биохимия атеросклероза

and atherogenicity. These phenotypes have been assigned as pattern A when larger, more buoyant LDL and pattern B when smaller, more dense LDL predominate [1–5].

LDL size correlates positively with plasma high-density lipoproteins (HDL) levels and negatively with plasma triglyceride concentrations and the combination of small dense LDL, decreased HDL-cholesterol, and increased triglycerides has been called as the “atherogenic lipoprotein phenotype” [6] (Fig. 9.1). This partially heritable trait is a feature of the metabolic syndrome and is associated with increased cardiovascular risk.

LDL size seems also to be an important predictor of cardiovascular events and progression of coronary artery disease (CAD) and the predominance of small dense LDL have been accepted as an emerging cardiovascular risk factor by the National Cholesterol Education Program Adult Treatment Panel III [1, 2, 7].

Genetic and Environmental Influences

The predominance of small dense LDL is approximately 30% in adult men, 5–10% in young men and women less than 20 years, and approximately 15–25% in postmenopausal women [1, 2]. It has been shown that LDL size is genetically influenced with a heritability ranging from 35% to 45% based on an autosomal dominant or codominant model with varying additive and polygenic effects [8]. Thus, nongenetic and environmental factors influence

larger smaller

FIGURE 9.1. LDL heterogeneity and plasma triglyceride and HDL-cholesterol concentrations [2, 26]. ALP, atherogenic lipoprotein phenotype.

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the expression of this phenotype and an increase of small dense LDL has been shown for abdominal adiposity and oral contraceptive use [9–11].

Dietary factors are also important. It has been shown that a very low-fat, high-carbohydrate diet can induce the pattern B phenotype in persons genetically predisposed to this phenotype [12]. In addition, the predominance of small dense LDL is commonly found in conjunction with familial disorders of lipoprotein metabolism that are associated with increased risk of premature CAD, including familial combined hyperlipidemia, hyperbetalipoproteinemia, and hypoalphalipoproteinemia [13–15].

Heterogeneity of apoB-Containing Particles

It is commonly accepted today that apolipoprotein B (apoB) particles do not comprise a population with continuously variable size; instead there are a multiple subclasses with discrete size and density, different physicochemical composition, and different metabolic behavior. Based on their characteristic appearance in analytical ultracentrifugation and gradient gel electrophoresis distinct subclasses of very low-density lipoprotein (VLDL), intermediatedensity lipoprotein (IDL), and LDL particles have been defined [3, 16–32].

There are at least two subclasses of VLDL, two subclasses of IDL, and seven subspecies of LDL from large LDL I to very small LDL IVB [3]. Size differences of VLDL particles are mainly due to changes in core triglycerides while the coat consisting of apolipoproteins, free cholesterol, and phospholipids is of relatively constant thickness. LDL subclasses show differences in the surface lipid content and certain features of the apolipoprotein B100 structure probably contribute to changes in the size of these particles [3].

Metabolism

It has been suggested that there are parallel metabolic channels within the delipidation cascade from VLDL to LDL [3]. A metabolic relationship between large VLDL particles and small LDL particles has been demonstrated using stable isotopes in subjects with a predominance of small dense LDL [33]. Kinetic analysis of tracer studies in humans demonstrate that LDL particles show an initial rapid plasma decay which is due to both intra–extravascular exchange and catabolism of LDL These studies have not yet identified the specific precursors of individual LDL subclasses, however there are data from animal models suggesting that separate pathways may be responsible for the generation of distinct LDL particles [3, 34]. Inverse correlation’s of changes in large LDL (LDL-I) and small LDL (LDL-III) and of changes of medium-sized LDL (LDL II) and very small LDL (LDL IV) in dietary intervention studies raise the possibility of precursor–product relationships between distinct LDL subclasses [3].

Low HDL

FIGURE 9.2. The metabolic origins of atherogenic lipoprotein phenotype [2, 5]. VLDL, very low-density lipoprotein; IDL, intermediate-density lipoprotein; CE, cholesterol esters; TG, triglycerides; LpL, lipoprotein lipase; CIII, apolipoprotein CIII; CETP, cholesteryl ester transfer protein; HL, hepatic lipase.

(0.09 M)–boric acid (0.08 M)–Na2 EDTA(0.003 M) buffer (pH 8.3) [1, 2]. In detail, plasma is adjusted to 20% sucrose, and 3 to 10 L is applied to the gel. Potentials are set at 40 (15 min), 80 (15 min), and 125 mV (24 h). Gels are fixed and stained for lipids in a solution containing oil red O in 60% ethanol at 55˚C, for proteins in a solution containing 0.1% Coomassie brilliant blue R-250, 50% ethanol and 9% acetic acid and then scanned at 530 nm with a densitometer. Molecular diameters are determined on the basis of migration distance by comparison with standards of known diameter [4, 9, 34].

Assignment of LDL subclass phenotypes is based on particle diameter of the major plasma LDL peak: LDL phenotype A (larger, more buoyant LDL) is defined as an LDL subclass pattern with the major gradient gel peak at a particle diameter of 258 Å or greater, whereas the major peak of LDL phenotype B (small, dense LDL) is at a particle diameter of less than 258 Å [1, 2, 9, 40] (Figs. 9.3 and 9.4).

Atherogenicity of Small Dense LDL

Several reasons have been suggested for atherogenicity of small dense LDL: smaller denser LDL are taken up more easily by arterial tissue than larger LDL [41], suggesting greater transendothelial transport of smaller particles. In addition, smaller LDL particles may also have decreased receptormediated uptake and increased proteoglycan binding [42]. Sialic acid,

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perhaps because of its exposure at the LDL surface, plays a determinant role in the in vitro association of LDL with the polyanionic proteoglycans [43] and it has been shown that sialic acid content of LDL particles of subjects with the pattern B phenotype is reduced.

Further, it has been shown that oxidative susceptibility increases and antioxidant concentrations decreases with decreasing LDL size [44]. Altered properties of the surface lipid layer associated with reduced content of free cholesterol [45] and increased content of polyunsaturated fatty acids [46] might contribute to enhanced oxidative susceptibility of small dense LDL.

Recently [47] we have chosen the model of apoB transgenic mice to evaluate the kinetic behavior of human LDL particles of different size in vivo in a genetically homogeneous recipient avoiding other metabolic differences that could influence LDL metabolism. We found that small LDL particles have intrinsic features that lead to retarded metabolism and decreased intra–extravascular equilibration compared to medium-sized LDL; these properties could contribute to greater atherogenicity of small dense LDL

LDL size and Coronary Heart Disease

The predominance of atherogenic small dense LDL has been associated with an approximately threefold increased risk for CAD [48]. This has been demonstrated in case–control studies of myocardial infarction [48–50] and of angiographically documented coronary disease [51]. However, in most, but not all [50] of these studies, the disease risk associated with small dense LDL was no longer significant after adjusting for covariates, including triglycerides [48, 51] or other cardiovascular risk factors [49].

In a nested case–control study of myocardial infarction during 7 years in patients of the physicians health study cases had significantly smaller LDL size than controls matched for age and smoking. However, LDL size was not an independent risk predictor after adjustment for triglycerides [52].

In the prospective Stanford Five City Project the association of the incidence of fatal and nonfatal CAD with LDL diameter has been investigated. The significant difference in LDL size between cases and controls was independent of levels of HDL-cholesterol, non-HDL-cholesterol, triglycerides, smoking, systolic blood pressure, and body mass index, but not independent from the ratio of total cholesterol to HDL-cholesterol. In this study LDL size was the best differentiator of CAD status in logistic regression analysis [53].

In the Quebec Cardiovascular Study the association between LDL particle size and incident ischemic heart disease has been analyzed on the basis of data from the entire population-based, prospective cohort of men initially free from coronary heart disease (CHD) with a follow up of 5 years. In this study, small dense LDL particles predicted the rate of CHD independent of LDL-cholesterol, triglycerides, HDL-cholesterol, apoB and the total cholesterol to HDL-cholesterol ratio. Further, the increase in cardiovascular risk

attributed to lipid risk factors was modulated to a significant extent by variations in LDL particle size [54].

In addition, the cholesterol concentration in small dense LDL particle may give even more precise information. Again, in the Quebec heart study, St-Piere et al. [55] demonstrated that the cholesterol concentration in small dense LDL particles showed the strongest association with the risk of CHD. Multivariate logistic and survival models indicated that the relationship between LDLcholesterol levels in particles with a diameter less than 255 Å and CHD risk was independent of all nonlipid risk factors and of LDL-cholesterol, HDLcholesterol, triglycerides, and lipoprotein(a) level [55]. These data suggest that the cholesterol within small dense LDL is particularly harmful. Measurement of LDL particle size and possibly in addition cholesterol content within these particles may enhance our capability to predict cardiovascular events.

In a population of 98 men less than 50 years and 100 women less than 50 years who underwent elective diagnostic coronary arteriography, smaller denser LDL were associated with CAD independently of traditional risk factors (age, sex, smoking, diabetes, LDL and HDL-cholesterol concentrations), other than plasma triglycerides. These results stress the importance of triglycerides and small dense LDL in premature CAD [51]. Taken together these studies suggest that LDL size is an important predictor of CAD. However, in most studies LDL size was not completely independent of traditional lipid especially triglycerides. This is not surprising as these parameters are obviously metabolically linked.

Mykkanen et al. [56] found that LDL size was not a predictor of CHD events in elderly men and women after controlling for diabetes status. The main reason for the discrepancies might be a survival bias due to old age in these Finnish subjects. Further, Finnish subjects had relatively high LDLcholesterol and total cholesterol. Therefore the power to detect effects of LDL size on CHD events might have been diminished.

Interestingly, a recent study analyzing data from the CARE trial in a prospective nested case–control study found that larger LDL size after adjustment for other variables was an independent predictor of recurrent coronary events in a population with CAD [57]. However, in this study cases and controls were closely matched for prevalence of LDL subclass pattern B (approximately 40%). Thus, the population was one in which the atherogenic lipoprotein phenotype did not discriminate risk for recurrent events, and in this context a strong risk associated with larger LDL was detected.

Interestingly, significantly larger LDL and HDL particles have been found in Ashkenazi Jews with exceptional longevity compared to an age-matched control group. Larger LDL particles in this study were associated with a lower prevalence of hypertension, cardiovascular disease, and metabolic syndrome [58].

Finally, we have recently found that small dense LDL may be associated with premature CAD [59] and with the extension of coronary atherosclerosis [60].

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LDL Size and Acute Myocardial Infarction

Acute myocardial infarction is accompanied by profound plasma lipid and lipoprotein modifications that have a great clinical relevance since they must be taken into account in making therapeutic decisions [61].

The common lipid alterations observed during the acute phase include a rise of triglyceride levels and a fall of total, LDLand HDL-cholesterol concentrations [62–64]. These lipoprotein modifications may be linked to other changes occurring in lipid metabolism during the acute phase of myocardial infarction: either upor downregulation of cholesterol synthesis, an increased LDL-receptor activity, and an impairment of lipolytic enzymes [65–68]. Also glucose metabolism is severely impaired [69].

Therefore, the acute myocardial infarction and the atherogenic lipoprotein phenotype seems to share a similar array of interrelated metabolic aberrations, including increased levels of triglyceride-rich lipoproteins, reduced levels of HDL, and a relative resistance to insulin-mediated glucose uptake. However, despite a number of data regarding the modifications of total plasma lipoprotein fractions during a myocardial infarction, it is less defined whether or not the LDL peak particle size is also modified by the acute phase and therefore the best time to measure it [62–64].

We recently found [70] in a group of subjects admitted to hospital for a myocardial infarction, and followed until the discharge and 3 months after the event, that the reduction of LDL peak particle size is premature and persist during the hospitalization, with a significant increase 3 months after the myocardial infarction. In addition, the timing of these changes seems to precede those of all other lipoproteins. Therefore, we suggest that the evaluation of the LDL peak particle size should be extended to patients with acute myocardial infarction as much as possible.

LDL Size and Vascular Diseases

It has been recently stated by the National Cholesterol Education Program Adult Treatment Panel III that clinical forms of noncoronary atherosclerosis carry a risk for CHD equal to those with established CHD [7]. These conditions include peripheral arterial disease, symptomatic (transient ischemic attack or stroke of carotid origin) and asymptomatic (>50% stenosis on angiography or ultrasound) carotid artery disease, and abdominal aortic aneurysm [7].

However, despite a huge number of data on the relation between LDL size and atherosclerosis in patients with CHD, very few authors have investigated such relation in patients with noncoronary forms of atherosclerosis and we need to wait for further studies with larger number of patients. Yet, the available data suggest a strong association between small dense LDL and noncoronary forms of atherosclerosis.

It has been found that smaller denser LDL particles represent a risk factor for peripheral arterial disease, in absence or presence of diabetes [71]. In addition,

some studies have showed that common features of peripheral arterial disease are represented by increased triglyceride levels and lower HDL-cholesterol concentrations [71] and it is known that patients with such lipid abnormalities consist primarily of subjects with predominantly atherogenic small dense LDL particles [1–3].

The association between carotid disease and small dense LDL has been found in healthy subjects [72–76] as well as in different categories of patients: with familial combined hyperlipidemia, familial hypercholesterolemia, vascular dementia, Alzheimer’s disease, insulin resistance, or type 2 diabetes [77–81]. In addition, it has been recently suggested that carotid atherosclerosis regression or progression may be linked to LDL size [78, 82].

Regarding patients with abdominal aortic aneurysms, no published studies directly examined the association of LDL size with the presence of such disease. However, it has been recently found [83] that patients with abdominal aortic aneurysms show an elevated prevalence of the metabolic syndrome, which is known to be associated with the predominance of small dense LDL [1, 2, 6] and, therefore, it is likely that such patients may have reduced LDL size.

LDL Size and Diabetes

Patients with diabetes represent a category of high-risk individuals that need stronger measures of lipid-lowering therapies [7, 84, 85]. Hypertriglyceridemia, low HDL-cholesterol, and an increased fraction of small dense LDL particles characterize diabetic dyslipidemia [86–88], whereas LDL or total cholesterol are generally not increased in diabetes patients except for a slight increase of LDL-cholesterol in women (UKPDS) [89]. A predominance of small dense LDL particles has been identified as being associated with a higher risk of CHD in the Physician’s Health Study [52], the population based on Stanford Five Cities Project [53] and the Quebec Cardiovascular Study [54, 55].

In addition, small dense LDL is associated with the cluster of risk factors that characterize the insulin resistance syndrome [90] and it has been suggested that the small dense LDL particles can be added to the group of changes described as the metabolic syndrome [87]. Interestingly, subjects with predominance of small dense LDL have a greater than twofold increased risk for developing type 2 diabetes mellitus, independent from age, sex, glucose tolerance, and body mass index. An increase of peak LDL size was associated with a 16% decrease in the risk of developing type 2 diabetes mellitus [91].

Recently, we investigated the clinical significance of LDL size and LDL subclasses in diabetes type 2 patients [81]. Diabetes patients with manifest CHD had decreased LDL particle sizes and altered LDL subclass distributions, i.e., specifically more LDL III B and less LDL I and LDL II as compared to diabetes patients without established CHD. Multivariate analysis revealed that LDL size was the strongest marker of CHD as compared to