis and lowers serum LDL cholesterol. The mechanism by which hepatic cholesterol controls LDL receptor synthesis is through a regulatory protein called sterol regulatory element-binding protein (SREBP). When the hepatic cholesterol content declines, SREBP is activated and stimulates the synthesis of LDL receptors.
The regulatory form of cholesterol in the liver cell is unesterified cholesterol, not cholesterol ester. The hepatic content of unesterified cholesterol depends on several factors including the amounts of cholesterol derived from chylomicrons and other lipoproteins, hepatic synthesis of cholesterol, secretion of cholesterol into bile, conversion of cholesterol into bile acids, esterification of cholesterol, and secretion of cholesterol into serum with VLDL. Factors that influence each of these processes can alter serum LDL cholesterol concentrations by modifying the hepatic content of unesterified cholesterol and thereby the expression of LDL receptors.
HDL consist of a series of lipoprotein particles of relatively high density, all of which contain apo A-I. A proportion of HDL particles also contain apo A-II. Some HDL species (HDL3) are denser than others (HDL2). HDL particles are composed largely of by-products of catabolism of TAG-rich lipoproteins. The surface coats of HDL particles contain phospholipids and unesterified cholesterol, apo A-I with or without apo A-II, and other apolipoproteins (apo Cs and apo Es). Their particle cores consist largely of cholesterol esters, although small amounts of TAG are also present. The cholesterol esters of HDL are formed by esterification with a fatty acid through the action of an enzyme, lecithin cholesterol acyl transferase; the substrates for this reaction derive either from unesterified cholesterol released during lipolysis of TAG-rich lipoproteins or from the surface of peripheral cells. After esterification of cholesterol, the cholesterol esters of HDL are transferred back to TAG-rich lipoproteins and are eventually removed by the liver through direct uptake of remnant lipoproteins or LDL. Whether whole HDL particles can be directly removed from the circulation is uncertain. Some investigators believe that the HDL components are dismantled and removed piecemeal.
Dietary Regulation of Serum Lipoproteins
A large body of research has shown that diet has a major impact on the concentrations and composition of serum lipoproteins, and hence on serum cholesterol concentrations. Three major factors affect cholesterol and lipoprotein concentrations:
- dietary cholesterol,
- the macronutrient composition of the diet, particularly dietary fatty acids, and
- energy balance, as reflected by body weight. The influence of each of these factors can be considered.
All dietary cholesterol is derived from animal products. The major sources of cholesterol in the diet are egg yolks, products containing milk fat, animal fats, and animal meats. Many studies have shown that high intakes of cholesterol will increase the serum cholesterol concentration. Most of this increase occurs in the LDL cholesterol fraction. When cholesterol is ingested, it is incorporated into chylomicrons and makes its way to the liver with chylomicron remnants. There, it increases the hepatic cholesterol content and suppresses LDL receptor expression. The result is an increase in serum LDL cholesterol concentrations. Excess cholesterol entering the liver is removed from the liver either by direct secretion into bile or by conversion into bile acids; also, dietary cholesterol suppresses hepatic cholesterol synthesis. There is considerable variability in each of these steps in hepatic cholesterol metabolism; for this reason, the quantitative effects of dietary cholesterol on serum LDL cholesterol levels vary from one person to another. For every 200 mg of cholesterol per day in the diet, serum LDL cholesterol is increased on average by approximately 6 mg per dl (0.155 mmol per litre).
Macronutrient Composition of the Diet
Dietary Fat and Fatty Acids
Most of the fat in the diet consists of TAG that are composed of three fatty-acid molecules bonded to glycerol. The contribution of TAG to the total energy intake varies among individuals and populations, ranging from 15% to 40% of the total nutrient energy. The fatty acids of TAGs are of several types: Saturated, cis-monounsaturated, trans-monounsaturated, and polyunsaturated fatty acids. All fatty acids affect lipoprotein levels in one way or another.
Table 2 lists the major fatty acids of the diet and shows their effects on serum lipoproteins. The effects of carbohydrates, which also influence serum lipoprotein metabolism are also shown. It should be noted that all lipoprotein responses are compared with and related to those of cis-monounsaturated fatty acids, which are widely accepted to be neutral or baseline.
Saturated Fatty Acids
The saturated fatty acids are derived from both animal fats and plant oils. Rich sources of dietary saturated fatty acids include butter fat, meat fat, and tropical oils (palm oil, coconut oil, and palm kernel oil). Saturated fatty acids are straight-chain organic acids with an even number of carbon atoms (Table 2). All saturated fatty acids that have from eight to 16 carbon atoms increase the serum LDL cholesterol concentration when they are consumed in the diet. In the USA and much of Europe, saturated fatty acids make up 12–15% of the total nutrient energy intake.
The mechanisms by which saturated fatty acids increase LDL cholesterol levels are not known, although available data suggest that they suppress the expression of LDL receptors. The predominant saturated fatty acid in most diets is palmitic acid (C16:0); it is cholesterol-increasing when compared with cis-monounsaturated fatty acids, specifically oleic acid (C18:cis1, n-9), which is considered to be ‘neutral’ with respect to serum cholesterol concentrations. In other words, oleic acid is considered by most investigators to have no effect on serum cholesterol or lipoproteins. Another saturated fatty acid, myristic acid (C14:0), apparently increases LDL cholesterol concentrations somewhat more than does palmitic acid, whereas other saturates – lauric (C12:0), caproic (C10:0), and caprylic (C8:0) acids – have a somewhat lesser cholesterol increasing effect. On average, for every 1% of total energy consumed as cholesterol-increasing saturated fatty acids, compared with oleic acid, the serum LDL cholesterol level is increased approximately 2 mg dl1 (0.025 mmol l1).
One saturated fatty acid, stearic acid (C18:0), does not increase serum LDL cholesterol concentrations. The main sources of this fatty acid are beef tallow and cocoa butter. The reason for its failure to increase LDL cholesterol concentrations is uncertain, but may be the result of its rapid conversion into oleic acid in the body.
Trans-Monounsaturated Fatty Acids
These fatty acids are produced by industrial hydrogenation of vegetable oils or by biohydrogenation in the rumen of cows and sheep. The largest source of trans-monounsaturates is processed vegetable oils, with small contributions from animal sources. In many countries they contribute between 2% and 4% of the total nutrient energy intake. A series of trans acids are produced by hydrogenation: Most are monounsaturated. The trans–monounsaturated fatty acids increase LDL cholesterol concentrations to a level similar to that of palmitic acid when substituted for dietary oleic acid. In addition, they cause a small reduction in serum HDL cholesterol concentrations.
There has been some debate on whether plant- and animal- derived trans fatty acids are equivalent in terms of their effects on blood cholesterol levels. Although additional studies are expected to yield a definitive answer, a recent review of the literature found no consistent evidence of a differential effect of animal trans fatty acids.
Cis-Monounsaturated Fatty Acids
The major fatty acid in this category is oleic acid (C18:cis1, n-9). It is found in both animal and vegetable fats, and typically is the major fatty acid in diet. Intakes commonly vary between 10% and 20% of the total energy. Oleic acid intake is particularly high in the Mediterranean region where large amounts of olive oil are consumed. Other sources rich in oleic acid are rapeseed oil (canola oil) and high-oleic forms of safflower and sunflower oils. Peanuts and pecans are also high in oleic acid. Animal fats likewise contain a relatively high percentage of oleic acid among all their fatty acids; even so, these fats also tend to be rich in saturated fatty acids. When high-carbohydrate diets are consumed, the human body can synthesize fatty acids; among these, oleic acid is the predominant fatty acid produced.
As indicated before, oleic acid is generally considered to be the baseline fatty acid with respect to serum lipoprotein levels, i.e., it does not increase (or lower) LDL cholesterol or VLDL cholesterol concentrations, nor does it lower (or increase) HDL cholesterol concentrations. It is against this neutral fatty acid that the responses of other fatty acids are defined (Table 2). For example, if oleic acid is substituted for cholesterol-increasing fatty acids, the serum LDL cholesterol concentration will decline. Nonetheless, oleic acid is not considered a cholesterol lowering fatty acid, but instead, this response defines the cholesterol-increasing potential of saturated fatty acids.
Polyunsaturated Fatty Acids
There are two categories of polyunsaturated fatty acids: n-6 and n-3. The major n-6 fatty acid is linoleic acid (C18:2, n-6). It is the predominant fatty acid in many vegetable oils, for example, corn oil, soya bean oil, and high linoleic forms of safflower and sunflower seed oils. Intakes of linoleic acid typically vary from 4% to 10% of nutrient energy, depending on how much vegetable oil is consumed in the diet. The n-3 fatty acids include linolenic acid (C18:3, n-3), docosahexanoic acid (DHA) (C22:6, n-3), and eicosapentanoic acid (EPA) (C20:5, n-3). Linolenic acid is high in linseed oil and present in smaller amounts in other vegetable oils. DHA and EPA are enriched in fish oils.
For many years, linoleic acid was thought to be a unique LDL cholesterol-lowering fatty acid. Recent investigations suggest that earlier findings overestimated the LDL-lowering potential of linoleic acid. Even though substitution of linoleic acid for oleic acid in the diet may reduce LDL cholesterol levels in some individuals, a difference in response is not consistent. Only when intakes of linoleic acid become quite high do any differences become apparent. At high intakes, however, linoleic acid also lowers serum HDL cholesterol concentrations. Moreover, compared with oleic acid, it may reduce VLDL cholesterol levels in some individuals. Earlier enthusiasm for high intakes of linoleic acid to reduce LDL cholesterol levels has been dampened for several reasons: For example, its LDL-lowering ability does not offset the potential disadvantages of HDL lowering, and other concerns include possible untoward side effects such as promoting oxidation of LDL and suppressing cellular immunity to cancer.
The n-3 fatty acids in fish oils (DHA and EPA) have a powerful action to reduce serum VLDL levels. Diets rich in n-3 fatty acids also reduce peripheral LDL delivery and specific uptake, and affect LPL expression in the arterial wall.
When carbohydrates are substituted for oleic acid in the diet, serum LDL cholesterol levels remain unchanged. However, VLDL cholesterol concentrations usually increase and HDL cholesterol concentrations decline on high-carbohydrate diets. Thus, a lack of difference in the total serum cholesterol concentrations during the exchange of carbohydrate and oleic acid is misleading. The two categories of nutrients have different actions on lipoprotein metabolism. The differences in response to dietary carbohydrate and oleic acid provide a good example of how measurements of serum total cholesterol fail to reveal all of the changes that are occurring in the lipoprotein fractions.
Other Diet Constituents
Phytosterols are naturally occurring plant sterols and may reduce serum cholesterol levels by reducing intestinal cholesterol absorption. Proposed mechanisms include an inhibition of luminal cholesterol uptake or esterification, or increasing re-excretion of cholesterol into the lumen by enterocytes. Carotenoids, such as capsanthin, have been shown to increase HDL and possibly cholesterol flux into HDL particles. Peanuts, wheat antioxidants, black tea, garlic, and ginseng are among the several diet compounds showing cholesterol-lowering effects under defined experimental conditions.
When energy intake exceeds energy expenditure, the balance of energy is stored in adipose tissue in the form of TAG. When the TAG content of adipose tissue becomes excessive (body mass index 30 or above), a state of obesity is said to exist. In some obese persons, excessive accumulations of TAG occur in tissues other than adipose tissue. Two such tissues are skeletal muscle and liver. High contents of TAG in muscle and liver arise primarily because of continuous leakage of excessive quantities of NEFA from adipose tissue. In the presence of a desirable body weight, normal insulin levels are sufficient to suppress hydrolysis of TAG in adipose tissue, and NEFA release is low. On the other hand, in obese persons NEFA release is excessive, and skeletal muscle and liver are flooded with high serum NEFA concentrations. The result is engorgement of these organs with TAG. When skeletal muscle is overloaded with TAG, insulin-mediated glucose uptake is impaired. This condition is called insulin resistance. When the liver is packed with TAG, hepatic metabolism is altered and insulin action on the liver is deranged. As a result, there is an overproduction of VLDL; this leads to high VLDL cholesterol concentrations and, because LDL is a product of VLDL, to higher LDL cholesterol levels. In addition, obesity is accompanied by a reduction in HDL cholesterol concentrations. Thus obesity is responsible for multiple alterations in lipoprotein metabolism; it has significant effects on three major lipoprotein species – VLDL, LDL, and HDL. These changes appear to be the result of a combination of excessive hepatic TAG as a substrate for VLDL formation and failure of insulin to exert its usual action to curtail VLDL secretion.
That being said, the epidemiological association between obesity and high cholesterol or TAG blood levels remains elusive to demonstrate. In many countries, cholesterol levels continue to decrease, in spite of the ongoing obesity epidemic.
Many of the adverse metabolic effects of obesity are reversed by exercise. Increased energy expenditure through regular and sustained exercise helps to prevent the accumulation of excessive quantities of TAG in adipose tissue. In addition, increased muscle metabolism produced by exercise burns off NEFA and prevents TAG accumulation in the liver. Hence, increased and sustained energy expenditure favorably modifies the lipoproteins, particularly by lowering VLDL cholesterol concentrations and increasing serum HDL cholesterol. The effects of exercise on LDL cholesterol concentrations are more modest, but in some persons, exercise produces a reduction.
Other Factors Affecting Serum Lipoproteins
Between the ages of 20 and 50 years, there is a gradual increase in serum cholesterol concentrations. In the USA, for example, the serum cholesterol increases on average approximately 50 mg per dl (1.295 mmol per litre). This change may be related in part to increasing obesity, according to the mechanisms described above. However, even in persons who do not gain weight with advancing age, serum cholesterol concentrations usually increase to some extent. Available evidence indicates that this increase results from a decrease in the expression of LDL receptors. The reasons for a decline in receptor synthesis with aging are not known, but may reflect metabolic aging. Recent studies have reported an association between high serum cholesterol levels and reduced brain glucose utilization in aging men.
In women, there is a further increase in serum cholesterol concentrations after the age of 50 years. This rise is believed to be due largely to loss of estrogens after the menopause. Estrogens are known to stimulate the synthesis of LDL receptors, and, consequently, receptor expression declines after menopause. This increment in cholesterol levels can be largely reversed by estrogen replacement therapy.
Family studies and research in twins indicate that approximately 50% of the variation of serum cholesterol concentrations in the general population can be explained by genetic polymorphisms. Presumably this variation is related to factors that regulate lipoprotein concentrations. In some cases, specific genetic defects are severe, resulting in marked changes in lipoprotein concentrations. When this occurs, the affected individual is said to have a monogenic disorder. In other cases, multiple genetic modifications are present that combine to change lipoprotein concentrations. When a few modifications are present, the condition is called oligogenic, but when many modifications combine to change lipoprotein concentrations, the condition is named polygenic. Several monogenic disorders have been identified; a few oligogenic conditions have been described, but there are very few instances in which complex polygenic traits have been unraveled. Genome-wide analyses have identified chromosome loci associated with LDL levels. Similarly, genotype analyses suggest that a group of identifiable genetic variants in key genes responsible for cholesterol metabolism could help identify individual risk. Some of these variants have been described (e.g., PCSK9 in the Italian population).
A question of great interest is whether nutritional and genetic factors ever interact synergistically to alter lipoprotein concentrations. Undoubtedly, dietary factors and genetic changes can be additive in their effects on serum lipoproteins, but a synergistic interaction has been difficult to prove. In what follows, consideration will be given to the impact of modification of some of the key gene products regulating lipoprotein metabolism.
The most severe elevations in LDL cholesterol levels occur in patients who have mutations in the gene encoding for LDL receptors. Approximately 1 in 500 persons are heterozygous for these mutations. Their condition is called heterozygous familial hypercholesterolemia. LDL cholesterol concentrations are essentially twice the normal level in this condition. Very rarely patients are homozygous for mutation in the LDL receptor gene and thus have homozygous familial hypercholesterolemia. Their LDL cholesterol levels are approximately four times the normal level. Individuals with this condition develop severe premature atherosclerosis.
Many other individuals appear to have a reduction in LDL receptor expression on a genetic basis, but they do not have as severe elevations of serum LDL cholesterol as patients with familial hypercholesterolemia. Presumably, these individuals have genetic modifications in factors that regulate transcription of the LDL receptor gene. Although such genetic modifications may be relatively common, they are poorly defined. Again, an important but unanswered question is whether some persons are genetically susceptible to the cholesterol increasing effects of dietary cholesterol and saturated fatty acids. If so, they may possess modifications in the genetic control of LDL receptor expression.
Apolipoprotein B-100 Structure
Approximately 1 in 500 persons also have a mutation in the primary structure of apo B that interferes with its binding to LDL receptors. This mutation gives rise to the disorder called familial defective apolipoprotein B-100. The consequence is an elevation of LDL cholesterol concentrations, and the clinical pattern resembles that of familial hypercholesterolemia.
Apolipoprotein B Synthesis
Rare patients have mutations in the gene encoding for apo B that impair the synthesis of this apolipoprotein. Such patients usually have very low LDL cholesterol concentrations. These individuals are said to have familial hypobetalipoproteinemia. In other rare cases, the intracellular TAG transport protein called MCT is genetically absent; when this occurs, no lipoprotein particles containing apo B can be formed. LDL cholesterol is absent from serum, and the disorder is called familial abetalipoproteinemia.
Some researchers speculate that serum elevations in VLDL cholesterol and LDL cholesterol can result from excessive synthesis and secretion of apo B-containing lipoproteins by the liver. When this occurs on a genetic basis, the disorder is designated familial combined hyperlipidemia. However, a monogenic basis of this clinical phenotype has not yet been identified. Therefore, most investigators have concluded that familial combined hyperlipidemia probably represents an oligogenic or a polygenic disorder. In this disorder, lipoprotein elevations appear to be worsened by nutritional factors – particularly by obesity.
This apolipoprotein is present on TAG-rich lipoproteins and it facilitates the removal of remnant lipoproteins by LDL receptors in the liver. When apo E is affected by mutation, this enabling action is curtailed and hepatic uptake of remnant lipoproteins is impaired. The result is an accumulation of chylomicron remnants and VLDL remnants in the circulation. The accumulation is accentuated by the coexistence of other disorders of metabolism of TAG-rich lipoproteins. When remnant accumulation occurs on a genetic basis, the disorder is called familial dysbetalipoproteinemia.
There are two forms of apo C – apo C-II and apo C-III. Apo CII is required for the activation of LPL; when it is genetically absent, affected patients develop severe elevations of TAG-rich lipoproteins. Apo C-III inhibits the activity of LPL. In certain metabolic disorders, notably insulin resistance, the synthesis of apo C-III is increased; an elevated apo C-III can lead to impaired function of LPL and increases in serum concentrations of TAG-rich lipoproteins.
This is the major apolipoprotein of HDL. Rare patients have mutations in apo A-I that result in very low concentrations of HDL cholesterol. However, most individuals in whom HDL cholesterol concentrations are moderately reduced show increased catabolism of apo A-I. The mechanism for this change has not been fully determined, but one important cause may be an overexpression of HTGL.
This enzyme is required for lipolysis of TAG in TAG-rich lipoproteins. Rare patients are homozygous for mutations in LPL that impair its function. In such patients, serum concentrations of chylomicrons are markedly increased. The accumulation of chylomicrons in serum is greatly accentuated by the presence of fat in the diet. Only by severe dietary fat restriction is it possible to prevent severe TAG elevations in serum.
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