Lipids, lipoproteins, apolipoproteins and their disease association Henry O. Ogedegbe, Ph.D.

, BB, C(ASCP)SC, CC(NRCC) Department of Environmental Health, Molecular and Clinical Sciences, Florida Gulf Coast University, 10501 FGCU Blvd. South, Fort Myers, FL 33965-6565 Tel: 941-590-7486 Fax: 941-590-7474 E-mail: hogedegb@fgcu.edu Abstract Lipids are ubiquitous molecules, which are present in all tissues of the body and play major roles in a variety of biological processes. They are primary source of fuel and are components of cell membranes and cell structures where they provide stability and are involved in transmembrane transportation. Lipoproteins are the transport vehicles for lipids. The apoliporoteins are the protein moieties of lipoproteins. Measurements of lipids, lipoproteins and apolioproteins have been employed to determine susceptibility of individuals to coronary artery diseases. Increased levels of total cholesterol, low-density lipoprotein cholesterol and apolipoprotein B-100 are directly related to risk for coronary heart disease while increased levels of high-density lipoprotein and apolipoprotein A-I are negatively associated with coronary heart disease risk. Apo E 2 and 4 has been reported to be independent risk factors for coronary artery disease and as predictors for the development of atherosclerosis. Apo E 4 allele is associated with very high levels of low-density lipoprotein cholesterol and total cholesterol while 2 allele is associated with decreased levels of low-density lipoprotein cholesterol and higher triglyceride levels. An association between Alzheimers disease and the apo E 4 allele has been reported in both familial and sporadic late-onset Alzheimers disease. The apo E 4 allele increases the risk and lowers the age of onset distribution of Alzheimers disease in a close dependent fashion. The inheritance of each dose of apo E 4 increases risk of disease and decreases age of onset; conversely, the apo E 2 allele appears to be protective, by lowering the risk of disease and increasing age of onset. Genotyping of apo E may be helpful in determining individuals who may be at risk for developing coronary artery disease and/or Alzheimers disease. Introduction Artherosclerotic vascular disease is a major cause of morbidity and mortality in the Western world.1-3 The atherosclerotic lesion usually comprises of fatty streaks that eventually develop into fibrous plaques.1 Foam cells consisting mainly of macrophages laden with lipids characterize the initial fatty streak which may regress or progress via a transitional lesion to fibrous plaques. In the US, cardiovascular disease and osteoporosis together account for most of the morbidity and mortality in the aging population in spite of improved treatment modalities. Both diseases often appear together, especially in the elderly and are usually regarded as independent entities.4 There is evidence however, which suggest that both diseases share etiologic factor because hyperlipidemia contributes to atherosclerotic plaque formation and osteoporosis through similar biologic mechanism involving lipid oxidation.5 Even though advances in lifestyle, risk modification, pharmacotherapy, endovascular interventions and surgery have considerably improved clinical outcome, 4050% of adverse cardiovascular events continue to occur despite current strategies.6 Genetic and environmental factors contribute to the development and progression of atherosclerotic coronary heart disease (CHD) processes. Major independent risk factors for CHD, including adverse levels of plasma lipids and lipoproteins, are also influenced by genetic and potentially modifiable environmental factors.7 Not much is currently known about lipoprotein metabolism in neurodegenerative diseases, but lipid alterations have been repeatedly reported in Alzheimer brains in which neuronal loss and deafferentation are major features. Although the mechanism underlying the link between the epsilon4 allele of the apolipoprotein E gene and 1

Alzheimer's disease (AD) is presently unclear, it may well be postulated that it is related to disturbances in brain lipoprotein metabolism.8 In order to understand the significance of lipids, lipoproteins and apolipoproteins in relations to atherosclerosis, arterial diseases and Alzheimers disease, their biochemistry must be understood. Lipids are a diverse group of chemicals related primarily because they are insoluble in water, soluble in nonpolar solvents and found in animal and plant tissue. They are ubiquitous molecules found in all tissues and cells of the body where they play important functional roles in all aspects of biological life. They serve as hormones or hormone precursors, aid in digestion, and provide fuel for metabolism and store energy. In addition they act as functional and structural components in biomembranes, and form insulation for nerve cell membranes.9 There are five broad groups of lipids, based on their chemical properties: fatty acids, sterol derivatives, glycerol esters, sphingosine and terpenes Table 1. Dietary lipids are 98 to 99% triglycerides (TG), the remainder consists of cholesterol, phospholipids, di and monoglycerides, fat-soluble vitamins, steroids, and terpenes.10 In order for the body to utilize lipid molecules, they must first be processed through biochemical reactions that are catalyzed by specific enzymes. This process takes place in three phases known as digestion, absorption, and transportation. Table 1 Classification of Lipids Fatty Acids Short Chain (2 - 4 carbon) Medium chain (6 10 carbon) Long chain (12 - 20 carbon ) Prostaglandins Sterols Cholesterols and Cholesteryl esters Steroid hormones Bile acids Vitamin D Glycerols Triglycerides Diglycerides Monoglycerides Phosphoglycerides Plasmalogens Sphingosines Sphingomelins Glycosphingolipids Cerebrosides Terpenes Vitamin A Vitamin E Vitamin K

The digestive phase takes place in the lumen of the intestine and it involves the action of several enzymes such as lipoprotein lipase (LPL), pancreatic lipase, lecithin cholesterol acyl transferase (LCAT), and acyl cholesterol acyl transferase (ACAT). Emulsification of dietary fats with bile secretions to form small particles known as micelles increases their surface area, rendering them more susceptible to digestion by pancreatic lipase. Micelles are negatively charged polymolecules which have diameters of about 5 nm and which are able to access the intestinal microvilli where they penetrate the mucosal wall.10 Triglycerides are hydrolyzed into diand monoglycerides, free fatty acids, and glycerol by the action of lipid digestive enzymes. Also during digestion, cholesterol esters are hydrolyzed to free cholesterol and free fatty acids via a reaction that is catalyzed by cholesterol esterase. Bile and lecithin are essential for solubilization of fat prior to digestion.10 Absorbed micelles invariably reach the circulatory system via the lymphatic system and the thoracic duct. Transportation of lipids throughout the bloodstream is accomplished through the use of specialized particles known as lipoproteins Table 2. They are complex transport vehicles for fatty acids, cholesterol and their esters. Lipoproteins include chylomicrons, very low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL). The different lipoproteins combined with protein molecules ferry the various hydrophobic lipid molecules enclosed within the core of their biomembranes. The protein molecules are known as apolipoproteins and are the purified protein components of lipoproteins.11 There are five main types of apolipoproteins known as apolipoprotein A, B, C, D, and E and each of the main groups have sub groups. Minor apolipoproteins include apo J, H, F and G. They function as cofactors such as C-II for LPL, A-I for LCAT, and they act as lipid transfer proteins. In addition, they act as ligands for interaction with lipoprotein receptors in tissues such as apo B-100 and apo E for the LDL receptor, apo E for the remnant receptor, and apo A-I for the HDL receptor.12 The liver plays a vital role in lipid metabolism. The endogenous lipids produced by the liver 2

account for about 1.5 g of lipids daily and normal diet contributes about 150-300 mg of lipids per day. The lipids produced in the liver are transported by lipoproteins to various sites in the body where they are needed. The liver has both apo B and apo E receptors, which are involved in the catabolic clearance of the cholesterolladen LDL particles. The liver is also involved with the production of several of the enzymes that are used in the biosynthesis and catabolism of the various lipid molecules. Bile salts, which are required for lipid emulsification during the digestion of fats, are produced in the liver from cholesterol molecules.11 Table 2 Association of Human Plasma Lipids, Lipoproteins and Apolipoproteins Lipoprotein Chylomicrons VLDL IDL LDL HDL Major Lipids Exogenous triglycerides Endogenous triglycerides Endogenous triglycerides , cholesterol esters Cholesterol esters, free cholesterol Phospholipids, cholesterol esters Major Apolipoproteins A-I, B-18, C-I, C-II, C-III B-100, C-I, C-II, C-III, E B-100, E B-100 A-, A-II

Triglyceride The majority of fatty acids form esters known as glycerides with glycerol of which TG is the most abundant.. Plant triglycerides have linoleic acids, which are long chain fatty acids and are liquid at 4oC. Animal triglycerides are solid at room temperature. For over three decades, epidemiological studies have investigated the association of high TG with increased risk for CHD. Although the debate continues on the epidemiological impact of TG on CHD, many surveys report that elevated serum triglyceride is an important risk factor. At the same time, other studies have found inconclusive evidence of the association of TG with CHD. A potential bias being that it cannot be determined if the increased TG preceded the onset of the disease.13 Consequently, hypertriglyceridemia per se is probably not an appropriate therapeutic target for the prevention of atherosclerotic cardiovascular disease because it is a poor marker of atherogenic risk and because there have been no clinical trials that have directly addressed the question of whether lowering the triglyceride level reduces the number of clinical events.14 In contrast to patients with hypercholesterolemia, no national guidelines have been proposed for the treatment of patients with hypertriglyceridemia. However, high plasma triglyceride concentrations in diabetic subjects have been found to increase their risk for developing CHD.15 More recent case-control studies in patients with premature coronary artery disease have shown that total triglyceride and VLDL levels discriminate better between subjects with and without coronary artery disease (CAD). Angiographic studies demonstrate that elevated serum triglyceride is found in CAD patients and that elevated VLDL or IDL is associated with severity.16 Cholesterol Little was known about the structure of cholesterol until the pioneering research of A. Windaus and H. Wieland in the first part of the 20th century. Its structure was completely elucidated in 1932.17 Cholesterol is implicated as a risk factor for developing CHD and atherosclerosis. In spite of their atherogenic properties, sterols are necessary for the synthesis of steroid hormones, manufacture of bile salts in the liver and maintenance of membrane fluidity. Plant sterols are known as phytosterols and they may compete with cholesterol for uptake by the mucosal cells of the gut. This competitive property of phytosterol may be used therapeutically to lower cholesterol concentration in the blood. Lowering blood cholesterol levels decreases risk for CHD, especially in the absence of other risk factors.18 According to the National Cholesterol Education Program (NCEP) guidelines, total cholesterol of less than 200 mg/dl is desirable while levels between 200-239 mg/dl is borderline risk for CHD. Levels greater than 240 mg/dl is considered high risk.19 Serum cholesterol traditionally 3

has been considered a poor predictor of total stroke risk; however, it is associated positively with ischemic stroke risk and associated negatively with hemorrhagic stroke risk.20 Chylomicrons Chylomicrons are produced exclusively in the intestine and are involved with the transportation of dietary fats, especially TG. Thus the lipid content of chylomicrons is derived solely from dietary fat intake. The chylomicrons usually traverse the lymphatic system to the thoracic duct and then move on to the systemic circulation but do not enter the portal venous system. They pick up apo C-II from HDL, which then activates the LPL hydrolysis of TG into monoglycerides and free fatty acids. The chylomicron particle contains exogenous TG combined with cholesterol, small amounts of phospholipid and specific apolipoproteins A-I, AII, B-48, C-I, C-II, C-III, and E. An outer shell of phospholipids, free cholesterol and proteins surrounds the particle.9,10 It has been shown that n-3 Fatty acids treatment effectively lowers chylomicrons and VLDLs, but their effect on chylomicron remnants was observed only in the late postprandial phase.21 Low-Density Lipoproteins Low-density lipoprotein particles are the major cholesterol carriers in circulation and their physiological function is to carry cholesterol to the cells. The LDL particle consists of a lipid core composed of 38% cholesterol ester and 11% triglyceride. The outer coat of the molecule consists of 22% phospholipids, 8% unesterified cholesterol and 21% apolipoprotein.10 It contains one molecule of apo B-100 and some variable quantity of apo C-III and apo E. The receptor-mediated catabolism of the particle is an important determinant of the concentration of LDL-C in plasma. Epidemiological, pathological and genetic studies show a strong positive correlation between elevated plasma concentrations of LDL-cholesterol (LDL-C) and the risk of premature CHD.22 Atherogenesis modifies the particles resulting in their accumulation in arterial walls.23 Further delipidation of intermediate-density lipoprotein (IDL) by hepatic lipases results in the formation of LDL particle. It is catabolized in the liver and the tissues and derived from VLDL. The particle is recognizable and its breakdown is receptor-dependent. Extensive biochemical and genetic evidence indicate that the LDL receptor (LDL-R) is a protein (most likely a glycoprotein) molecule.5 Endocytosis of the LDL particle is followed by its degradation. The major LDL subclasses include LDL1, LDL2, LDL3, LDL4 and LDL5. The particle may be separated from VLDL and HDL by methods based on physical characteristics such as density, size, charge, or apolipoprotein content. Estimation of LDL concentration may be obtained directly or with the Friedewalds formula.24 According to the NCEP guidelines; an LDL-C concentration of less than 130 mg/dl is desirable while concentration between 130-159 mg/dl is considered borderline risk for CHD. Concentrations above 160 mg/dl are considered high risk.19 Oxidative modification of LDL plays a key role in the pathophysiology of atherosclerosis. Isolevuglandins (isoLGs) are extremely reactive gamma-ketoaldehydes that avidly bind covalently with proteins and cause protein-protein as well as DNA-protein crosslinking. IsoLG-protein adducts are generated upon oxidation of LDL, and may contribute to atherogenesis since such adducts cause recognition and endocytosis of the modified LDL by macrophage cells.25 Myeloperoxidase (MPO), an abundant heme enzyme released by activated phagocytes, catalyzes the formation of a number of reactive species that can also modify LDL to a form that converts macrophages into lipid-laden or 'foam' cells, the hallmark of atherosclerotic lesions.26 Familial hypercholesterolemia (FH), familial defective apoliporotein B-100 (FDB), familial combined hyperlipidemia (FCHL) and cholesterol ester storage disease result in elevation of serum LDL-C. On the other hand, serum LDL-C is decreased in abetalipoproteinemia and familial hypobetalipoproteinemia. Patients with CHD may be managed by lowering their LDL-C concentration. Many patients with FH are unable to reach targets because of drug intolerance or extremely high baseline LDL-C levels. Consequently, LDL apheresis has become a useful modality for the treatment of patients with severe refractory hypercholesterolemia. Commonly used LDL apheresis systems utilize immunoadsorption columns, dextran sulfate cellulose columns, or heparin 4

precipitation.27 In a study to determine the effects of estrogen use on lipoproteins by postmenopausal women, Campos et al.28 showed that estrogen use in postmenopausal women is associated with significantly elevated plasma apo A-l levels and decreased LDL-C concentrations. Thus estrogen use by postmenopausal women may be a negative risk for CHD. Lipoprotein(a) Lipoprotein(a) is also known as the sinking pre- lipoprotein and was first described in 1963 by Kare Berg as lipoprotein antigen or a genetic variant of LDL that was more prevalent in the plasma of myocardial infarction survivors than in age-matched control group of Scandinavian men.29-31 It belongs to a class of apo B containing lipoproteins that is highly associated with atherosclerotic diseases, stroke and myocardial infarction.32 Lp(a) is a macromolecular complex which is genetically determined, and has been identified as an independent risk factor for premature CAD. It is elevated in diabetic and non-diabetic android obese subjects, and aggravates the atherogenic effect of diabetes mellitus.33 It is an LDL molecule linked to apoprotein(a) and further linked by a disulfide bond to apo B.34,35 It is a heterogeneous glycoprotein molecule, which shares at least 75% homology with plasminogen including multiple tandem repeats of the kringle 4 of plasminogen and one repeat of kringle 5 and the protease domain which unlike plaminogen lacks enzyme activity.1,30 There are 10 subclasses of kringle 4 numbered 1 to 10 exhibiting differences in amino acid composition. All the kringles occurs as a single copy except kringle 4-2 whose number varies among individuals and accounts for the size polymorphism of apo(a).36 The number of kringle 4 repeats in apo(a) is genetically determined and highly variable and apo(a) polymorphs are inherited as an autosomal codominant genetic trait. The molecular mass of apo(a) which ranges from 420 to 840 kd according to the number of kringle 4 repeats is inversely related to Lp(a) concentration in serum.37 Both genes are located on chromosome 6.1 It is highly glycosylated with numerous O-glycosidic linkages in the regions between the kringle domains.38 Lipoprotein(a) is a Risk Factor fpr Coronary Heart Disease The physiology and function Lp(a) are poorly understood but there is suggestion that it might contribute to the thrombotic as well as to the atherogenic, aspects of CAD and mitogenesis.38,39 Both Lp(a) and apo(a) stimulate smooth muscle cell proliferation in vitro. It appears to have a role in fibrinolytic system since myocardial infarctions without obstructive CAD have a high frequency of Lp(a) concentration above 300 mg/dL, similar to what is seen in myocardial infarction with angiographically documented CAD. High concentration of Lp(a) in serum is associated with increased risk for CHD. Lp(a) is an independent risk factor for recurrent atherosclerotic heart disease in men and women after menopause.40,41 Many studies have now demonstrated increased Lp(a) concentrations in patients with CAD.31,42,43 High Lp(a) concentration may not be proportional to the risk of CHD in African Americans.1 The distribution of Lp(a) concentrations varies in different population groups.1,44 Lipoprotein(a) concentrations are lower in Caucasians than in people whose ancestry originated in Africa or the Indian subcontinent.45 Gupta et al.44 investigated the relationship of plasma levels of Lp(a) and other lipid values in patients undergoing coronary arteriography in India. The result showed that Lp(a) concentration was higher in CAD group compared to normal coronary artery group. Plasma values of TC, TG, apo A-1, apo-B, LDL-C, LDL/HDL-C ratio and apo A-1/B ratio were not significantly different in the groups with normal coronary arteries and CAD. This may indicate that the measurement of Lp(a) provides a better marker for predicting the presence of angiographically defined CAD as compared to traditional measures.44 Similar to increased Lp(a) concentrations seen in adults who are at risk for CAD, above-normal concentrations of Lp(a) can be also be detected in five- to seven-day-old newborns.46 Vella et al.47 studied several risk factors in relation to parental CAD in which TC, TG, HDL-C, LDL-C Apo A-I, Apo B and Lp(a) were determined in subjects with and without parental CAD. The Lp(a) concentrations were similar to those seen in other white populations with higher frequencies at low values. Subjects whose parents reported CAD had higher mean Lp(a) values than than those who did not report CAD. The result indicated that Lp(a) is an important risk factor for CHD and also that Lp(a) is more strongly related to the risk of CHD than HDL-C and LDL-C and apo A-I and B.47 5

Screening for Lp(a) Screening for Lp(a) should be considered under the following circumstances: (a) patient or family history of premature atherosclerotic heart disease, (b) familial history of hyperlipidemia, (c) established atherosclerotic heart disease with a normal routine lipid profile, (d) hyperlipidemia refractory to therapy, and (e) history of recurrent arterial stenosis.40 Oxidized - Lp(a) can induce P-selectin expression in cultured human umbilical vein endothelial cells (HUVECs), which may thereby influence the pathogenesis of athersclerosis.48 In a study to investigate the effect of hormone replacement therapy (HRT) on Lp(a) levels in postmenopausal women with CAD, Falco et al.49 showed that HRT induced a significant decrease in Lp(a) level. In a study to examine the correlation of Lp(a) to the extent and severity of CAD and its relation to unstable clinical events, Zampoulakis et al.50 found that elevated Lp(a) predisposes to the extent of CAD and total occlusion but not to the severity of lesion. Thus patients with increased Lp(a) levels and unstable angina are at increased risk of suffering myocardial infarction. In a prospective study to establish whether elevated Lp(a), detected as a sinking pre-beta-lipoprotein band on electrophoresis of fresh plasma, is an independent risk factor for the development of premature CHD in men, Bostom et al.51 discovered that elevated plasma Lp(a) is an independent risk factor for the development of premature CHD in men, comparable in magnitude and prevalence to a TC level of 240 mg/dL or more, or an HDL-C level less than 35 mg/dL. LDL receptor does not appear to have a role in the clearance of Lp(a) thus HMG-CoA which upregulates LDL-R does not have appreciable effect on Lp(a) concentration. Lipoprotein(a) Isoforms There is an inverse correlation between the size of apo(a) isoform and Lp(a) concentration: the largest isoforms are associated with the lowest concentrations. As a result of the observed differences in the distribution of Lp(a) levels across apo(a) sizes in different racial and ethnic groups, Paultre et al.52 tested the hypothesis that apo(a) isoform size determines the association between Lp(a) and CAD.They related Lp(a) levels, apo(a) isoforms, and the levels of Lp(a) associated with different apo(a) isoforms to the presence of CAD. They concluded from their study that elevated levels of Lp(a) with small apo(a) isoforms independently predict risk for CAD in African American and white men. The results further suggested that small apo(a) size confers atherogenicity to Lp(a).104 In a study to test the hypothesis that elevated Lp(a) concentrations may be associated with recurrence of symptoms and restenosis after balloon angioplasty, Desmarais et al.39 evaluated 240 consecutive patients undergoing coronary balloon angioplasty with measurements of Lp(a), TC, HDL-C, LDL-C, TG, Apo A-I, Apo B-100 concentrations from fresh specimens. The result showed that patients with recurrence had significantly greater Lp(a) concentrations compared with those without. Thus they concluded that an elevated Lp(a) concentration was a risk factor for clinical recurrence after percutaneous transluminal balloon coronary angioplasty. Other lipid levels were not significantly associated with recurrence.39 High-Density Lipoproteins High-density lipoprotein is involved with its major protein constituent apo A-I in the reverse cholesterol transport (RCT). The efficiency of RCT depends on the specific ability of apo A-I to promote cellular cholesterol efflux, bind lipids, activate LCAT.53 Thus the levels of HDL-C and composition of HDL subclasses in plasma are regulated by many factors, including apolipoproteins, lipolytic enzymes, lipid transfer proteins, receptors, and cellular transporters.54 The HDL molecule usually contains 50% protein and 50% lipids. About 30% of the lipids of the HDL particles are phospholipids while the remaining 70% is made up of cholesterol.10 It is the smallest of the lipoproteins (9 to 12 nm) but has the highest density (1.063 to 1.21 g/ml) of any of the lipoproteins, due to its high protein content. It may be fractionated further into subfractions known as HDL2 and HDL3.55 HDL2 is present in premenopausal women at about three times the concentration found in men. Low HDL2 has been implicated in the predisposition to development of CHD. The HDL2 are larger in size and richer in lipids than HDL3 and are more efficient vehicles for transfer of cholesterol from peripheral tissue to the liver.56 The major apolipoproteins found in HDL are A-I and A-II, some apo C and a small amount of apo E. A major function of HDL is to act as a repository for apo C and E that are needed in the metabolism of chylomicrons and VLDL.12 Both the liver and the intestine are responsible for HDL production. Only HDL-C, 6

particularly HDL2-C, show an independent inverse relationship to the incidence of atherosclerotic CAD.57 Studies employing laboratory animals have suggested that HDL administration not only inhibits progression of, but even reduces, established atherosclerotic lesions.58 It has been shown that HDL isolated from subjects with non-insulin-dependent diabetes mellitus (NIDDM) exhibits a decreased capacity to induce cholesterol efflux. In a study Gowri et al.15, examined the effect of HDL2 and HDL3 subfractions from poorly controlled NIDDM and control subjects on THP-1 macrophagemediated LDL-oxidation and found that the composition and protective effects of HDL2 but not HDL3, differed significantly between control and diabetic subjects. They concluded that compositional alterations in HDL2 from poorly controlled NIDDM subjects might reduce its antiatherogenic properties. High-density lipoproteincholesterol concentration greater than 60 mg/dL is a negative risk factor associated with CHD. Women generally have higher levels of HDL-C and lower TC than men due to higher estrogen levels. However, after menopause, this difference disappears due to decreased estrogen.59 Both HDL and LDL transport cholesterol in either the free form or as a fatty acid ester in the blood. Genetic abnormalities in either the apo B, E, LDL receptor may lead to an increased plasma LDL-C concentration.60 High-density lipoproteins are involved with the transport of excess cholesterol from the peripheral tissues back to the liver, either directly via the HDL receptor, which recognizes and binds to apo A-I or via remnant receptors, which recognize apo E. Other lipoprotein receptors, termed 'scavenger receptors', are found on the surface of macrophages. Expression of these receptors is not regulated by intracellular cholesterol concentration, as are LDL receptors, thus allowing the buildup of large amounts of cholesterol in the macrophages. This can lead to the formation of foam cells and a build up of plaque in blood vessels, which might set in motion an almost irreversible process of lumen closure, or atherosclerosis.1 Apolipoprotein B Apo B-100 is associated with IDL, VLDL, and LDL. The LDL and IDL particle are directly related to the development of atherosclerosis. It is the sole protein component of LDL and most circulating apo B is associated with LDL. It is the ligand responsible for the receptor-mediated uptake and clearance of LDL from the circulation.22 Apo B-100 is possibly the longest single polypeptide chain known having 4536 amino acids.12 Apo B is recognized by the LDL-R which functions in the delivery of cholesterol to peripheral tissues for membrane and or steroid hormone synthesis and to the liver for removal or reuse.61 Measurement of apo B-100 and LDL-concentrations in serum or plasma may be employed to assess the presence of CHD risk in individuals. Both apo B and LDL are increased in patients with CHD. Apolipoprotein B is the least well characterized of the apolipoproteins and the presence of at least four major molecular forms in circulating plasma has been reported. The LDL contains three of these forms: apo B-100, apo B-74 and apo B-26.62 A fourth form, apo B-48, is produced by the intestine and is the major apolipoprotein found in chylomicrons. Apo B-100, which is the protein component of LDL and apo E, which is present in intermediate lipoproteins and some forms of HDL, are the two proteins through which particular lipoproteins bind to the LDL-R.1 The domain of apo B-100 that interacts with the LDL-R comprise two clusters [A(3147-3157) and B (3359-3367)] of basic amino acids linked through a disulfide bond between residues 3167 and 3297.63 Several point mutations of the putative receptor binding domain of apo B-100 have been identified. The first and most frequent substitution to be identified is apo B-100 (Arg3500Gln). The other two substitutions; apo B-100 (Arg3500Trp) and apo B-100 (Arg3531Cys) occur less frequently.63 Effect of Apolipoprotein B on Diabetes Mellitus and Coronary Heart Disease In a study by Mero et al.64, to examine if there is a relationship between the severity of CAD and postprandial lipemia in patients with NIDDM they found that postprandial apo B-48 and apo B-100 metabolism in triglyceride rich lipoproteins is distorted in NIDDM patients, even in those with only mild CAD. Igua et al.65 compared the structural and biological characteristics of apo B-100 containing particle subfractions isolated from poorly controlled diabetic patients with insulin dependent diabetes mellitus (IDDM) and healthy controls and found that IDDM is not associated with any significant abnormalities in the apo B containing lipoprotein 7

particles. Therefore they concluded that the excessive occurrence of CHD and other atheroslerotic vascular diseases in patients with IDDM must have other causes. In adults, a low level of LDL-C/apo B-100 ratio is an indicator of apo B-enriched small dense LDL, which is associated with premature CAD.66 Islam et al.66 investigated whether a low LDL-C/apo B-100 ratio was associated with a positive family history of premature CAD in young children. The result showed that in young children, a low LDL-C/apo B-100 ratio and high apo B-100 levels were associated with a positive family history of CAD in white girls suggesting that they were at increased risk of genetically mediated CAD. Apo B concentration less than 1.04 g/L indicates low risk for CHD, from 1.04 g/L to less than 1.22 g/L is moderate risk, from 1.22 g/L to less than 1.40 g/L is high risk and 1.40 g/L or greater is very high risk.67 Apolipoprotein A Apolipoprotein A-I is the major apolipoprotein of HDL. It sets the plasma level of HDL and appears to confer protection against the development of atherosclerosis. Measurement of apo A-I may be a better marker than HDL-C in CHD risk assessment.68 A small amount of apo A-I can be found unassociated with HDL, some of which may be associated with nascent HDL that are newly secreted from the liver and intestine. It plays a role in the mobilization of excess cholesterol from cells that cannot metabolize or otherwise dispose of it. The transportation process of HDL and apo A-I may involve both passive and second messenger pathways.61 The products of apo A-I, A-II, C-III and A-IV genes are the major proteins components of HDL. Each of them modifies the activities of LCAT in vitro. The genes coding for apo A-I, C-III, and A-IV occur in a tight cluster spanning ~15 kb on the long arm of human chromosome 11 where the apo C-III is transcribed in the opposite direction to the apo A-I and A-IV genes.69 More than 10 common polymorphisms within the apo A-I, C-III, and A-IV have been detected. Tangier Disease Lipid-poor apolipoproteins remove cellular cholesterol and phospholipids by an active transport pathway controlled by an ATP binding cassette transporter called ABCA1. Mutations in ABCA1 cause Tangier disease, a severe HDL deficiency syndrome characterized by a rapid turnover of plasma apolipoprotein A-I, accumulation of sterol in tissue macrophages, and prevalent atherosclerosis. This implies that lipidation of apolipoprotein A-I by the ABCA1 pathway is required for generating HDL particles and clearing of sterol from macrophages. Thus, the ABCA1 pathway has become an important therapeutic target for mobilizing excess cholesterol from tissue macrophages and protecting against atherosclerosis.70 To test this hypothesis, that apo A-I in combination with apo A-II and C-I remove free cholesterol from the extrahepatic tissues to the liver for subsequent elimination, Eriksson et al.71 measured fecal steroid excretion before and after intravenous infusion of human proapo A-I liposome complexes in subjects. The result showed that infusion of proapo A-I liposomes in humans promotes net cholesterol excretion from the body, implying a stimulation of reverse cholesterol transport, a mechanism that may prove useful in treatment of artherosclerosis. Apo A is produced in the intestine and liver and appears to play structural roles in the HDL. The action on apo A-I is inhibited by apo A-II and 50% of HDL mass is protein with apo A-I and apo A-II constituting almost 90%. The ratio of apo A-I and apo A-II is approximately 3:1 and apo A-I contains 243 amino acids while apo A-II contains 154 amino acids.72 Apolipoprotein A is a Negative Risk for Coronary Heart Disease Apo A-I is inversely related to a predisposition to CHD.73 A study by Schaefer and associates75 showed that treatment of patients with 3-hydroxyl-3-methyl-glutaryl CoA (HMG-CoA) reductase inhibitors such as pravastatin increases fractional synthetic rate of apo A-I as a result of increased production rate. Their result demonstrated that increased production of HDL apo A-I is the metabolic cause of the increase in HDL and apo A-I levels under inhibition of HMG-CoA reductase in man.74 Therefore treatment with HMG-CoA reductase inhibitors are effective in lowering LDL-C levels and increasing the level of HDL-C and A-I in patients with hypercholesterolemia. In a study to compare pre- and post-Ramadan lipid and lipoprotein profiles in stable Kuwaiti hyperlipidemic subjects attending a Lipid Clinic, Akanji et al.75 found that the most consistent changes 8

post-Ramadan were increased levels of apo A-1, apo A-1/apo B and apo A-1/HDL ratios and reduced uric acid levels which suggest that Ramadan fasting in hyperlipidemic subjects might favorably influence CHD risk. Apolipoprotein A Mutants A novel mutant of apo A-I associated with low HDL has been identified in a Japanese family. Apo A-I (Glu235-->0) Nichinan is caused by a 3-bp deletion of nucleotides 1998 through 2000 in exon 4 of the apo A-I gene. This induces a critical structural change in the carboxyl-terminal domain of apo A-I for cellular cholesterol efflux and increases the catabolism of apo A-I resulting in low HDL-C levels.76 A Finnish family with premature CHD and decreased HDL-C levels was identified as having an apo A-I variant, apo A-I (Lys107-->0), caused by a 3-bp deletion of nucleotides 1396 through 1398 in exon 4 of the apo A-I gene. These subjects were heterozygous for this mutation. The heterozygotes had reduced apo A-I and apo A-II concentrations compared with normal family members. However LDL-C, TG, VLDL, IDL, LDL subclasses were similar in both groups. The low HDL-C in the heterozygotes results from the decreased concentration of apo A-I and A-II containing particles and the smaller size and reduced cholesterol content.77 This puts the heterozygotes at greater risk for CHD. ApoA-I (R151) Paris is a natural apo A-I variant that is associated with low levels of HDL-C and the partial deficiency of LCAT in the plasma of heterozygous carriers. Defective LCAT cofactor activity but normal lipid binding and cholesterol-efflux-promoting abilities characterize it.78 Carriers of the apo A-IMilano (A-IM) mutant have a severe hypoalphalipoproteinemia but are not at increased risk for premature CHD. The high efficiency of apo A-IM containing HDL for cell cholesterol uptake would result in an improved reverse cholesterol transport in the apo A-IM carriers which may explain the low susceptibility to atherosclerosis development.79 Apo A-I of less than 1.20 g /L is a risk factor and 1.65 g/L or greater is an antirisk factor for CHD.67 Various hormones influence apo A-I expression such as those belonging to the thyroid and steroid family. Thyroid hormone, glucocorticoids, and estradiol enhance activity of the gene while retinoic acid and androgens decrease it.80 Apolipoprotein E Apolipoprotein E was first isolated from plasma in 1973 and originally was known as arginine-rich apolipoprotein. It plays a role in the transport and redistribution of lipids, including cholesterol in the liver and is implicated in growth and repair of injured neurons in the nervous system. The apo E gene is located on chromosome 19q13.2 in a cluster of related apolipoprotein genes that span approximately 48 kb, and which includes the apo C-I and C-II loci which code for activating cofactors of LCAT and LPL. Apo E is most likely synthesized in the liver and initially enters the plasma as part of nascent HDL. It is a 299-amino acid glycoprotein with a molecular weight of 34,200 and has a central role in lipid metabolism in both physiological and pathophysiological processes. Levels of apo E in plasma range from 2-6 mg/dl.9 Apo E promotes the binding of lipoproteins (LDL, VLDL, and apo E-HDL) to the LDL-R and a specific chylomicron remnant receptor, and is also associated both with transport of cholesterol ester in plasma and with the redistribution of cholesterol in the tissues. It is a major protein constituent of TG rich lipoproteins, including chylomicrons, VLDL and their remnants. A major function of apo E is to serve as a high affinity ligand for several hepatic lipoproteins receptors, including LDL-R and LDL receptor-related protein and for cell surface HSPG. By interacting with these receptors, or with HSPG, apo E mediates the clearance of chylomicrons, VLDL and their remnants from circulation.81,82 Apolipoprotein E Isoforms Apo E phenotype is an important variable that has been used in large-scale population studies.68 There are three major isoforms of apo E, namely apo E2, E3, and E4. Individuals may have any one or a combination of any two of these isoforms.57 Apo E3 and E4 both bind well to the LDL-R whereas E2 is defective in binding. Apo E is genetically polymorphic and has 3 codominant alleles (apo 2, 3, and 4) at the apo E gene locus. The three 9

alleles give rise to six genotypes, of which three are homozygous (2/2, 3/3, and 4/4) and three are heterozygous (2/3, 2/4, and 3/4). The 2 allele is associated with lower levels of serum TC, LDL-C, and apo B compared to the 3 allele. The 4 allele is associated with higher levels of TC, LDL-C and apo B and higher risk for cardiovascular disease and AD.83,84 The 2 allele is present in type III hyperlipoproteinemia. Epidemiological evidence suggest that the apo E4 phenotype conveys a higher cardiovascular risk than does apo E2 or apo E3.85 In addition, an 2/3 genotype appears to confer relative protection compared with an 3/3 genotype.86 Effect of Apolipoprotein E Genotype on Drug Treatment There is evidence, which suggest that individual with different genotypes have inter-individual response to diets and lipid lowering drugs. For example, there is indication that apo E2 individuals are more likely to respond favorably to gemfibrozil and cholestyramine therapy.87 With probucol, apo E4 genotype individuals may show improved plasma lipoprotein-lipid profiles more than apo E3 individuals. Apo E2 and E3 genotype perimenopausal women appear to show improved plasma lipoprotein-lipid profiles more with HRT than apo E4 women.119 Low-fat diet interventions tend to reduce plasma LDL cholesterol and, perhaps, plasma total cholesterol levels more in apo E4 than in apo E2 or E3 individuals.87 Most studies generally indicate that apo E2 and E3 individuals improve plasma lipoprotein-lipid profiles more with exercise training than apo E4 individuals. Thus the future utility of apo E genotype to determine optimal therapy to improve plasma lipoprotein-lipid profiles and stratify CAD risk is very possible.87 In a study, Ballantyne et al.88 examined the association of apo E genotypes with baseline plasma lipid levels and severity of CAD, and response to treatment with fluvastatin in the Lipoprotein and Coronary Atherosclerosis Study (LCAS). The result indicated that subjects with the 3/4 or 4/4 genotype had an increased frequency of previous angioplasty, but other measures of baseline CAD severity and baseline lipids did not differ significantly among the genotypes, nor did CAD progression or clinical events. They concluded that although subjects with the epsilon4 allele had less reduction in LDL-C with fluvastatin, they had similar benefit in terms of CAD progression. Apolipoprotein E Genotype and Atherosclerosis Atherosclerosis involves the hardening of arteries due to the deposit of cholesterol in the cells of the smooth muscle layers of the vascular tissue and may be age and/or environment related. Apo E 2 and 4 has been reported to be independent risk factors for CAD and as predictor for the development of atherosclerosis.89 Apo E 4 allele is associated with very high levels of LDL-C and TC while 2 allele is associated with decreased levels LDL-C and higher TG levels. The common apo E isoforms are the result of amino acid substitutions in codons 112 and 158 (cysteine to arginine). The presence of these isoforms leads to differences in the affinity of the resulting individual phenotypes for LDL and apo E receptors, LDL-R activity, apo E distribution among lipoproteins, LDL formation rate and cholesterol absorption, all of which may promote atherosclerosis and increase the risk of developing CAD. Thus, given the similarity between risk factor for heart disease and risk factor for stroke, a positive association between apo E and stroke would be expected. Patients with apo E 4 allele who have hemorrhagic strokes have poorer prognosis, and after head injury 4 patients seem to be associated with poorer recovery than non-4 patients.90 Apolipoprotein E Genotype and Coronary Heart Disease Risk In a study to determine whether Apo E 4 allele is an independent predictor of coronary event, Scuteri et al.121 measured apoE genotypes in 730 participants in the Baltimore Longitudinal Study of Aging who were free of preexisting CHD. They concluded that the apo E 4 genotype is a strong independent risk factor for coronary events in men, but not women. The association does not appear to be mediated by differences in total cholesterol levels. In a meta-analysis to assess the impact of apo E alleles in coronary disease in 14 published 10

observational studies, Wilson et al.91 observed that 4 allele was associated with greater odds for CHD in both men and women compared with 3 allele or 2 allele. Apolipoprotein E and Alzheimers Disease The ability of Apo E to modulates the risk for AD, CHD and cerebral atherosclerosis is well established.92,93 Alzheimers disease is an age-associated neurodegenerative disorder, which is characterized by a progressive decline of the cognitive functions. It is the most common cause of dementia in elderly people. Apo E 4 allele has been implicated in AD. Most cases of AD are sporadic and the factors involved are unknown. However, a small percentage of cases are hereditary and are typically due to mutations in one or more of the three separate genes. Mutation in the amyloid precursor protein (APP) gene on chromosome 21, and the presenilin-1 (PS1) gene on chromosome 14 are fully penetrant and cause early-onset AD. Mutations in the presenilin-2 (PS2) gene a PS1 homologue on chromosome 1 cause partially penetrant autosomal dominant AD with onset age beginning at 40 years and extending past 75 years. A fourth gene which encodes the protein apo E is implicated in lateonset AD.94,95 An association between AD and the apo E 4 allele has been reported in both familial and sporadic late-onset AD. The apo E 4 allele increases the risk and lowers the age of onset distribution of AD in a close dependent fashion. The inheritance of each dose of apo E increases risk of disease and decreases age of onset; conversely, the apo E 2 allele appears to be protective, by lowering the risk of disease and increasing age of onset.96 To evaluate the genetic factors for AD among a Chinese population in Taiwan, Hu et al.97 studied the polymorphisms of six candidate genes of AD, including the regulatory region of apo E (Apo-E, G-186T), the promoter of apo E (Apo-E, A-491T), the bleomycin hydrolase gene (BH, A1450G), a mutation of alpha(2)macroglobulin gene (A2M G2998A), LDL-R-related protein gene (LRP, C766T), and alpha(1)antichymotrypsin gene (ACT, -15Ala/Thr) in AD patients and non-affected elder individuals among Taiwanese Chinese. Eighty-two AD patients and 110 non-affected individuals were recruited for the study. Their result showed that among the nine candidate genes of AD that they studied the apo E 4 allele was the only independent genetic risk factor for AD. The other candidate genes in the study were not associated with the occurrence of AD. In addition, there was no gene-gene interactions.97 The apo E is a multifunctional molecule with potential roles in amyloid deposition and clearance, microtubule stability, intracellular signaling, immune modulation, glucose metabolism oxidative stress and other cellular process.98 However, the association with AD is not absolute since only about 50% of all patients with AD have the apo E 4 allele and a substantial number of persons with the apo E 4 allele do not develop AD.98 Consequently, another gene in combination with the apo E 4 allele may be involved in sporadic AD. Furthermore, the link of apo E 4 allele to both sporadic and familial late-onset AD raises the possibility that a dysfunction of the lipid transport system could seriously affect lipid homeostasis in the brain. The abnormally low concentration of apo E observed in the brains of some AD subjects with the apo E 4 allele could compromise cholesterol, fatty acid, and phospholipid transport in the central nervous system. This in turn could impair the cholinergic system, which, in contrast to other neurotransmitters in the central nervous system, relies heavily on lipids to synthesize acetylcholine. An inverse relationship has been observed between apo E 4 allele copy number and residual brain choline acetyltransferase activity and nicotinic-receptor binding sites in the brains of subjects with AD.99 Apo E in the presence of the protein amyloid-beta may also promote the development of hair-shaped fibrils and neuritic plaques containing arms of damaged nerve cells in the brain. These plaques, which contain damaged nerve cells, are the hallmark of AD. Thus, when combined with clinical criteria, apo E genotyping improves specificity of the diagnosis of AD.100 Apolipoprotein D Apolipoprotein D is a 29-kDa glycoprotein that is primarily associated with HDL in human plasma. It is an atypical apolipoprotein and, based on its primary structure, it is predicted to be a member of the lipocalin family. Lipocalins adopt a beta-barrel tertiary structure and transport small hydrophobic ligands. Although apo 11

D can bind cholesterol, progesterone, pregnenolone, bilirubin and arachidonic acid, it is unclear if any, or all of these, represent its physiological ligands.101 The apo D gene is expressed in many tissues, with high levels of expression in spleen, testes and brain. It is present at high concentrations in the cyst fluid of women with gross cystic disease of the breast, a condition associated with increased risk of breast cancer.101,102,103 It also accumulates at sites of regenerating peripheral nerves and in the cerebrospinal fluid of patients with neurodegenerative conditions, such as AD. It may, therefore, participate in maintenance and repair within the central and peripheral nervous systems. While its role in metabolism has yet to be defined, it is likely to be a multi-ligand, multi-functional transporter. It could transport a ligand from one cell to another within an organ, scavenge a ligand within an organ for transport to the blood or could transport a ligand from the circulation to specific cells within a tissue.101 Apo D is present in the human brain, especially in glial cells, and has increased abundance in the elderly and AD subjects.104 It is a protein component of the human plasma lipid transport system but is also associated with a more favorable prognosis in women with breast cancer.102 Vazquez et al.102 examined the tumoral expression of apo D in epithelial ovarian cancer so as to analyze the possible correlation with tumor and patient characteristics as well as to androgen receptors and their prognostic significance. They found the existence of apo D expression to be significant in ovarian carcinomas, and and that the protein expression might be of clinical usefulness for identifying lesions with different evolution.102 Apo D is expressed in normal human prostate and elevated Apo D staining is associated with advanced prostate cancer.105 Apo D has been implicated in the transport of several small hydrophobic molecules including sterols and steroid hormones. Defects in apo D metabolism in NiemannPick disease type C (NPC) appears to be linked to the known defects in cholesterol homeostasis in the disorder.106 Apolipoprotein C The primary activity of apolipoprotein C is to activate LPL leading to lipolysis of TG. There are three major forms namely apo C-I, C-II, C-III and they vary in amino acid content having 57, 78, and 79 amino acids respectively. They are synthesized in the liver and associate with VLDL and HDL particles.55 The metabolic function of apo C-I is its inhibitory action on the uptake of VLDL via hepatic receptors, particularly the LDL receptor-related protein. Consequently, the presence of apo C-I on the lipoprotein particle may prolong its residence time in the circulation and subsequently facilitate its conversion to LDL. Apo C-II, on the other hand, is a major activator of LPL, which is required for an efficient processing of triglyceride-rich lipoproteins (TRL) in the circulation. However, an excess of apo C-II on the lipoprotein particle has been sugg suggested to inhibit the LPL-mediated hydrolysis of triglycerides. Apo C-III inhibits the lipolysis of TRL by hampering the interaction of these lipoproteins with the heparan sulfate proteoglycan-lipoprotein lipase complex.107 Poorly lipolyzed apo C-III-containing lipoprotein particles may accumulate in plasma because of their lower binding affinity towards hepatic receptors due to a change in lipid composition, particle size or the presence of apo C-III on the particle itself. All apo C specifically modulate the metabolism of TRL, which may contribute to the development of hyperlipidemia and other lipoprotein abnormalities in humans.107 Apo C-III plays an important role in the metabolism of plasma triglyceride, which can delay the catabolism of TRL by interfering with apo E-mediated receptor clearance of remnant particles from plasma. Apo C-III and apo E play a central role in controlling the plasma metabolism of TRL and apo C-III has been implicated as a potential determinant of the TG lowering effect of fibrates, which down-regulate its expression.108 Li et al.109 investigated the protective effect of apoA-I, A-II, C-I and C-II, the main proteins in HDL on the morphology and function of human umbilical vein endothelial cells (HUVEC) injured with LDL in vitro. They cultured human endothelial cells derived from umbilical veins and exposed them to LDL, HDL, and apo A-I, AII, C-I and C-II. They then examine the morphology of the endothelial cells and measured the amount of lactate dehydrogenase (LDH) and 6-keto-prostaglandin F1 alpha (PGF1 alpha) released. The result showed contration of the endothelial cells, increased release of LDH and decreased secretion of prostacyclin (PGI2).109 However, 12

on addition of HDL, and apo A-I, A-II, C-I and C-II before incubation with LDL, cellular injury induced by LDL was inhibited. They concluded that apo A-I, A-II, C-I and C-II, as well as HDL, may play an important role in combating atherogenesis by protecting endothelial cells from damages induced by LDL.109 Hyperlipoproteinemia Hyperlipoproteinemia is the result of malfunction in the synthesis and/or catabolism of lipoproteins. The Fredrickson classification of hyperlipoproteinemia is based on the appearance of plasma as well as the total cholesterol and triglyceride values in the plasma (Table 3). The first comprehensive classification of the dyslipoproteinemias,which was formulated in 1965 and 1966 and adopted by the World Health Organization (WHO) described hyperlipoproteinemia as plasma phenotypes. The Fredrickson type are important because they allow focus on metabolic diseases related to hyperlipoproteinemia, identify hyperlipoproteinemia as disorders that affect particular lipoproteins, associate lipoprotein types with distinctive clinical features and provide the basis for successful diet and drug therapy. Often the only way to tell a type I from type V was the plasma appearance or a type III from type IV was to use a lipoprotein electrophoresis pattern. It is now more convenient to talk about where the metabolic defect is in lipoprotein metabolism. Lipoprotein disorders may be classified as primary or secondary. The primary disorders are either genetic or nongenetic. The secondary disorders may have their origin in diet, use of alcohol or drugs or disease of metabolic hormonal, infectious or malignant etiology.9,10 The origin of CAD which causes a high morbidity and mortality in most western countries involves a significant genetic component. The identity of some of these genetic mutations include those of LDL-R, apo B-100 and Lp(a). Other genetic loci that have been implicated include LPL, apo CII, cholesteryl ester transfer protein (CETP), apo A-I and LCAT.110 Hypertriglyceridemia Hypertriglyceridemia can have a primary or secondary etiology. In primary forms the origin is essentially genetic, while the secondary ones are consequent upon metabolism of various pathologies including renal, thyroid, diabetes mellitus etc.111 Diabetic dyslipoproteinemia characterized by hypertriglyceridemia, low HDL-C, and often elevated LDL-C with predominance of small, dense LDL is a strong risk factor for atherosclerosis.112 Hypertriglyceridemia is not a common finding in well controlled patients with IDDM; however, in NIDDM, hypertriglyceridemia and coronary heart disease are a well recognized clinical triad.113 In the latter setting, hypertriglyceridemia is usually the result of an associated inherited hyperlipidemia, most commonly familial hypertriglyceridemia but also familial combined hyperlipidemia (FCHL). In the former, one sees elevated TG and a low HDL-C, in the latter the same phenotype may be present but often there is a high LDL-C.113 Irrespective of the pathogenesis of the primary hypertriglyceridemic disorder, the occurrence of poorly controlled diabetes will enhance the hypertriglyceridemia and even in the NIDDM subject, with triglycerides in the thousands, dietary and glycemic control, alone, will strikingly ameliorate the hypertriglyceridemia.111,113 Familial hypertriglyceridemia (FHT) has been suggested to be an autosomal dominant condition with age-dependent penetrance, but so far the underlying defective gene has not been elucidated. Patients with type IV hyperlipoproteinemia, particularly those with FHT, have impaired absorption of bile acid, which may contribute to the hypertriglyceridemia. Increased production of cholesterol has been associated with type IV hyperlipidemia, but the influence of the confounding variable of obesity has been difficult to ascertain. Moreover, cholesterol metabolism has not been systematically evaluated in patients with FHT, one of the two major subsets of type IV patients.114 Familial Hyperchylomicronemia The familial hyperchylomicronemia may be divided into type I and V hyperlipoproteinemia.10 The familial hyperchylomironemia syndrome is a hereditary disorder of lipoprotein metabolism caused by LPL deficiency, apo-CII deficiency or LPL inhibition.115 The syndrome is characterized by hyperchylomicronemia, hypertriglyceridemia, pancreatitis and attacks of epigastric pain. The presence of eruptive xanthomas, may eventually lead to necrotizing pancreatitis or pancreatic insufficiency.116 Treatment consists of lifelong 13

adherence to a low-fat diet to prevent hyperchylomicronemia and its sequel. Apolipoprotein C-II plays a major role as a cofactor for lipoprotein lipase, the enzyme involved in the hydrolysis of triglyceride-rich particles. Human apo C-II consists of 79 amino acid residues and the amino-terminal two thirds of the molecule binds to lipid through the formation of amphipathic helixes, while the carboxy-terminal third is engaged in activation of LPL.117 Most cases of type I hyperlipoproteinemia are due to genetic defects in the LPL gene or in its activator, the apo C-II gene. Several cases of acquired type I hyperlipoproteinemia have also been described in the course of autoimmune diseases due to the presence of circulating inhibitors of LPL.118 Primary hyperchylomicronemia is known as a syndrome in which the accumulation of chylomicron occurs in the circulation. The main clinical symptoms of this disorder are the large increase in plasma triglyceride and cholesterol, and the presence of xanthomatous eruption, lipemia retinalis, hepatosplenomegaly, and the complication of acute pancreatitis. With gene analysis, a deficiency of LPL or apolipoprotein C-II is revealed as a main cause of primary chylomicronemia. In addition, in some cases, abnormalities of remnant receptors, the presence of antibody against LDL, apolipoprotein C-II, and LDL-R are seen as causes of chylomicronemia syndrome.119 Most individuals presenting with chylomicronemia have the familial forms of hypertriglyceridemia in combination with secondary acquired disorders.115 Type V hyperchylomicronemia is characterized by the presence of increased VLDL and chylomicrons in the plasma of fasting individuals on normal diet. Triglyceride levels similar to type I hyperlipoproteinemia may be present and the patients may experience abdominal syndromes including pancreatitis, eruptive xanthomas, lipemia retinalis and hepatosplenomegaly due to the increased TG level.10 Most cases occur in adulthood with females presenting later than men. Plasma cholesterol levels are usually slightly increased while LDL-C and HDL-C are usually normal to low. The defect appears to be associated with inadequate clearance of chylomicrons. Familial Hyperlipoproteinemia Familial type III hyperlipoproteinemia is a rare disorder, which affects 1 to 10 in 10,000 people in the general population.120 People with this disorder have premature atherosclerosis in peripheral vessels and coronary arteries. In addition, they have accumulations of chylomicron remnant and VLDL in the fasting state. Homozygosity for apolipoprotein 2 and accumulation of TRL in plasma are characteristic of this disease. Compared with other hyperlipoproteinemias, type III hyperlipoproteinemia usually responds to therapy. As a result of its clinical significance, the apo E polymorphism has been studied in several different human populations where, despite the wide variation observed in allele frequency distribution, apo E 3 allele was found to be the most common apo E allele.121 In addition to the common apo E isoforms, several rare apo E variants have been identified in hyperlipidemic patients and their kindred. Most of these variants are characterized by replacements of one or more charged amino acids by uncharged amino acids or vice versa.92 Some replacements in the LDL receptor-binding region (positions 136 to 150) cause defective binding to the LDL receptor and are associated with the recessive form of familial type III hyperlipoproteinemia. In addition, some replacements of basic amino acids with neutral or acidic amino acids lead to heparan sulfate proteoglycan (HSPG) binding which is associated with the dominant form of familial type III hyperlipoproteinemia Familial Combined Hyperlipidemia Familial combined hyperlipidemia (FCHL) is a complex disorder with several environmental factors, which interact with multiple genes. Elevated levels of total serum cholesterol and/or TG characterize it. The disorder is estimated to be common in Western populations with a prevalence of 1% to 2%. In addition, 14% of patients with premature CHD have FCHL which makes the disorder one of the most common genetic dyslipidemias underlying premature CHD.122 Familial combined hypertriglyceridemia has been suggested to be an autosomal dominant condition with age-dependent penetrance, but so far the underlying defective gene has not been elucidated. Lipoprotein lipase gene and apo A-I/C-III/A-IV gene cluster might be involved in familial clustering 14

of hypertriglyceridemia. Heterozygous LPL deficiencies caused by several types of gene mutation are known to result in a partial defect in catabolism of VLDL, causing mild to moderate hypertriglyceridemia. However, although the mutation of LPL gene results in reduced lipolytic activity, this type of dyslipidemia appears to manifest only if VLDL-TG production is also increased. These suggest that overproduction of VLDL-TG is a more important cause of hypertriglyceridemia than is the LPL deficiency.123 Familial Hypercholesterolemia Familial defective apolipoprotein B-100 (FDB) and familial hypercholesterolemia (FH) are the common causes of monogenic primary hypercholesterolemia. They are both associated with severe hypercholesterolemia and cannot always be distinguished from one another phenotypically. Familial defective apolipoprotein B-100 is the most common known mutation causing primary hypercholesterolemia.124 More than 150 mutations exist in the LDL-R gene, associated with FH an autosomal dominant inherited disorder characterized by severe hypercholesterolemia, frequent presence of tendon xanthomas and an elevated risk of premature CAD.125 Familial hypercholesterolemia is caused by different mutations in the LDL-R gene or by a guanine to adenine mutation in exon 26 of the apolipoprotein B gene which causes FDB.126 Familial defective apo B-100 is an autosomal codominant disorder leading to plasma LDL-C elevation and CAD. It is caused by substitution of glutamine for arginine at amino acid residue 3500 of apo B-100, in the putative LDL-R binding domain of the mature protein. This results in reduced affinity of LDL for the LDL-R. The amino acid substitution decreases the binding capacity of the LDL for the LDL-R, which then leads to an increase in levels of plasma TC and LDL-C. The frequency of the mutation may be as high as 1 in 600 in the normal population and 1 in 500 to 1 in 700 in Europe and in North America.127,128 Table 3 Fredricksons Classification of Hyperlipoproteinemia Fredricksons Cholesterol Classification I Normal to increased Iia Increased to very increased Increased Triglycerides Very increased Normal Lipoprotein Pattern Changes Chylomicrons very increased LDL very increased VLDL and LDL increased VLDL and LDL very increased VLDL very increased, LDL increased Chylomicrons, VLDL and LDL very increased Appearance of Specimen Milky top layer Some Causes of hyperlipoproteinemia Insuliopenic diabetes mellitus, lupus erythematosus, Pancreatitis Clear Nephrotic syndrome, hypothyroidism, porphyria stress Slightly turbid Nephrotic syndrome, hypothyroidism, porphyria stress Turbid hypothyroidism, myxedema, diabetic acidosis, primary biliary cirrhosis Turbid Diabetes mellitus, nephrotic syndrome, pregnancy, alcoholism Milky Insuliopenic diabetes mellitus, nephrotic syndrome, alcoholism, 15

Iib

Increased

III

Normal to increased Normal to increased Normal to increased

Increased

IV

Increased

Very increased

myeloma, pancreatitis The vast majority of affected heterozygotes have TC and LDL-C levels well above the 95th percentile for age and gender; in contrast, HDL-C, VLDL-C and plasma TG are not affected by the mutation. 128 In FDB heterozygotes, about 70% of the LDL particles are mutant, which may alter their atherogenicity relative to LDL containing normal apo B.129 Maher et al.129 compared CAD in heterozygous FDB with CAD in heterozygous FH and found that there was no significant difference between the FDB and FH patients in the type of cardiac symptoms or their age of onset and coronary angiographic appearance was similar in both groups. They then surmised that the LDL particles with the R3500Q mutation in apo B have the same atherogenicity as the LDL particles with normal apo B. To compare the phenotypic expression of either defects, Brugger et al.126 studied patients with FH and FDB from Germany and found that the average TC level in plasma was 413.7 mg/dL in FH and 321.8 mg/dL in FDB patients. Patients with FH had a significantly higher risk of myocardial infarction, coronary artery bypass graft, positive coronary angioplasty, atherosclerotic plaques in the carotid arteries and CAD than patients with FDB. This finding is contrary to those of Maher et al.129 who found no significant difference between similar groups of patients. Because FDB is one of the independent causes of early onset CHD, the R3500Q mutation should be considered in families with a high frequency of cardiovascular diseases. Abetalipoproteinemia Abetalipoproteinemia is a rare disease in which apo B is not synthesized and as a result, lipoproteins containing this apolipoprotein are not formed resulting in the accumulation of lipid droplets in the intestine and the liver, due to an inability to produce chylomicrons and VLDL in the intestine and liver, respectively. The disease results from mutations in the gene encoding the 97-kd subunit of the microsomal triglyceride transfer protein, which plays a central role on secretion of lipoprotein from the liver and the intestine. It catalyzes the transfer of TG, cholesteryl ester and phosphatidylcholine between membranes and lipoproteins. Downstream effects resulting from this defect include malnutrition, very low plasma cholesterol and TG levels, altered lipid and protein compositions of membranes and lipoprotein particles, and vitamin deficiencies. Unless treated, abetalipoproteinemic subjects develop gastrointestinal, neurological, ophthalmological, and hematological abnormalities.130,131 Hypobetalipoproteinemia Familial hypobetalipoproteinemia (FHBL) is a co-dominant disorder characterized by reduced plasma levels of LDL-C. It can be caused by mutations in the gene encoding apo B-100, leading to the formation of truncated apo Bs which have a reduced capacity to export lipids from the hepatocytes as lipoprotein constituents.132 It is characterized by less than fifth percentile age- and sex-specific levels of apo B and LDL-C. In a minority of cases, FHBL is due to truncation-producing mutations in the apo B gene on chromosome 2p23-24.133 Low LDLC and apo B levels in plasma cosegregate with mutations of apo B in some kindreds with FHBL. Approximately 35 apo B mutations, many specifying apo B truncations, have been described. Based on the percentile nomenclature where the full-length nature apo B consisting of 4536 amino acids is designated as apoB-100, only those truncations of apo B >25% of normal length are detectable in plasma.133 Familial hypoalphalipoproteinemia Isolated deficiency of HDL-C is genetic in presentation and is known as familial hypoalphalipoproteinemia (FHA). Hypoalphalipoproteinemia (HA) is a common finding in patients with premature CAD. It is present in about 5% of patients with premature CHD. Some patients with the disease have decreased production of HDL. Plasma half-life of HDL in normal individuals ranges from 3.3 to 5.8 days. Catabolism of HDL is enhanced in nephrotic patients but decreased in hypertriglyceridemic subjects and greatly enhanced in familial HDL deficiency (Tangier disease).10 Familial hypoalphalipoproteinemia syndromes are phenotypically heterogeneous. One form is associated with abnormal cellular cholesterol efflux caused by heterozygous mutations at the ABCA1 gene, that defines familial HDL deficiency while homozygous mutations or compound heterozygosity causes Tangier disease.134 Tangier disease is characterized by deficiency of HDL and their major 16

protein constituent apo A-I as well as presence of low molecular mass lipoproteins and a high concentration of apo C-III in the lipoprotein fraction. ABCA1 heterozygotes have decreased HDL-C and increased TG. Age is an important modifier of the phenotype in heterozygotes with higher proportion of them aged 30-70 years having HDL-C greater than the 5th percentile for age and sex compared with carriers less than 30 years if age.135 Hyperalphaliporoteinemia Cholesteryl ester transfer protein is a plasma glycoprotein that mediates the transfer of cholesteryl ester from HDL to triglyceride-rich lipoproteins in exchange for triglycerides.136 Since CETP regulates the plasma levels of HDL cholesterol and the size of HDL particles, it is considered to be a key protein in RCT, which is a protective system against atherosclerosis. Cholesteryl ester transfer protein and plasma phospholipid transfer protein belong to members of the lipid transfer and lipopolysaccharide-binding protein gene family, which include the lipopolysaccharide-binding protein (LBP) and bactericidal and permeability-increasing protein. The proteins possess different physiological functions, but share marked biochemical and structural similarities. The importance of plasma CETP in lipoprotein metabolism was demonstrated by the discovery of CETP-deficient subjects with a marked hyperalphalipoproteinemia.137 There is no agreement about whether plasma cholesteryl ester transfer protein (CETP) deficiency is associated with an antiatherogenic state or not, although this disorder was reported to be one of the major causes of marked hyperalphalipoproteinemia.138 However, hyperalphalipoproteinemia is regarded by some as a beneficial state accompanied by a longevity syndrome.139 Hirano et al.138 performed a large population based study concerning the atherogenicity of markedly elevated HDL-C levels in a genetically homogeneous population to determine whether CETP deficiency is associated with decreased atherogenicity. The study revealed that the frequency of the CETP gene mutation was higher in patients with CHD than in control patients. This indicated that marked hyperalphalipoproteinemia caused by CETP gene mutation may not represent a longevity syndrome. There might therefore be a need to revisit and reevaluate the clinical significance and pathophysiology of a marked hyperalphalipoproteinemia.138 Familial hyperalphalipoproteinemia Familial hyperalphalipoproteinemia (FHA) is a heritable trait associated with elevated plasma concentrations of HDL-C and possibly with longevity and protection against CHD. Individual with this condition are known to have elevated plasma levels of HDL-C and apo A-I, which may be due to a selective upregulation of apo A-I production, which might have antiantherogenic properties. Rader et al.140 studied the production rate of apo A-I and apo A-II in FHA individuals and control subjects and found that the production rate of apo A-I was markedly increased in FHA subjects than in control subjects while the production rate of apo A-II was not substantially increased. Hyperalphalipoproteinemia due to complete deficiency of cholesteryl ester transfer activity is characterized by the presence of both small polydisperse LDL and markedly large HDL enriched with cholesteryl ester and apo E. Laboratory Measurements of Lipids Phospholipids Measurement phospholipids have in the past been done by thin-layer chromatography. Theseparation of the phospholipids is based on the side chains groups and involves extraction of the of the phospholipids into solvent, drying for concentration, spotting onto silica gel plates, separation with a chloroform-methanolwater solvent and visualizing through charring and iodination.141 Spectrophotometric methods can also be employed to quantitate the phospholipid but inorganic phosphorus are used as indicators.141 Cholesterol Measurement of cholesterol includes both the ester and the free forms of the steroid. In serum or plasma, two thirds of the total cholesterol exist in the esterified form with the rest in the free form.10 This can result in greater intensity in the color produced with the cholesterol ester than with the free cholesterol. The implication of which is a large positive bias. In enzymatic reactions, the hydrolysis of the longer-chain cholesterol esters, 17

such as cholesterol arachidonate is not complete which may lead to a negative bias.10 The methods of cholesterols are usually modifications of (1) Liebermann-Burchard, (2) iron-salt-acid, (3) p-toluene-sulfonic acid, or (4) enzymatic end point. The Lierbermann-Burchard procedure is the most widely used method and early analytical methods used strong acids such as sulfuric and acetic acids and chemicals such as acetic anhydride or ferric chloride, which produced a measurable color with cholesterol. The current reference method uses hexane extraction after hydrolysis with alcoholic potassium hydroxide followed by reaction with Liebermann-Burchard color reagent, which comprises sulfuric and acetic acid and acetic anhydride.56 An enzymatic method for cholesterol employs the enzyme cholesteryl ester hydrolase, which cleaves the fatty acid residue and thus convert cholesteryl ester to unesterified or free cholesterol. The free cholesterol is reacted by cholesterol oxidase, which produces cholest-4en-3-on and hydrogen peroxide. The hydrogen peroxide produced is a substrate for an enzymatic color reaction, which employs horseradish peroxidase to couple two colorless chemicals into a colored compound.56 The enzymatic method, has been applied to automated procedures including dry-chemistry approach. Use of electrode systems is another approach for quantifying cholesterol. The oxygen selective membrane is used to measure the rate of oxygen consumption when the serum is reacted with a reagent containing cholesterol oxidase.141 Triglycerides The measurement of triglycerides and cholesterol is usually used to detect genetic and other metabolic problem such as hyperlipoproteinemias. The measurement of triglycerides is also required for the estimation of LDL cholesterol concentration by the Friedewald equation.68 Most triglyceride methods employ lipases and proteases to cleave fatty acids from glycerol and the glycerol is converted to glycerol-3-phosphate and adenosine diphosphate (ADP) in the presence of adenosine triphosphate (ATP) and glycerol kinase. The ADP is reacted with phosphoenolpyruvate in the presence of pyruvate kinase to ATP and pyruvate. The pyruvate is reacted with nicotinamide adenine dinucleotide (NADH) in the presence of lactate dehydrogenase and converted to lactate and reduced NADH and the absorbance is measured at 340 nm. Other methods using fluorometric measurement in which the disappearance of NADH fluorescence is read at 460 nm after excitation at 355 nm. The method is usually direct, rapid, and specific one of several enzymatic sequences. Lipoproteins The lipoproteins HDL and LDL are usually quantified based on their cholesterol content. The lipoproteins may be separated and quantified based on their densities, sizes, charge, and apolipoprotein contents. The range of observed densities among lipoprotein classes are a function of their lipid and protein content and they allows for fractionation by ultracentrifugation. Separation by electrophoresis is made possible by the differences in charge and size. Antibodies that are specific for particular apolipoproteins can be used to bind and separate lipoprotein classes.56 Apolipoproteins The apolipoproteins may be measured by immunoassay methods using turbidimetric or nephelometric assays. Other methods that have been used to quantify apolipoproteins include radioimmunoassay (RIA) radioimmunodetection (RID) and Enzyme labeled immunosorbent immunoassay (ELISA).56 Either polyclonal antibodies or monoclonal ones may be employed in these assays. Various molecular methods may be employed to genotype apolipoprotein E including genotyping of the apo E locus by isoelectric focusing, by restriction digestion of a PCR fragment of the gene, and by allele-specific oligonucleotide hybridization. The isoelectric focusing method is long and cumbersome, while earlier restriction digestion methods using the enzyme HhaI produce too many small fragments to permit easy interpretation. Conclusion Epidemiological, clinical, genetic, experimental and pathological studies have established primary roles for lipids, lipoprotein and apolipoproteins in atherogenesis.142,143 It is well established that lipids, lipoproteins and apoliporoteins are useful in diagnosing and prognosticating therapeutic intervention in the management of hyperlipidemic conditions in patients.144,145 There is increased interest in the measurement of apolipoproteins 18

because of the suggestion that they might be better predictors of coronary heart disease than lipid parameters currently used.68,146 According to Jungner et al.145 apo B appears to give more accurate predictive estimate of atherogenic risk than TC or LDL-C. Apo B and A-I can be determined with high precision and accuracy and with automation can reduce cost. Low levels of apo A-I/B ratios in individuals has been found to be indicative of susceptibility to CHD.68 Thus measurement of apo A-I/B ratio may be useful in the assessment of CHD risk in individuals. Numerous clinical studies have indicated the usefulness of Lp(a) as a risk marker for atherosclerotic diseases.39,48,50,51 Therefore patients with increased levels of Lp(a) and unstable angina may be at greater risk of suffering a myocardial infarction. Studies have shown that apo E genotyping might be helpful in predicting susceptibility to AD.98-100 Apo E has also been implicated in CHD. 83,90,91 Low-density lipoproteincholesterol and apo B-100 concentrations in serum have direct relationship with risk for CAD.146-148 At the same time, elevated levels of HDL-C and apo A-I are inversely related to CHD risk.74,75,76 It is therefore obvious that information about lipid, lipoprotein and apolipoprotein and their biochemistry and metabolism are very relevant to the health of the population at large. This information may be applied to successfully diagnosis and management of CAD cases. The desire to reduce lipid levels in individuals should be embraced by all as part of the overall program designed to achieve the healthy people 2010 initiative.149 People should be aware of the relationships between the various lipid components and the disease processes. References: 1. Jialal I. Evolving lipoprotein risk factors: lipoprotein(a) and oxidized low-density lipoprotein Clin Chem 1998;44:1827-1832. 2. Orford JL, Kinlay S, Ganz P, Selwyn AP. Treating Ambulatory Ischemia in Coronary Disease by Manipulating the Cell Biology of Atherosclerosis. Curr Atheroscler Rep 2000;2(4):321-326 3. Parhami F, Garfinkel A, Demer LL. Role of lipids in osteoporosis. Arterioscler Thromb Vasc Biol 2000;20(11):2346-8 4. Stulc T, Ceska R, Horinek A, Stepan J. Hyperlipoproteinemia, the Apo-E genotype and bone density. Cas Lek Cesk 200010;139(9):267-71 5. Mormando RM. Applying the second National Cholesterol Education Program report to geriatric medicine. Geriatrics 2000;55(8):48-53 6. Shah PK. Focus on HDL: a new treatment paradigm for athero-thrombotic vascular disease. Expert Opin Investig Drugs 2000;9(9):2139-46 7. Hay man LL. Abnormal blood lipids: is it environment or is it genes? J. Cardiovascular Nursing 2000;14:39-49 8. Danik M, Champagne D, Petit-Turcotte C, Beffert U, Poirier J. Brain lipoprotein metabolism and its relation to neurodegenerative disease. Crit Rev Neurobiol 1999;13(4):357-407 9. Tietz NW. Editor. Textbook of clinical chemistry. 1986. W. B. Saunders Company Philadelphia. 10. Kaplan LA, Pesce AJ. Clinical Chemistry; Theory, Analysis, and Correlation. 1989. The C. V. Mosby Company, St Louis pp. 454-483 11. Devlin TM. Textbook of Biochemistry with Clinical Correlations. 1992. Wiley-Liss, New York pp. 387-473 12. Murray RK, Granner DK, Mayes PA, Rodwell VW. Harpers Biochemistry, 23rd ed. Appleton and Lange, Norwalk. pp. 212-278 1993 13. Austin MA. Plasma triglyceride and coronary heart disease. Arteriosclerosis and Thrombosis 1991;11:2-14 14. Rubins HB Triglycerides and coronary heart disease: implications of recent clinical trials. J Cardiovasc Risk 2000;7(5):339-45 15. Gowri MS, Van der Westhuyzen DR, Bridges SR, Anderson JW. Decreased protection by HDL from poorly controlled type 2 diabetic subjects against LDL oxidation may Be due to the abnormal composition of HDL. Arterioscler Thromb Vasc Biol 1999;19(9):2226-2233 16. Chanu B. Hypertriglyceridemia: danger for the arteries. Presse Med 1999;28(36):2011-7 17. Vance DE, Van den Bosch H. Cholesterol in the year 2000. Biochim Biophys Acta 2000;1529(1-3):1-8 18. Gregory L C, Duh S, Christenson RH. Eight compact analysis systems evaluated for measuring total cholesterol. Clin Chem. 1994;40:579-585 19

Bachorik PS. Ross JW. National Cholesterol Education Program recommendations for measurement of lowdensity lipoprotein cholesterol: executive summary. The National Cholesterol Education Program Working Group on Lipoprotein Measurement. Clin Chem 1995;41:1414-1420. 20. Ansell BJ. Cholesterol, Stroke Risk, and Stroke Prevention. Curr Atheroscler Rep 2000;2(2):92-96 21. Westphal S, Orth M, Ambrosch A, Osmundsen K, Luley C. Postprandial chylomicrons and VLDLs in severe hypertriacylglycerolemia are lowered more effectively than are chylomicron remnants after treatment with n-3 fatty acids. Am. J. Clin. Nutr. 2000;71:914-920 22. Knott TJ, Pease RJ, Powell LM, Wallis SC, Rall SC Jr, Innerarity TL, Blackhart B, Taylor WH, Marcel Y, Milne R, et al Complete protein sequence and identification of structural domains of human apolipoprotein B. Nature 1986;323(6090):734-8 23. Hevonoja T, Pentikainen MO, Hyvonen MT, Kovanen PT, Ala-Korpela M. Structure of low density lipoprotein (LDL) particles: basis for understanding molecular changes in modified LDL. Biochim Biophys Acta 2000 ;1488(3):189-210 24. Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low density cholesterol in plasma without the use of the preparative ultracentrifugation. Clin. Chem. 1972;18:499-502 25. Salomon RG, Kaur K, Batyreva E. Isolevuglandin-protein adducts in oxidized low density lipoprotein and human plasma. A strong connection with cardiovascular disease. Trends Cardiovasc Med 2000;10(2):53-9 26. Carr AC, Myzak MC, Stocker R, McCall MR, Frei B. Myeloperoxidase binds to low-density lipoprotein: potential implications for atherosclerosis. FEBS Lett 2000;487(2):176-180 27. Gordon BR. Incorporation of Low-density Lipoprotein Apheresis into the Treatment Program of Patients with Severe Hypercholesterolemia. : Curr Atheroscler Rep 2000;2(4):308-313 28. Campos H, Wilson PW, Jimenez D, McNamara JR, Ordovas J, Schaefer EJ. Differences in apolipoproteins and low-density lipoprotein subfractions in postmenopausal women on and off estrogen therapy: results from the Framingham Offspring Study. Metabolism 1990;39(10):1033-8 29. Nauk M, Winkler K, Wittmann C, Mayer H, Luley C, Marz W, Wieland H. Direct determination of lipoprotein(a) cholesterol by ultracentrifugation and agarose gel electrophoresis with enzymatic staining for cholesterol. Clin. Chem. 1995;41:731-738 30. Seman LJ, DeLuca C, Jenner JL, Cupples LA, McNamara JR, Wilson PW, Castelli WP, Ordovas JM, Schaefer EJ. Lipoprotein(a)-cholesterol and coronary heart disease in the Framingham Heart Study. Clin Chem 1999 Jul;45(7):1039-46 31. Berg K, Dahlen G, Frick MH. Lp(a) lipoprotein and pre-beta1-lipoprotein in patients with coronary heart disease. Clin Genet 1974;6(3):230-5 32. Kostner GM, Ibovnik A, Holzer H, Grillhofer H. Preparation of a stable fresh frozen primary lipoprotein[a] (Lp[a]) standard. J. Lipid Res. 1999;40:2255-2263 33. Wassef GN. Lipoprotein (a) in android obesity and NIDDM: a new member in 'the metabolic syndrome'. Biomed Pharmacother 1999;53(10):462-5 34. Seman LJ, Jenner JL, McNamara, JR, Schaefer EJ. Quantification of lipoprotein(a) in plasma by assaying cholesterol in lecithin bound plasma fraction. Clin Chem 1994;40:400-403 35. Kamboh MI, McGarvey ST, Aston CE, Ferrell RE, Bausserman L. Plasma lipoprotein(a) distribution and its correlates among Samoans. Human Biology 2000;72(2):321-336 36. Klezovitch O, Edelstein C, Zhu L, Scanu AM. Apoliporotein(a) binds via its C-terminal domain to the protein core of the proteoglycan decorin. Implications for the retention of lipoprotein(a) in atherosclerotic lesions. J Biol Chem 1998;273(37):23856-23865 37. Min W, Lee JO, Huh JW. Relation between lipoprotein(a) concentrations in patients with acute-phase response and risk analysis for coronary heart disease. Clin Chem 1997;43:1891-1895. 38. Craig WY, Neveux LM, Palomaki GE, Cleveland MM, Haddow JE. Lipoprotein(a) as a risk factor for ischemic heart disease: metaanalysis of prospective studies. Clin Chem 199844:2301-2306. 39. Desmarais RL, Sarembock IJ, Ayers CR, Vernon SM., Powers ER, Gimple LW. Elevated Serum Lipoprotein(a) Is a Risk Factor for Clinical Recurrence After Coronary Balloon Angioplasty. Circulation 1995;91:1403-1409
19.

20

40. Futterman LG, Lemberg L. Lp(a) lipoprotein--an independent risk factor for coronary heart disease after menopause. Am J Crit Care 2001;10(1):63-7 41. Seed M, Ayres KL, Humphries SE, Miller GJ. Lipoprotein (a) as a predictor of myocardial infarction in middle-aged men. Am J Med 2001;110(1):22-7 42. Armstrong VW, Cremer P, Eberle E, Manke A, Schulze F, Wieland H, Kreuzer H, Seidel D. The association between serum Lp(a) concentrations and angiographically assessed coronary atherosclerosis. Dependence on serum LDL levels. Atherosclerosis 1986;62(3):249-57 43. Murai A, Miyahara T, Fujimoto N, Matsuda M, Kameyama M. Lp(a) lipoprotein as a risk factor for coronary heart disease and cerebral infarction. Atherosclerosis 1986;59(2):199-204 44. Gupta R, Vasisht S, Bahl VK, Wasir HS. Correlation of lipoprotein (a) to angiographically defined coronary artery disease in Indians. Int J Cardiol 1996;57(3):265-70 45. Cobbaert C, Arentsen JC, Mulder P, Hoogerbrugge, N Lindemans J. Significance of various parameters derived from biological variability of lipoprotein(a), homocysteine, cysteine, and total antioxidant status. Clin Chem 1997;43:1958-1964. 46. Biervliet JPV, Michiels G, Rosseneu M. Quantification of lipoprotein(a) in dried blood spots and screening for above-normal lipoprotein(a) concentrations in newborns. Clin Chem 1991.37:706-708 47. Vella JC, Jover E. Relation of lipoprotein(a) in 11- to 19-year-old adolescents to parental cardiovascular heart disease. Clin Chem 1993;39:477-480. 48. Zhao S, Xu D. Oxidized lipoprotein(a) enhanced the expression of P-selectin in cultured human umbilical vein endothelial cells. Thromb Res 2000;100(6):501-510 49. Falco C, Tormo G, Estelles A, Espana F, Tormo E, Gilabert J, Velasco JA, Aznar J. Fibrinolysis and lipoprotein(a) in women with coronary artery disease. Influence of hormone replacement therapy. Haematologica 2001;86(1):92-98 50. Zampoulakis JD, Kyriakousi AA, Poralis KA, Karaminas NT, Palermos ID, Chimonas ET, Cokkinos DV. Lipoprotein(a) is related to the extent of lesions in the coronary vasculature and to unstable coronary syndromes. Clin Cardiol 2000;23(12):895-900 51. Bostom AG, Cupples LA, Jenner JL, Ordovas JM, Seman LJ, Wilson PW, Schaefer EJ, Castelli WP. Elevated plasma lipoprotein(a) and coronary heart disease in men aged 55 years and younger. A prospective study. JAMA 1996 Aug 21;276(7):544-8 52. Paultre F, Pearson TA, Weil HF, Tuck CH, Myerson M, Rubin J, Francis CK, Marx HF, Philbin EF, Reed RG, Berglund L. High levels of lp(a) with a small apo(a) isoform are associated with coronary artery disease in african american and white Men. Arterioscler Thromb Vasc Biol 2000;20(12):2619-24 53. Frank PG, Marcel YL. Apolipoprotein A-I: structure-function relationships. J Lipid Res 2000;41(6):853-72 54. von Eckardstein A, Nofer JR, Assmann G. Acceleration of reverse cholesterol transport. Curr Opin Cardiol 2000;15(5):348-54 55. Anderson SC, Cockayne S. Clinical Chemistry, Concepts and Applications. W.B. Saunders and Company, Philadelphia pp. 165-187 1993 56. Bishop ML, Duben-Engelkirk JL, Fody EP. Clinical chemistry, principles, procedures, correlations. 3rd ed. Lippincott Williams and Wilkins, Philadelphia 2000 57. Jayakumari N, Raghu K, Kumari VA, Balakrishna KG, Iyer KS. Distribution of cholesterol in HDL and its subfractions in patients with coronary atherosclerotic heat disease. Indian heart J. 1993;45:265-268 58. Badimon JJ, Badimon L, Fuster V. Regression of atherosclerotic lesions by high density lipoprotein plasma fraction in the cholesterol-fed rabbit. J. Clin. Invest. 1990;85:1234-1241 59. Campos H, McNamara JR, Wilson PW, Ordovas JM, Schaefer EJ. Differences in low-density lipoprotein subfractions and apolipoproteins in premenopausal and postmenopausal women. J Clin Endocrinol Metab 1988;67(1):30-5 60. Van den Broek AJC, Hollaar L, Schaefer HIMB, Van der Laarse A, Schuster H, Defesche JC, Kastelein JJP, vant Hooft FM. Screening of familial defective apolipoprotein B-100 with improved U937 monocyte proliferation assay. Clin. Chem. 1994;40:395-399

21

Bachorik PS, Lovejoy KL, Carroll MD, Johnson CL. Apolipoprotein B and AI distributions in the United States, 19881991: results of the National Health and Nutrition Examination Survey III (NHANES III). Clin Chem 1997;43:2364-2378. 62. Kane JP, Hardman DA, Paulus H. Heterogeneity of apolipoprotein B isolation of a new species from human chylomicrons. Proc. Natl. Acad. Sci. USA. 1980;77:2465-2469 63. Fisher E, Scharnagl H, Hoffmann MM, Kusterer K, Wittmann D, Wieland H, Gross W, Winfried Mrz W. Mutations in the Apolipoprotein (apo) B-100 Receptor-binding region: Detection of apo B-100 (Arg3500 Trp) Associated with Two New Haplotypes and Evidence That apo B-100 (Glu3405 Gln) Diminishes Receptor-mediated Uptake of LDL. Clin Chem1999;45:1026-1038. 64. Mero N, Malmstrom R, Steiner G, Taskinen MR, Syvanne M. Postprandial metabolism of apolipoprotein B48- and B-100-containing particles in type 2 diabetes mellitus: relations to angiographically verified severity of coronary artery disease. Atherosclerosis 2000;150(1):167-77 65. Igau B, Lestavel S, Clavey V, Slomianny C, Drouin P, Bresson R, Fruchart JC, Duriez P, Fievet C. Apo Bcontaining lipoprotein particles in poorly controlled insulin-dependent diabetes. Atherosclerosis 1996;120(1-2):209-19 66. Islam S, Gutin B, Manos T, Smith C, Treiber F. Low density lipoprotein cholesterol/apolipoprotein B-100 ratio: interaction of family history of premature atherosclerotic coronary artery disease with race and gender in 7 to 11 year olds. Pediatrics 1994;94(4 Pt 1):494-9 67. Connelly PW, Poapst M, Davignon J, Lussier-Cacan S, Reeder B, Lessard R, Hegele RA, Csima A. Reference values of plasma apolipoproteins A-I and B, and association with nonlipid risk factors in the populations of two Canadian provinces: Quebec and Saskatchewan. Canadian Heart Health Surveys Research Group. Can J Cardiol 1999;15(4):409-18 68. Ogedegbe HO. Measurement of low-density lipoprotein cholesterol, apolipoprotein B-100 and apolipoprotein A-I concentrations in serum in coronary heart disease risk assessment. Diss. The Union Institute Cincinnati. UMI 1996 9625452 69. Hong SH, Park WH, Lee CC, Song JH, Kim JQ. Association between genetic variations of apo AI-CIII-AIV cluster gene and hypertriglyceridemic subjects. Clin Chem 1997;43:13-17. 70. Oram JF, Vaughan AM. ABCA1-mediated transport of cellular cholesterol and phospholipids to HDL apolipoproteins. Curr Opin Lipidol 2000;11(3):253-60 71. Eriksson M, Carlson LA, Miettinen TA, Angelin B. Stimulation of fecal steroid excretion after infusion of recombinant proapolipoprotein A-I. Potential reverse cholesterol transport in humans. Circulation 199910;100(6):594-8 72. Anderson SC, Cockayne S, Clinical Chemistry, Concepts and Applications. W.B. Saunders and Company, Philadelphia pp. 165-187 1993 73. Nakaya Y, Hiasa Y, Motimoto S. Plasma lipids and coronary atherosclerosis in Japanese men. Tokushima J. Exp. Med 1993;40:177-182 74. Schaefer JR, Schweer H, Ikewaki K, Stracke H, Seyberth HJ, Kaffarnik H, Maisch B, Steinmetz A. Metabolic basis of high density lipoproteins and apolipoprotein A-I increase by HMG-CoA reductase inhibition in healthy subjects and a patient with coronary artery disease. Atherosclerosis 1999;144(1):17784 75. Akanji AO, Mojiminiyi OA, Abdella N. Beneficial changes in serum apo A-1 and its ratio to apo B and HDL in stable hyperlipidaemic subjects after Ramadan fasting in Kuwait. Eur J Clin Nutr 2000;54(6):50813 76. Han H, Sasaki J, Matsunaga A, Hakamata H, Huang W, Ageta M, Taguchi T, Koga T, Kugi M, Horiuchi S, Arakawa K A. novel mutant, ApoA-I nichinan (Glu235-->0), is associated with low HDL cholesterol levels and decreased cholesterol efflux from cells. Arterioscler Thromb Vasc Biol 1999;19(6):1447-55 77. Tilly-Kiesi M, Zhang Q, Ehnholm S, Kahri J, Lahdenpera S, Ehnholm C, Taskinen MR. ApoA-IHelsinki (Lys107-->0) associated with reduced HDL cholesterol and LpA-I:A-II deficiency. Arterioscler Thromb Vasc Biol 1995;15(9):1294-306 78. Daum U, Langer C, Duverger N, Emmanuel F, Benoit P, Denefle P, Chirazi A, Cullen P, Pritchard PH, Bruckert E, Assmann G, von Eckardstein A. Apolipoprotein A-I (R151C)Paris is defective in activation of 22
61.

lecithin: cholesterol acyltransferase but not in initial lipid binding, formation of reconstituted lipoproteins, or promotion of cholesterol efflux. J Mol Med 1999;77(8):614-22 79. Franceschini G, Calabresi L, Chiesa G, Parolini C, Sirtori CR, Canavesi M, Bernini F. Increased cholesterol efflux potential of sera from ApoA-IMilano carriers and transgenic mice. Arterioscler Thromb Vasc Biol 1999;19(5):1257-62 80. Hargrove GM, Junco A, Wong NC. Hormonal regulation of apolipoprotein AI. J Mol Endocrinol 1999;22(2):103-11 81. Huang Y, Ji Z, et al. 1999. Overexpression of apolipoprotein E3 in transgenic rabbits causes combined hyperlipidemia by stimulating hepatic VLDL production and impairing VLDL lipolysis. Arterioscler. Thromb. Vasc. Biol. 1999;19:2952-2959 82. Willems van Dijk k., van Vlijmen BJM. Hyperlipidemia of apo E2 (Arg158-Cys) and apo E3-Leiden transgenic mice is modulated predominantly by LDL receptor expression. Thromb Vasc Biol. 19999;19:2945-2951 83. Dzimiri N, Meyer BF, Hussain SS, Basco C, Afrane B, Halees Z. Relevance of apolipoprotein E polymorphism for coronary artery disease in the Saudi population. Arch. Pathol. Lab. Med. 1999;123:12411245 84. Kardaun JWPF, White L, Resnick HE, Petrovitch H, Marcovina SM, Saunders AM, Foley DJ, Havlik RJ. Genotypes and Phenotypes for Apolipoprotein E and Alzheimer Disease in the Honolulu-Asia Aging Study. Clin Chem 2000;46:1548-1554. 85. Decarli C, Reed T, et al. Impact of apolipoprotein E 4 and vascular disease on brain morphology in men from the NHLBI twin study. Stroke. 1999;30:1548-1553 86. Dart AM, Cooper B. Independent effects of apo E phenotype and plasma triglyceride on lipoprotein particle sizes in the fasting and postprandrial states. Arterioscler Thromb vasc Biol. 1999;19:2465-2473 87. Hagberg JM, Wilund KR, Ferrell RE. APO E gene and gene-environment effects on plasma lipoproteinlipid levels. Physiol Genomics 200018;4(2):101-108 88. Ballantyne CM, Herd JA, Stein EA, Ferlic LL, Dunn JK, Gotto AM, Marian AJ. Apolipoprotein E genotypes and response of plasma lipids and progression-regression of coronary atherosclerosis to lipidlowering drug therapy. J Am Coll Cardiol 2000;36(5):1572-8 89. Scuteri A, Bos AJ, Zonderman AB, Brant LJ, Lakatta EG, Fleg JL. Is the apoE4 allele an independent predictor of coronary events? Am J Med 2001;110(1):28-32 90. Evangelou N, Jackson M, Beeson D, Palace J. Association of the apo E 4 allele with disease activity in multiple sclerosis. J. Neurol. Neurosurg. Psychiatry. 1999;67:203-205 91. Wilson PW, Schaefer EJ, Larson MG, Ordovas JM. Apolipoprotein E alleles and risk of coronary disease. A meta-analysis. Arterioscler Thromb Vasc Biol 1996;16(10):1250-5 92. Fernandes MAS, Oliveira CR. et al. Apolipoprotein E 4 allele is a risk factor for Alzheimers disease: The Central Region of Portugal (Coimbra) as a case study. Eur Neurol 1999;42:183-184 93. Danet S, Brousseau T, Richard F, Amouyel P, Berr C. Risk of dementia in parents of probands with and without the apolipoprotein E4 allele. The EVA study. J. Epidemiol. Community Health .1999;53:393-398 94. Orth M, Weng W, et al. Effects of a frequent apolipoprotein E isoform, apo E4Freiburg (Leu28 to Pro), on lipoproteins and the prevalence of coronary artery disease in whites. Arterioscler. Thromb. Vasc. Biol. 1999;19:1306-1315 95. Schellenberg GD, D'Souza I, Poorkaj P. The Genetics of Alzheimer's disease. Curr Psychiatry Rep 2000;2(2):158-164 96. Saunders AM. Apolipoprotein E and Alzheimers disease: an update on genetic and functional analyses. J Neuropathol Exp Neurol 2000;59(9):751-758 97. Hu C, Sung S, Liu H, Hsu W, Lee L, Lee C, Tsai C, Chang J. Genetic risk factors of sporadic Alzheimer's disease among Chinese in Taiwan. J Neurol Sci 2000 1;181(1-2):127-31 98. Yasuda M, Hirono N, et al. Case-control study of presenilin-1 intronic polymorphism in sporadic early and late onset Alzheimers disease. J. Neurol. Neurosurg. Psychiatry. 1999;66:722-726 23

99. Poirier, J. Apolipoprotein E: A pharmacogenic target for treatment of Alzheimers disease. Mol. Diagn. 1999;4:335-341 100. Small GW, Chen ST. et al. Memory self-appraisal in middle-aged and older adults with the apolipoprotein E4 allele. Am J Psychiatry. 1999;156:1035-1038 101. Rassart E, Bedirian A, Do Carmo S , Guinard O, Sirois J, Terrisse L, Milne R. Apolipoprotein D. Biochim Biophys Acta 200018;1482(1-2):185-98 102. Vazquez J, Gonzalez L, Merino A, Vizoso F. Expression and clinical significance of apolipoprotein D in epithelial ovarian carcinomas. Gynecol Oncol 2000;76(3):340-7 103. Lane DM, Boatman KK, McConathy WJ. Serum lipids and apolipoproteins in women with breast masses. Breast Cancer Res Treat 1995;34(2):161-9 104. Kalman J, McConathy W, Araoz C, Kasa P, Lacko AG. Apolipoprotein D in the aging brain and in Alzheimer's dementia. Neurol Res 2000;22(4):330-6 105. Aspinall JO, Bentel JM, Horsfall DJ, Haagensen DE, Marshall VR, Tilley WD. Differential expression of apolipoprotein-D and prostate specific antigen in benign and malignant prostate tissues. J Urol 1995;154(2 Pt 1):622-8 106. Suresh S, Yan Z, Patel RC, Patel YC, Patel SC. Cellular cholesterol storage in the Niemann-Pick disease type C mouse is associated with increased expression and defective processing of apolipoprotein D. J Neurochem 1998;70(1):242-51 107. Jong MC, Havekes LM. Insights into apolipoprotein C metabolism from transgenic and gene-targeted mice. Int J Tissue React 2000;22(2-3):59-66 108. Attia N, Durlach V, Cambilleau M, Roche D, Girard-Globa A. Postprandial concentrations and distribution of apo C-III in type 2 diabetic patients. Effect Of bezafibrate treatment. Atherosclerosis 2000;149(2):427-33 109. Li J, Jiang L, Liu Q Protective effect of apolipoprotein A I, A II, C I and C II on endothelial cells injury induced by low density lipoprotein.) Chin Med J (Engl) 1998;111(1):78-81 110. Ozturk IC, Killeen AA An overview of genetic factors influencing plasma lipid levels and coronary artery disease risk. Arch Pathol Lab Med 1999;123(12):1219-22 111. Malaguarnera M, Giugno I, Ruello P, Vinci E, Panebianco MP, Motta M. Treatment of hypertriglyceridemia. Current aspects. Recenti Prog Med 2000 Recenti Prog Med 2000 Jul-Aug;91(78):379-87;91(7-8):379-87 112. Frost RJ, Otto C, Geiss HC, Schwandt P, Parhofer KG. Effects of atorvastatin versus fenofibrate on lipoprotein profiles, low-density lipoprotein subfraction distribution, and hemorheologic parameters in type 2 diabetes mellitus with mixed hyperlipoproteinemia. Am J Cardiol 2001 Jan 1;87(1):44-48 113. Fisher WR. Hypertriglyceridemia in diabetes. An approach to management. J Fla Med Assoc 1991;78(11):747-50 114. Duane WC. Cholesterol metabolism in familial hypertriglyceridemia: effects of obesity versus triglyceride level. J Lab Clin Med 1997;130(6):635-42 115. Santamarina-Fojo S. The familial chylomicronemia syndrome. Endocrinol Metab Clin North Am 1998;27(3):551-67, viii 116. de Graaf J, Hoffer MJ, Stuyt PM, Frants RR, Stalenhoef AF. Familial chylomicronemia caused by a novel type of mutation in the APOE-CI-CIV-CII gene cluster encompassing both the APOCII gene and the first APOCIV gene mutation: APOCII-CIV(Nijmegen). Biochem Biophys Res Commun 2000;273(3):1084-7 117. Olivecrona G, Beisiegel U. Lipid binding of apolipoprotein CII is required for stimulation of lipoprotein lipase activity against apolipoprotein CII-deficient chylomicrons. Arterioscler Thromb Vasc Biol 1997;17(8):1545-9 118. Garcia-Otin AL, Civeira F, Peinado-Onsurbe J, Gonzalvo C, Llobera M, Pocovi M. Acquired lipoprotein lipase deficiency associated with chronic urticaria. A new etiology for type I hyperlipoproteinemia. Eur J Endocrinol 1999;141(5):502-5 119. Iwasaki M, Tada N. Primary hyperchylomicronemia and gene defects. Nippon Rinsho 1999;57(12):2759-64 120. Wang T, Nakajima K, Leary ET, Warnick GR, Cohn JS, Hopkins PN, Wu LL, Cilla DD, Zhong J, Havel RJ. Ratio of Remnant-like Particle-Cholesterol to Serum Total Triglycerides Is an Effective Alternative to 24

Ultracentrifugal and Electrophoretic Methods in the Diagnosis of Familial Type III Hyperlipoproteinemia. Clin Chem 1999;45:1981-1987. 121. Seixas, S., Trovoda, M. J., and Rocha, J. 1999. Haplotype analysis of apolipoprotein E and apolipoprotein C1 loci in Portugal and So Tom e Prncipe (Gulf of Guinea): Linkage disequilibrium evidence that Apo E 4 is the ancestral apo E allele. Human Biology. Wayne State University Press Detroit 1999;70(6):1001-1008 122. Pajukanta P, Porkka KV Genetics of Familial Combined Hyperlipidemia. Curr Atheroscler Rep 1999;1(1):79-86 123. Kotake H, Oikawa S. Primary hypertriglyceridemia. Nippon Rinsho 1999;57(12):2782-8 124. Geisel J, Schleifenbaum T, Oette K, Weisshaar B. Familial defective apolipoprotein B-100 in 12 subjects and their kindred. Eur J Clin Chem Clin Biochem 1992;30(11):729-36 125. Choong M, Koay ESC, Khoo K, Khaw M, Sethi SK. Denaturing gradient-gel electrophoresis screening of familial defective apolipoprotein B-100 in a mixed Asian cohort: two cases of arginine3500 tryptophan mutation associated with a unique haplotype. Clin Chem 1997;43:916-923. 126. Brugger D, Schuster H, Zollner N. Familial hypercholesterolemia and familial defective apolipoprotein B-100: comparison of the phenotypic expression In 116 cases. Eur J Med Res 1996;1(8):383-6 127. Friedl W. Familial defective apolipoprotein B-100: molecular basis, prevalence and clinical features. Wien Klin Wochenschr 1991;103(20):621-5 128. Tybjaerg-Hansen A, Humphries SE. Familial defective apolipoprotein B-100: a single mutation that causes hypercholesterolemia and premature coronary artery disease. Atherosclerosis 1992;96(2-3):91-107 129. Maher VM, Gallagher JJ, Thompson GR, Myant NB. Does the presence of the 3500 mutant apolipoprotein B-100 in low density lipoprotein particles affect their atherogenicity? Atherosclerosis 1995;118(1):105-10 130. Berriot-Varoqueaux N, Aggerbeck LP, Samson-Bouma M, Wetterau JR. The role of the microsomal triglygeride transfer protein in abetalipoproteinemia. Annu Rev Nutr 2000;20:663-97 131. Wang J, Hegele RA. Microsomal triglyceride transfer protein (MTP) gene mutations in Canadian subjects with abetalipoproteinemia. Hum Mutat 2000;15(3):294-5 132. Tarugi P, Lonardo A, Ballarini G, Erspamer L, Tondelli E, Bertolini S, Calandra S A study of fatty liver disease and plasma lipoproteins in a kindred with familial hypobetalipoproteinemia due to a novel truncated form of apolipoprotein B (APO B-54.5). J Hepatol 2000;33(3):361-70 133. Wu J, Kim J, Li Q, Kwok PY, Cole TG, Cefalu B, Averna M, Schonfeld G. Known mutations of apoB account for only a small minority of hypobetalipoproteinemia. J Lipid Res 1999;40(5):955-9 134. Mott S, Yu L, Marcil M, Boucher B, Rondeau C, Genest J. Decreased cellular cholesterol efflux is a common cause of familial hypoalphalipoproteinemia: role of the ABCA1 gene mutations. Atherosclerosis 2000;152(2):457-68 135. Clee SM, Kastelein JJ, van Dam M , Marcil M, Roomp K, Zwarts KY, Collins JA, Roelants R, Tamasawa N, Stulc T, Suda T, Ceska R, Boucher B, Rondeau C, DeSouich C, Brooks-Wilson A, Molhuizen HO, Frohlich J, Genest J, Hayden MR. Age and residual cholesterol efflux affect HDL cholesterol levels and coronary artery disease in ABCA1 heterozygotes. J Clin Invest 200015;106(10):1263-1270 136. Ordovas JM Genetic polymorphisms and activity of cholesterol ester transfer protein (CETP): should we be measuring them? Clin Chem Lab Med 2000;38(10):945-9 137. Yamashita S, Hirano K, Sakai N, Matsuzawa Y. Molecular biology and pathophysiological aspects of plasma cholesteryl ester transfer protein Biochim Biophys Acta 2000;1529(1-3):257-275 138. Hirano K, Yamashita S, Nakajima N, Arai T, Maruyama T, Yoshida Y, Ishigami M, Sakai N, KamedaTakemura K, Matsuzawa Y. Genetic cholesteryl ester transfer protein deficiency is extremely frequent in the Omagari area of Japan. Marked hyperalphalipoproteinemia caused by CETP gene mutation is not associated with longevity. Arterioscler Thromb Vasc Biol 1997;17(6):1053-9 139. Hirano K, Yamashita S, Kuga Y, Sakai N, Nozaki S, Kihara S, Arai T, Yanagi K, Takami S, Menju M, et al. Atherosclerotic disease in marked hyperalphalipoproteinemia. Combined reduction of cholesteryl ester transfer protein and hepatic triglyceride lipase. Arterioscler Thromb Vasc Biol 1995;15(11):1849-56 25

140. Rader

DJ, Schaefer JR, Lohse P, Ikewaki K, Thomas F, Harris WA, Zech LA, Dujovne CA, Brewer HB. Increased production of apolipoprotein A-I associated with elevated plasma levels of high-density lipoproteins, apolipoprotein A-I, and lipoprotein A-I in a patient with familial hyperalphalipoproteinemia. Metabolism 1993;42(11):1429-34 141. Bishop ML, Duben-Engelkirk JL, Fody EP. Clinical chemistry, principles, procedures, correlations. 2nd ed. Lippincott Williams and Wilkins, Philadelphia 1992 142. Havel RJ, Rapaport. Management of primary hyperlipidemia. The New Engl J Med 1995;332:14911498 143. Vander Heiden GL, Barboriak JJ, Sasse EA, Yorde DE. Correlation of the extent of coronary occlusion with apo B levels. Applications of a new enzyme immunoassay technique for apo B. Atherosclerosis. 1984;50: 29-33 144. NIH Consensus Development Conference. Lowering blood cholesterol to prevent heart disease. J Am Med Assoc. 1985;253:2080-2086 145. Jungner I, Santica M, Marcovina SM, Walldius G, Holme I, Kolar W, Steiner E. Apolipoprotein B and A-I values in 147 576 Swedish males and females, standardized according to the World Health Organization International Federation of Clinical Chemistry First International Reference Materials. Clin Chem 1997;43:1306-1310. 146. Riepponen P, Marniemi J, Rautaoja T. Immunoturbidimetric determination of apolipoprotein A-I and B in serum. Scand. J Clin Lan Invest. 1987;47:739-744 147. Rifai N, Warnick GR, McNamara JR, Belcher JD, Grinstead GF, Frantz ID. Measurement of lowdensity lipoprotein in serum: a status report. Clin Chem 1992;38:150-160 148. Marcovina SM, Adolphson JL, Pariavecchia M, Albers JJ. Effects of lyophilization of serum on the measurement of apolipoprotein A-I and B. Clin Chem 1990;36:366-369 149. Healthy People 2010. February 9, 2001. Available at URL: http://www.health.gov/healthypeople/

26