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Small, dense LDL cholesterol and apolipoprotein B: Relationship with serum lipids and LDL size
Atherosclerosis 207 (2009) 496–501
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Small, dense LDL cholesterol and apolipoprotein B: Relationship with serum lipids and LDL size Jelena Vekic a,∗ , Aleksandra Zeljkovic a , Zorana Jelic-Ivanovic a , Vesna Spasojevic-Kalimanovska a , Natasa Bogavac-Stanojevic a , Lidija Memon b , Slavica Spasic a a b
Institute of Medical Biochemistry, Faculty of Pharmacy, University of Belgrade, Belgrade, Serbia Clinical Chemistry Laboratory, Clinical Centre “Bezanijska Kosa”, Belgrade, Serbia
a r t i c l e
i n f o
Article history: Received 21 January 2009 Received in revised form 24 June 2009 Accepted 30 June 2009 Available online 8 July 2009 Keywords: Small dense LDL Apolipoprotein B Dyslipidemia Menopause
a b s t r a c t Objective: Small, dense low-density lipoprotein-cholesterol (sdLDL-C) is a recently recognised marker of cardiovascular disease risk. On the other hand, the usefulness of sdLDL-apoB concentration determination in clinical practice offers grounds for further exploration. This study investigates the associations of sdLDLC and sdLDL-apoB with serum lipid parameters and LDL size in healthy men and women. Methods: The concentrations of sdLDL-C and sdLDL-apoB were measured after heparin–magnesium precipitation of serum samples from ninety-five asymptomatic subjects (47 men, 30 premenopausal and 18 menopausal women). LDL size was determined by gradient gel electrophoresis and serum lipid and lipoprotein parameters were measured by routine laboratory methods. Results: Compared to premenopausal women, men had higher sdLDL-C (P < 0.001) and sdLDL-apoB concentrations (P < 0.001). No difference in the sdLDL-C concentration was found between men and menopausal women. Menopause status was associated with higher concentrations of both sdLDL-C (P < 0.01) and sdLDL-apoB (P < 0.05). Subjects with the LDL B phenotype had elevated sdLDL-C (P < 0.01) and sdLDL-apoB concentrations (P < 0.001). LDL size and triglycerides were independent determinants of both sdLDL-C and sdLDL-apoB concentrations. Conclusion: Gender and menopausal status have significant impact on sdLDL-C and sdLDL-apoB concentrations. The variability in sdLDL-C and sdLDL-apoB levels is considerably influenced by changes in LDL size and triglyceride concentration. Our results suggest that the characterisation of sdLDL particles by evaluating sdLDL-C could be complemented with sdLDL-apoB determination. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Elevated serum low-density lipoprotein (LDL)-cholesterol (LDLC) is one of the main risk factors for the development of cardiovascular disease (CVD). Numerous studies have demonstrated that the quality of LDL particles, rather than simply the level of LDL-C, exerts a potent influence on CVD risk [1,2]. Therefore, the identification of subjects with increased CVD risk should be based on the estimation of both LDL-C concentration and LDL particle characteristics. LDL plasma population is composed of heterogeneous subfractions that are different in size, density and protein/lipid content [3]. Two distinct LDL phenotypes have been described: phenotype A, with a predominance of large, buoyant LDL (lbLDL) particles,
∗ Corresponding author at: Institute of Medical Biochemistry, Faculty of Pharmacy, University of Belgrade, Vojvode Stepe 450, P. Box 146, 11000 Belgrade, Serbia. Tel.: +381 11 3951 265; fax: +381 11 39 72 840/39 74 349. E-mail address: [email protected] (J. Vekic). 0021-9150/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2009.06.035
and phenotype B, with a predominance of small, dense LDL (sdLDL) particles. It has been firmly established that sdLDL particles particularly contribute to the development of CVD [4–7]. We have recently demonstrated the abundance of sdLDL particles in subjects with CVD as well as in asymptomatic individuals with elevated CVD risk [8,9]. LDL particle characterisation requires either special equipment or a lengthy analytical time; therefore it is unsuitable for general clinical use. However, Hirano et al. [10] recently introduced a simple method for the determination of cholesterol and apolipoprotein B (apoB) concentration in sdLDL particles (sdLDL-C and sdLDL-apoB, respectively). Subsequently, the same authors demonstrated the clinical significance of sdLDL-C, but did not consider the importance of sdLDL-apoB determination [11]. To the best of our knowledge this method was used in Japan, whereas such LDL particle characterisation in Caucasians has barely been documented. Taking into account that substantial regional (worldwide) variations in the distribution of LDL phenotypes have already been recognised and described [9], it is also important to define ethnic-based differences in specific LDL particle characteristics.
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The aim of this study was to examine various characteristics of LDL particles in a sample of apparently healthy Serbian men and women with respect to menopausal status. In addition, we explored the interrelationship of LDL size, sdLDL-C and sdLDL-apoB concentrations, as well as their association with other serum lipid parameters. 2. Methods 2.1. Study participants We analysed samples from ninety-five apparently healthy volunteers (48 women and 47 men) aged 45.3 ± 10.4 years, currently employed at the Faculty of Pharmacy in Belgrade. Thirty women were of reproductive age, while the others reported at least 1 year of natural menopause. All participants were in good general condition with normal liver and kidney function and were not taking any medication. None of the menopausal women received hormonal replacement therapy. The study protocol included height and weight measurements for the determination of body mass index (BMI). Blood samples were collected into evacuated tubes containing EDTA after a 12-h fasting period. Plasma and serum were separated and multiple aliquots of each sample were stored at −80 ◦ C. The samples were thawed immediately before analyses. All the assays were performed blindly. The study was planned according to the ethical guidelines following the Declaration of Helsinki. All subjects involved in the study gave written informed consent before study entry and the Ethics Committee at the Faculty of Pharmacy, University of Belgrade, approved this study. 2.2. Lipid and apolipoprotein measurements Serum total cholesterol (TC) and triglyceride (TG) concentrations were assayed by routine enzymatic methods using an ILab 600 analyser. High-density lipoprotein-cholesterol (HDL-C) was measured using the same enzymatic method after precipitation of the plasma with phosphotungstic acid in the presence of magnesium ions. The concentration of LDL-C was determined by a direct homogeneous assay (Olympus Diagnostica GmbH, Hamburg, Germany). Apolipoprotein AI (apoAI) and apoB were measured by immunoturbidimetric assays (Dialab, Vienna, Austria).
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2.3. LDL particle size determination Plasma LDL particles were separated using a method previously described by Rainwater et al. [12]. A detailed description of the procedure has been published elsewhere [8,9]. In brief, electrophoresis was performed at 8 ◦ C in a Hoefer SE 600 Ruby electrophoresis unit (Amersham Pharmacia Biotech, Vienna, Austria) using Tris (90 mM)–boric acid (80 mM)–Na2 EDTA (2.7 mM) buffer, pH 8.35 for 20 h. Gels were calibrated using the Pharmacia High Molecular Weight protein standards, carboxylated polystyrene microsphere beads and human plasma with two LDL subclasses, standardised in Dr. David Rainwater’s laboratory (Southwest Foundation for Biomedical Research, San Antonio, TX, USA). After electrophoretic separation, the gels were stained for proteins with Coomassie brilliant blue G-250 and for lipids with Sudan black. Gels were analysed using Image Scanner (Amersham Pharmacia Biotech, Vienna, Austria) with Image Quant software (version 5.2; 1999; Molecular Dynamics). The migration distance for each absorbance peak was determined and the particle diameter corresponding to each peak was calculated from the calibration curve. The estimated diameter of the major peak in the LDL region of each scan was referred to as the dominant particle diameter. Based on the dominant diameter, we classified LDL phenotypes as phenotype A (>25.5 nm) or phenotype B (≤25.5 nm) [1,6,7]. 2.4. Measurement of sdLDL-C and sdLDL-apoB concentrations The sdLDL-C and sdLDL-apoB were measured using a heparin–magnesium precipitation method [10]. In brief, a serum sample (150 L) was mixed with precipitation reagent (150 L) containing 150 U/mL of heparin sodium salt and 90 mmol/L MgCl ◦ 2 , incubated for 10 min at 37 C, placed in an ice bath for 15 min and centrifuged at 15,000 rpm for 15 min at 4 ◦ C. An aliquot of the supernatant was used to measure LDL-C and apoB concentrations. The inter-assay coefficients of variation were 4.2% and 3.7% for sdLDL-C and sdLDL-apoB, respectively. The proportions of cholesterol and apoB in sdLDL particles (% sdLDL-C and % sdLDLapoB, respectively) were calculated as (sdLDL-C/serum LDL-C)·100 and (sdLDL-apoB/serum apoB)·100. The ratio sdLDL-C/sdLDL-apoB was used as an index of cholesterol content per sdLDL particle, whereas the ratio (serum LDL-C–sdLDL-C)/(serum apoB–sdLDL-
Table 1 Serum lipid and lipoprotein parameters, LDL size, sdLDL-C and sdLDL-apoB concentrations in healthy men and women.
Age (years) BMI (kg/m2 ) TC (mmol/L) HDL-C (mmol/L) LDL-C (mmol/L) TG (mmol/L) ApoAI (g/L) ApoB (g/L) LDL size (nm) sdLDL-C (mmol/L) sdLDL-C (%) sdLDL-apoB (g/L) sdLDL-apoB (%) sdLDL-C/sdLDL-apoB lbLDL-C/lbLDL-apoB
Premenopausal women (n = 30)
Pa
Menopausal women (n = 18)
Pb
Men (n = 47)
Pc
37.1 ± 6.8 22.77 ± 3.03 4.92 ± 0.77 1.55 ± 0.32 2.99 ± 0.78 0.82 (0.68–1.07) 1.60 ± 0.20 0.82 ± 0.22 28.22 ± 0.99 0.70 (0.48–1.12) 25.6 (17.1–33.4) 0.32 (0.25–0.41) 40.2 (33.3–48.6) 2.33 (1.83–2.67) 4.40 (4.00–4.67)
<0.01 <0.01 <0.001 0.832 <0.001 <0.001 0.191 <0.001 <0.001 <0.01 0.413 <0.05 0.573 0.458 0.063
52.8 ± 5.5 25.96 ± 2.75 6.74 ± 0.99 1.53 ± 0.41 4.54 ± 0.95 1.24 (0.80–2.38) 1.69 ± 0.27 1.27 ± 0.28 26.69 ± 1.69 1.46 (0.68–2.23) 30.8 (17.3–49.4) 0.48 (0.34–0.91) 41.7 (30.4–66.6) 2.30 (1.95–3.00) 4.67 (4.34–5.27)
0.065 0.073 <0.05 <0.01 0.056 0.076 <0.001 0.065 0.068 0.854 0.155 <0.01 <0.01 <0.01 0.657
47.7 ± 10.4 28.02 ± 4.45 5.78 ± 0.77 1.11 ± 0.26 3.90 ± 1.15 1.85 (1.28–2.38) 1.37 ± 0.21 1.09 ± 0.34 25.56 ± 2.37 1.40 (0.90–1.70) 37.7 (29.0–46.6) 0.86 (0.62–1.16) 66.7 (50.0–80.0) 1.44 (1.25–2.00) 5.00 (4.25–9.00)
<0.01 <0.01 <0.01 <0.001 <0.01 <0.001 <0.01 <0.01 <0.001 <0.001 <0.01 <0.001 <0.001 <0.001 0.153
Pa , statistical significance for the differences between premenopausal and menopausal women. Pb , statistical significance for the differences between menopausal women and men. Pc , statistical significance for the differences between men and premenopausal women. Normally distributed variables were compared using the Student’s t-test and presented as mean ± standard deviation. Skewed variables were compared by the Mann–Whitney U-test and presented as median (interquartile range). BMI, body mass index; TC, total cholesterol; HDL-C, high-density lipoprotein-cholesterol; LDL-C, low-density lipoprotein-cholesterol; TG, triglyceride; ApoAI, apolipoprotein AI; ApoB, apolipoprotein B; sdLDL-C, small, dense low-density lipoprotein-cholesterol; sdLDL-apoB, small, dense low-density lipoprotein-apolipoprotein B; lbLDL-C, large, buoyant low-density lipoprotein-cholesterol; lbLDL-apoB, large, buoyant low-density lipoprotein-apolipoprotein B.
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Table 2 Serum lipid and lipoprotein parameters, sdLDL-C and sdLDL-apoB concentrations according to the LDL phenotype.
TC (mmol/L) HDL-C (mmol/L) LDL-C (mmol/L) TG (mmol/L) ApoAI (g/L) ApoB (g/L) sdLDL-C (mmol/L) sdLDL-C (%) sdLDL-apoB (g/L) sdLDL-apoB (%)
LDL B phenotype (n = 28)
LDL A phenotype (n = 67)
P
5.98 ± 1.50 1.24 ± 0.31 3.92 ± 1.21 1.98 (1.27–3.16) 1.41 ± 0.27 1.12 ± 0.39 1.40 (1.00–2.00) 41.1 (30.0–53.6) 0.90 (0.60–1.10) 71.4 (53.3–80.0)
5.62 ± 1.28 1.45 ± 0.35 3.63 ± 1.12 1.09 (0.77–1.81) 1.57 ± 0.24 1.01 ± 0.32 0.80 (0.70–1.70) 29.2 (20.6–37.8) 0.40 (0.30–0.80) 42.8 (33.3–59.0)
0.247 <0.01 0.264 <0.001 <0.01 0.185 <0.01 <0.01 <0.001 <0.001
Normally distributed variables were compared using the Student’s t-test and presented as mean ± standard deviation. Skewed variables were compared by the Mann–Whitney U-test and presented as median (interquartile range).
apoB) was applied as a measure of cholesterol content per lbLDL particle. 2.5. Statistical analysis
women. There were no differences in HDL-C and apoAI concentrations between the two women’s groups. However, men had significantly lower levels of both parameters. We found the highest LDL size in premenopausal women and no significant differences between men and menopausal women. Similarly, we found no difference in the sdLDL-C concentration between men and menopausal women. However, the level of sdLDL-apoB was significantly higher in men. Premenopausal women had significantly lower concentrations of sdLDL-C and sdLDL-apoB than men and menopausal women. The observed differences persisted even after adjustment for LDL size, TG and total LDL-C and apoB concentrations (P < 0.05 for both sdLDL-C and sdLDL-apoB concentrations). The index of cholesterol content per sdLDL (median: 1.41 and interquartile range: 2.00–2.43) was significantly lower than the index of cholesterol content per lbLDL (median: 4.50 and interquartile range: 4.17–5.00) when determined in the entire study population (P < 0.001). Similar results were obtained in separate analyses for each age and gender group (P < 0.001 for all groups). 3.2. sdLDL-C and sdLDL-apoB concentrations according to LDL phenotype
Data are shown as mean ± standard deviation for normally distributed variables and as median value (interquartile range) for non-parametric variables. Differences between men and women were evaluated by the Student’s t-test. As the distributions of TG, sdLDL-C, sdLDL-apoB, % sdLDL-C and % sdLDL-apoB were skewed, the data were compared using the Mann–Whitney U-test. In accordance, the indexes of cholesterol content per sdLDL and lbLDL particles were also compared using the Mann–Whitney U-test. The adjusted mean levels of sdLDL-C and sdLDL-apoB were estimated by ANCOVA and group differences were compared while adjusting for LDL size, TG and total LDL-C and apoB concentrations. We used Spearman’s correlation analysis to assess univariate associations of sdLDL-C and sdLDL-apoB concentrations with LDL particle size and serum lipid parameters. Subsequent stepwise multiple regression analysis was used to estimate the contribution of independent predictors on the variance in sdLDL-C and sdLDL-apoB concentrations. Differences with P < 0.05 were considered to be statistically significant. 3. Results 3.1. Gender and menopausal status differences in sdLDL-C and sdLDL-apoB concentrations Table 1 indicates the mean levels of serum lipid and lipoprotein parameters, LDL size, sdLDL-C and sdLDL-apoB concentrations in men, premenopausal and menopausal women. The concentrations of LDL-C, apoB and TG were significantly lower in premenopausal women when compared to the other two groups. These parameters did not significantly differ between men and menopausal
The individuals with the LDL B phenotype had higher TG and lower HDL-C and apoAI concentrations. Moreover, we found that the LDL B phenotype was associated with higher sdLDL-C and sdLDL-apoB concentrations. Accordingly, carriers of the LDL B phenotype had higher values of both % sdLDL-C and % sdLDL-apoB (Table 2). 3.3. Associations of sdLDL-C and sdLDL-apoB with serum lipids and LDL size Our results (Table 3) showed significant correlations between LDL size and HDL-C, TG and apoAI concentrations. No significant associations were found between LDL size, TC, LDL-C and apoB. In contrast, sdLDL-C and sdLDL-apoB concentrations correlated with all the examined serum lipid parameters. Similar results were found for % sdLDL-C and % sdLDL-apoB, the exception being the relationships between % sdLDL-apoB and serum TC and LDL-C concentrations. We also examined associations between different features of LDL particles (Fig. 1). The strongest positive association was found between sdLDL-C and sdLDL-apoB concentrations and both parameters inversely correlated with LDL size. Variables identified by the multiple linear regression analysis as significant and independent determinants of sdLDL-C and sdLDLapoB are shown in Table 4. When age, gender and all examined lipid parameters were entered in the regression analysis the significant predictors of sdLDL-C concentration were sdLDL-apoB, serum apoB and TG concentrations, as well as LDL particle size. The variations in sdLDL-apoB concentrations were significantly associated with
Table 3 Spearman’s correlations between LDL size, sdLDL-C and sdLDL-apoB concentrations and serum lipid and lipoprotein parameters. LDL size (nm) TC (mmol/L) HDL-C (mmol/L) LDL-C (mmol/L) TG (mmol/L) ApoAI (g/L) ApoB (g/L) * ** ***
P < 0.05. P < 0.01. P < 0.001.
−0.155 0.353** −0.143 −0.400*** 0.369** −0.152
sdLDL-C (mmol/L) ***
0.686 −0.362** 0.688*** 0.649*** −0.295* 0.650***
sdLDL-C (%) *
0.256 −0.333** – 0.549*** −0.320** 0.265*
sdLDL-apoB (g/L) ***
0.570 −0.477*** 0.507*** 0.748*** −0.414*** 0.468***
sdLDL-apoB (%) 0.069 −0.360*** 0.101 0.487*** −0.391** –
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Fig. 1. Correlation between LDL size and sdLDL-C (A), LDL size and sdLDL-apoB (B) and sdLDL-C and sdLDL-apoB concentrations (C).
age, male gender and changes in sdLDL-C, LDL size and serum TG concentration. 4. Discussion Although the accumulation of sdLDL plays a pivotal role in the pathogenesis of atherosclerosis, the use of sdLDL in clinical practice remains a matter of debate [5,13]. Hirano et al. introduced a new precipitation method for the quantification of cholesterol and apoB in sdLDL particles and so far, it has been shown that elevated concentrations of sdLDL-C may contribute to the increased CVD Table 4 Stepwise multiple linear regression analysis for sdLDL-C and sdLDL-apoB concentrationsa . Variable
Predictors
sdLDL-C (mmol/L)
sdLDL-apoB (g/L) apoB (g/L) LDL size (nm) TG (mmol/L)
sdLDL-apoB (g/L)
sdLDL-C (mmol/L) TG (mmol/L) Gender (w/m) LDL size (nm) Age (years)
 (SE )
P
Adjusted R2 for model
0.791 (0.149)
<0.001
0.795
0.441 (0.155) −0.206 (0.022) −0.153 (0.057)
<0.001 <0.01 <0.05
0.599 (0.031)
<0.001
0.266 (0.023) 0.222 (0.048) −0.168 (0.011) 0.166 (0.002)
<0.001 <0.001 <0.01 <0.05
0.839
a Variables included in the models were: age, gender (0—woman, 1—man), TC, HDL-C, LDL-C, TG, apoAI, apoB, LDL size and sdLDL-apoB or sdLDL-C with respect to the analysed dependent variable. Values are given as  (SE ).
risk [11]. Moreover, high sdLDL-C, rather than smaller LDL particle size, has been found to be closely and independently associated with the severity of CVD [14]. The same authors also investigated the influence of circadian rhythm, fat and glucose intake on sdLDL-C concentration [15,16]. It has been suggested that sdLDLapoB determination provides no additional information beyond that of sdLDL-C [10,11]. Despite the increasing body of evidence that sdLDL-C determination is superior to that of LDL size with respect to CVD risk prediction, such conclusions have arisen exclusively from studies conducted in Japanese population. Whether these conclusions can be extrapolated to Caucasians remains to be clarified. Our present study has evaluated the relationship between LDL size and the concentrations of sdLDL-C and sdLDL-apoB in a sample of healthy subjects from a European population. 4.1. Effects of gender and menopausal status on sdLDL-C and sdLDL-apoB concentrations We found that non-modifiable cardiovascular risk factors, such as gender and menopausal status, influenced both sdLDL-C and sdLDL-apoB concentrations (Table 1). It is recognized that CVD risk is generally lower in women than in men, but increases progressively in women after menopause [17]. Accordingly, our results demonstrated that men and menopausal women had comparable sdLDL-C concentrations, both significantly higher compared with premenopausal women. Several studies have consistently shown more favourable lipoprotein profiles among premenopausal women than among men due to estrogen-related protective mechanisms. At menopause the LDL particle size distribution shifts toward sdLDL particles [18]. According to Carr et al. [19] such redistri-
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bution in favour of sdLDL occurs prior to the increase in LDL-C concentration. In our study menopausal women had both higher LDL-C and reduced LDL size, but also significantly increased sdLDLC and sdLDL-apoB concentrations than premenopausal women. Alterations in LDL size and composition during menopause are mainly attributable to increased hepatic lipase activity caused by reduced levels of circulating estrogens [20]. They could also be associated with ageing and age-related changes in lifestyle that have a significant impact on lipoprotein metabolism. The observed influence of gender and menopausal status on sdLDL-C and sdLDL-apoB concentrations persisted after taking into account the differences in TG, total LDL-C and apoB concentration and even in LDL size, a finding that supports the importance of advanced lipid testing in clinical practice. However, it is still unknown to what extent an elevated sdLDL-C contributes to increased CVD risk in men and menopausal women. In our present study these two groups were characterised by a cluster of unfavourable LDL features such are smaller LDL size and elevated sdLDL-C and sdLDL-apoB concentrations. Bearing in mind that sdLDL particles are more readily oxidised than larger LDLs [7], we can assume that higher levels of sdLDL-C and sdLDL-apoB could represent increased concentrations of substrates available for oxidation. We recently reported that individuals with smaller LDL particles exhibited evidence of increased oxidative stress [21]. Thus, the abundance of cholesterol combined with increased oxidative stress may lead to acceleration of the atherosclerotic process. 4.2. Effect of LDL phenotype on sdLDL-C and sdLDL-apoB concentrations We found that sdLDL-C was significantly higher in subjects with the LDL B phenotype (Table 2). According to Capell et al. [22], LDL particles lack cholesterol in subjects with the LDL B phenotype. We confirmed such findings by calculating the indexes of cholesterol contents per sdLDL and lbLDL. On the other side, we found that subjects possessing the LDL B phenotype had significantly higher sdLDL-apoB concentrations. Furthermore, the evaluation of % sdLDL-C and % sdLDL-apoB indicated a considerably higher proportion of sdLDL particles in carriers of the LDL B phenotype. Based on these findings, the above-mentioned difference in sdLDLC between the two LDL phenotype groups cannot be interpreted as increased cholesterol concentration per sdLDL particle, but rather as an overload of sdLDL particles in phenotype B subjects. Therefore, our results suggest that simply measuring sdLDL-C might be misleading if one neglects a proportion of sdLDLs in the total LDL mass. In a recent study, Shoji et al. [23] found strong positive association between sdLDL-C and carotid artery intima-media thickness. However, the authors postulated that the association depended on the number of sdLDL particles, rather than on the cholesterol content of sdLDL. They also concluded that the measurement of sdLDL-C offered indirect information on the number of sdLDL, but it did not provide direct evidence for the overproduction of apoB [23]. Therefore, measuring sdLDL-apoB could be as important as measuring sdLDL-C to characterise an individual’s LDL profile. 4.3. Relationships between LDL particle characteristics and serum lipids and lipoproteins A strong association was found between smaller LDL size, higher sdLDL-C and sdLDL-apoB levels and increased TG concentration (Table 3), underlining the crucial role of TG in LDL particle remodelling. It is generally admitted that the combined effects of lipases activities and lipid transfer between TG-rich lipoproteins and LDL enhance the conversion of larger LDL particles into smaller ones. Furthermore, sdLDL particles are cleared less effectively from the circulation in hypertriglyceridemia leading to an
additional increase in their concentration [4]. To gain further insight we performed multiple regression analysis and found strong independent association between reduced sdLDL-C and elevated TG concentration (Table 4). This finding completely fits into conclusion that hypertriglyceridemia generates cholesterol-poor LDL particles. Namely, although LDL particles serve as the acceptors of cholesterol esters from HDL in the reaction mediated by cholesterol ester transfer protein, in the case of hypertriglyceridemia cholesterol esters are preferentially transferred to TG-rich lipoproteins rather than to LDL [24]. 4.4. Study limitations The cross-sectional nature of the present study did not allow us to investigate a causal relationship between elevated sdLDL-C and sdLDL-apoB and the formation of atherosclerotic plaques. Secondly, age at commencement of menopause was self-reported and might have been subjected to recall bias. Even though a measurement of estrogen level is a more reliable approach for confirmation of menopausal status, self-reported ages at menopause are most commonly used in epidemiologic studies. 5. Conclusions The results of our study indicate that gender and menopausal status have significant impact on sdLDL-C and sdLDL-apoB concentrations. Furthermore, our findings confirmed that the protein/lipid composition of LDL particles is strongly influenced by other metabolic alterations, primarily by changes in TG concentration. Finally, we emphasise the importance of controlling for particle concentration in evaluating sdLDL-C, a task that can be accomplished by measuring sdLDL-apoB. Further longitudinal studies are needed to evaluate the relationship between elevated sdLDL-C and sdLDL-apoB levels and the development of atherosclerosis in later life. Conflict of interest statement None declared. Acknowledgements This work was supported by a grant from the Ministry of Science and Technological Development, Republic of Serbia (Project No. 145036B). The authors would also like to thank Dr. David R. Jones for the help in editing the manuscript. References [1] St-Pierre AC, Ruel IL, Cantin B, et al. Comparison of various electrophoretic characteristics of LDL particles and their relationship to the risk of ischemic heart disease. Circulation 2001;104:2295–9. [2] Rizzo M, Berneis K. Low-density-lipoproteins size and cardiovascular risk assessment. QJM-Int J Med 2006;99:1–14. [3] Krauss RM, Burke DJ. Identification of multiple subclasses of plasma low density lipoproteins in normal humans. J Lipid Res 1982;23:97–140. [4] Berneis KK, Krauss RM. Metabolic origin and clinical significance of LDL heterogeneity. J Lipid Res 2002;43:1363–79. [5] Sacks FM, Campos H. Low-density lipoprotein size and cardiovascular disease: a reappraisal. J Clin Endocrinol Metab 2003;88:4525–32. [6] Galeano NF, Al-Haideri M, Keyserman F, Rumsey SC, Deckelbaum RJ. Small dense low density lipoprotein has increased affinity for LDL receptor-independent cell surface binding sites: a potential mechanism for increased atherogenicity. J Lipid Res 1998;39:1263–73. [7] Tribble DL, Rizzo M, Chait A, et al. Enhanced oxidative susceptibility and reduced antioxidant content of metabolic precursors of small, dense lowdensity lipoproteins. Am J Med 2001;110:103–10. [8] Zeljkovic A, Spasojevic-Kalimanovska V, Vekic J, et al. Does simultaneous determination of LDL and HDL particle size improve prediction of coronary artery disease risk? Clin Exp Med 2008;8:109–16.
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Small, dense LDL cholesterol and apolipoprotein B: Relationship with serum lipids and LDL size Jelena Vekic a,∗ , Aleksandra Zeljkovic a , Zorana Jelic-Ivanovic a , Vesna Spasojevic-Kalimanovska a , Natasa Bogavac-Stanojevic a , Lidija Memon b , Slavica Spasic a a b
Institute of Medical Biochemistry, Faculty of Pharmacy, University of Belgrade, Belgrade, Serbia Clinical Chemistry Laboratory, Clinical Centre “Bezanijska Kosa”, Belgrade, Serbia
a r t i c l e
i n f o
Article history: Received 21 January 2009 Received in revised form 24 June 2009 Accepted 30 June 2009 Available online 8 July 2009 Keywords: Small dense LDL Apolipoprotein B Dyslipidemia Menopause
a b s t r a c t Objective: Small, dense low-density lipoprotein-cholesterol (sdLDL-C) is a recently recognised marker of cardiovascular disease risk. On the other hand, the usefulness of sdLDL-apoB concentration determination in clinical practice offers grounds for further exploration. This study investigates the associations of sdLDLC and sdLDL-apoB with serum lipid parameters and LDL size in healthy men and women. Methods: The concentrations of sdLDL-C and sdLDL-apoB were measured after heparin–magnesium precipitation of serum samples from ninety-five asymptomatic subjects (47 men, 30 premenopausal and 18 menopausal women). LDL size was determined by gradient gel electrophoresis and serum lipid and lipoprotein parameters were measured by routine laboratory methods. Results: Compared to premenopausal women, men had higher sdLDL-C (P < 0.001) and sdLDL-apoB concentrations (P < 0.001). No difference in the sdLDL-C concentration was found between men and menopausal women. Menopause status was associated with higher concentrations of both sdLDL-C (P < 0.01) and sdLDL-apoB (P < 0.05). Subjects with the LDL B phenotype had elevated sdLDL-C (P < 0.01) and sdLDL-apoB concentrations (P < 0.001). LDL size and triglycerides were independent determinants of both sdLDL-C and sdLDL-apoB concentrations. Conclusion: Gender and menopausal status have significant impact on sdLDL-C and sdLDL-apoB concentrations. The variability in sdLDL-C and sdLDL-apoB levels is considerably influenced by changes in LDL size and triglyceride concentration. Our results suggest that the characterisation of sdLDL particles by evaluating sdLDL-C could be complemented with sdLDL-apoB determination. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Elevated serum low-density lipoprotein (LDL)-cholesterol (LDLC) is one of the main risk factors for the development of cardiovascular disease (CVD). Numerous studies have demonstrated that the quality of LDL particles, rather than simply the level of LDL-C, exerts a potent influence on CVD risk [1,2]. Therefore, the identification of subjects with increased CVD risk should be based on the estimation of both LDL-C concentration and LDL particle characteristics. LDL plasma population is composed of heterogeneous subfractions that are different in size, density and protein/lipid content [3]. Two distinct LDL phenotypes have been described: phenotype A, with a predominance of large, buoyant LDL (lbLDL) particles,
∗ Corresponding author at: Institute of Medical Biochemistry, Faculty of Pharmacy, University of Belgrade, Vojvode Stepe 450, P. Box 146, 11000 Belgrade, Serbia. Tel.: +381 11 3951 265; fax: +381 11 39 72 840/39 74 349. E-mail address: [email protected] (J. Vekic). 0021-9150/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2009.06.035
and phenotype B, with a predominance of small, dense LDL (sdLDL) particles. It has been firmly established that sdLDL particles particularly contribute to the development of CVD [4–7]. We have recently demonstrated the abundance of sdLDL particles in subjects with CVD as well as in asymptomatic individuals with elevated CVD risk [8,9]. LDL particle characterisation requires either special equipment or a lengthy analytical time; therefore it is unsuitable for general clinical use. However, Hirano et al. [10] recently introduced a simple method for the determination of cholesterol and apolipoprotein B (apoB) concentration in sdLDL particles (sdLDL-C and sdLDL-apoB, respectively). Subsequently, the same authors demonstrated the clinical significance of sdLDL-C, but did not consider the importance of sdLDL-apoB determination [11]. To the best of our knowledge this method was used in Japan, whereas such LDL particle characterisation in Caucasians has barely been documented. Taking into account that substantial regional (worldwide) variations in the distribution of LDL phenotypes have already been recognised and described [9], it is also important to define ethnic-based differences in specific LDL particle characteristics.
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The aim of this study was to examine various characteristics of LDL particles in a sample of apparently healthy Serbian men and women with respect to menopausal status. In addition, we explored the interrelationship of LDL size, sdLDL-C and sdLDL-apoB concentrations, as well as their association with other serum lipid parameters. 2. Methods 2.1. Study participants We analysed samples from ninety-five apparently healthy volunteers (48 women and 47 men) aged 45.3 ± 10.4 years, currently employed at the Faculty of Pharmacy in Belgrade. Thirty women were of reproductive age, while the others reported at least 1 year of natural menopause. All participants were in good general condition with normal liver and kidney function and were not taking any medication. None of the menopausal women received hormonal replacement therapy. The study protocol included height and weight measurements for the determination of body mass index (BMI). Blood samples were collected into evacuated tubes containing EDTA after a 12-h fasting period. Plasma and serum were separated and multiple aliquots of each sample were stored at −80 ◦ C. The samples were thawed immediately before analyses. All the assays were performed blindly. The study was planned according to the ethical guidelines following the Declaration of Helsinki. All subjects involved in the study gave written informed consent before study entry and the Ethics Committee at the Faculty of Pharmacy, University of Belgrade, approved this study. 2.2. Lipid and apolipoprotein measurements Serum total cholesterol (TC) and triglyceride (TG) concentrations were assayed by routine enzymatic methods using an ILab 600 analyser. High-density lipoprotein-cholesterol (HDL-C) was measured using the same enzymatic method after precipitation of the plasma with phosphotungstic acid in the presence of magnesium ions. The concentration of LDL-C was determined by a direct homogeneous assay (Olympus Diagnostica GmbH, Hamburg, Germany). Apolipoprotein AI (apoAI) and apoB were measured by immunoturbidimetric assays (Dialab, Vienna, Austria).
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2.3. LDL particle size determination Plasma LDL particles were separated using a method previously described by Rainwater et al. [12]. A detailed description of the procedure has been published elsewhere [8,9]. In brief, electrophoresis was performed at 8 ◦ C in a Hoefer SE 600 Ruby electrophoresis unit (Amersham Pharmacia Biotech, Vienna, Austria) using Tris (90 mM)–boric acid (80 mM)–Na2 EDTA (2.7 mM) buffer, pH 8.35 for 20 h. Gels were calibrated using the Pharmacia High Molecular Weight protein standards, carboxylated polystyrene microsphere beads and human plasma with two LDL subclasses, standardised in Dr. David Rainwater’s laboratory (Southwest Foundation for Biomedical Research, San Antonio, TX, USA). After electrophoretic separation, the gels were stained for proteins with Coomassie brilliant blue G-250 and for lipids with Sudan black. Gels were analysed using Image Scanner (Amersham Pharmacia Biotech, Vienna, Austria) with Image Quant software (version 5.2; 1999; Molecular Dynamics). The migration distance for each absorbance peak was determined and the particle diameter corresponding to each peak was calculated from the calibration curve. The estimated diameter of the major peak in the LDL region of each scan was referred to as the dominant particle diameter. Based on the dominant diameter, we classified LDL phenotypes as phenotype A (>25.5 nm) or phenotype B (≤25.5 nm) [1,6,7]. 2.4. Measurement of sdLDL-C and sdLDL-apoB concentrations The sdLDL-C and sdLDL-apoB were measured using a heparin–magnesium precipitation method [10]. In brief, a serum sample (150 L) was mixed with precipitation reagent (150 L) containing 150 U/mL of heparin sodium salt and 90 mmol/L MgCl ◦ 2 , incubated for 10 min at 37 C, placed in an ice bath for 15 min and centrifuged at 15,000 rpm for 15 min at 4 ◦ C. An aliquot of the supernatant was used to measure LDL-C and apoB concentrations. The inter-assay coefficients of variation were 4.2% and 3.7% for sdLDL-C and sdLDL-apoB, respectively. The proportions of cholesterol and apoB in sdLDL particles (% sdLDL-C and % sdLDLapoB, respectively) were calculated as (sdLDL-C/serum LDL-C)·100 and (sdLDL-apoB/serum apoB)·100. The ratio sdLDL-C/sdLDL-apoB was used as an index of cholesterol content per sdLDL particle, whereas the ratio (serum LDL-C–sdLDL-C)/(serum apoB–sdLDL-
Table 1 Serum lipid and lipoprotein parameters, LDL size, sdLDL-C and sdLDL-apoB concentrations in healthy men and women.
Age (years) BMI (kg/m2 ) TC (mmol/L) HDL-C (mmol/L) LDL-C (mmol/L) TG (mmol/L) ApoAI (g/L) ApoB (g/L) LDL size (nm) sdLDL-C (mmol/L) sdLDL-C (%) sdLDL-apoB (g/L) sdLDL-apoB (%) sdLDL-C/sdLDL-apoB lbLDL-C/lbLDL-apoB
Premenopausal women (n = 30)
Pa
Menopausal women (n = 18)
Pb
Men (n = 47)
Pc
37.1 ± 6.8 22.77 ± 3.03 4.92 ± 0.77 1.55 ± 0.32 2.99 ± 0.78 0.82 (0.68–1.07) 1.60 ± 0.20 0.82 ± 0.22 28.22 ± 0.99 0.70 (0.48–1.12) 25.6 (17.1–33.4) 0.32 (0.25–0.41) 40.2 (33.3–48.6) 2.33 (1.83–2.67) 4.40 (4.00–4.67)
<0.01 <0.01 <0.001 0.832 <0.001 <0.001 0.191 <0.001 <0.001 <0.01 0.413 <0.05 0.573 0.458 0.063
52.8 ± 5.5 25.96 ± 2.75 6.74 ± 0.99 1.53 ± 0.41 4.54 ± 0.95 1.24 (0.80–2.38) 1.69 ± 0.27 1.27 ± 0.28 26.69 ± 1.69 1.46 (0.68–2.23) 30.8 (17.3–49.4) 0.48 (0.34–0.91) 41.7 (30.4–66.6) 2.30 (1.95–3.00) 4.67 (4.34–5.27)
0.065 0.073 <0.05 <0.01 0.056 0.076 <0.001 0.065 0.068 0.854 0.155 <0.01 <0.01 <0.01 0.657
47.7 ± 10.4 28.02 ± 4.45 5.78 ± 0.77 1.11 ± 0.26 3.90 ± 1.15 1.85 (1.28–2.38) 1.37 ± 0.21 1.09 ± 0.34 25.56 ± 2.37 1.40 (0.90–1.70) 37.7 (29.0–46.6) 0.86 (0.62–1.16) 66.7 (50.0–80.0) 1.44 (1.25–2.00) 5.00 (4.25–9.00)
<0.01 <0.01 <0.01 <0.001 <0.01 <0.001 <0.01 <0.01 <0.001 <0.001 <0.01 <0.001 <0.001 <0.001 0.153
Pa , statistical significance for the differences between premenopausal and menopausal women. Pb , statistical significance for the differences between menopausal women and men. Pc , statistical significance for the differences between men and premenopausal women. Normally distributed variables were compared using the Student’s t-test and presented as mean ± standard deviation. Skewed variables were compared by the Mann–Whitney U-test and presented as median (interquartile range). BMI, body mass index; TC, total cholesterol; HDL-C, high-density lipoprotein-cholesterol; LDL-C, low-density lipoprotein-cholesterol; TG, triglyceride; ApoAI, apolipoprotein AI; ApoB, apolipoprotein B; sdLDL-C, small, dense low-density lipoprotein-cholesterol; sdLDL-apoB, small, dense low-density lipoprotein-apolipoprotein B; lbLDL-C, large, buoyant low-density lipoprotein-cholesterol; lbLDL-apoB, large, buoyant low-density lipoprotein-apolipoprotein B.
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Table 2 Serum lipid and lipoprotein parameters, sdLDL-C and sdLDL-apoB concentrations according to the LDL phenotype.
TC (mmol/L) HDL-C (mmol/L) LDL-C (mmol/L) TG (mmol/L) ApoAI (g/L) ApoB (g/L) sdLDL-C (mmol/L) sdLDL-C (%) sdLDL-apoB (g/L) sdLDL-apoB (%)
LDL B phenotype (n = 28)
LDL A phenotype (n = 67)
P
5.98 ± 1.50 1.24 ± 0.31 3.92 ± 1.21 1.98 (1.27–3.16) 1.41 ± 0.27 1.12 ± 0.39 1.40 (1.00–2.00) 41.1 (30.0–53.6) 0.90 (0.60–1.10) 71.4 (53.3–80.0)
5.62 ± 1.28 1.45 ± 0.35 3.63 ± 1.12 1.09 (0.77–1.81) 1.57 ± 0.24 1.01 ± 0.32 0.80 (0.70–1.70) 29.2 (20.6–37.8) 0.40 (0.30–0.80) 42.8 (33.3–59.0)
0.247 <0.01 0.264 <0.001 <0.01 0.185 <0.01 <0.01 <0.001 <0.001
Normally distributed variables were compared using the Student’s t-test and presented as mean ± standard deviation. Skewed variables were compared by the Mann–Whitney U-test and presented as median (interquartile range).
apoB) was applied as a measure of cholesterol content per lbLDL particle. 2.5. Statistical analysis
women. There were no differences in HDL-C and apoAI concentrations between the two women’s groups. However, men had significantly lower levels of both parameters. We found the highest LDL size in premenopausal women and no significant differences between men and menopausal women. Similarly, we found no difference in the sdLDL-C concentration between men and menopausal women. However, the level of sdLDL-apoB was significantly higher in men. Premenopausal women had significantly lower concentrations of sdLDL-C and sdLDL-apoB than men and menopausal women. The observed differences persisted even after adjustment for LDL size, TG and total LDL-C and apoB concentrations (P < 0.05 for both sdLDL-C and sdLDL-apoB concentrations). The index of cholesterol content per sdLDL (median: 1.41 and interquartile range: 2.00–2.43) was significantly lower than the index of cholesterol content per lbLDL (median: 4.50 and interquartile range: 4.17–5.00) when determined in the entire study population (P < 0.001). Similar results were obtained in separate analyses for each age and gender group (P < 0.001 for all groups). 3.2. sdLDL-C and sdLDL-apoB concentrations according to LDL phenotype
Data are shown as mean ± standard deviation for normally distributed variables and as median value (interquartile range) for non-parametric variables. Differences between men and women were evaluated by the Student’s t-test. As the distributions of TG, sdLDL-C, sdLDL-apoB, % sdLDL-C and % sdLDL-apoB were skewed, the data were compared using the Mann–Whitney U-test. In accordance, the indexes of cholesterol content per sdLDL and lbLDL particles were also compared using the Mann–Whitney U-test. The adjusted mean levels of sdLDL-C and sdLDL-apoB were estimated by ANCOVA and group differences were compared while adjusting for LDL size, TG and total LDL-C and apoB concentrations. We used Spearman’s correlation analysis to assess univariate associations of sdLDL-C and sdLDL-apoB concentrations with LDL particle size and serum lipid parameters. Subsequent stepwise multiple regression analysis was used to estimate the contribution of independent predictors on the variance in sdLDL-C and sdLDL-apoB concentrations. Differences with P < 0.05 were considered to be statistically significant. 3. Results 3.1. Gender and menopausal status differences in sdLDL-C and sdLDL-apoB concentrations Table 1 indicates the mean levels of serum lipid and lipoprotein parameters, LDL size, sdLDL-C and sdLDL-apoB concentrations in men, premenopausal and menopausal women. The concentrations of LDL-C, apoB and TG were significantly lower in premenopausal women when compared to the other two groups. These parameters did not significantly differ between men and menopausal
The individuals with the LDL B phenotype had higher TG and lower HDL-C and apoAI concentrations. Moreover, we found that the LDL B phenotype was associated with higher sdLDL-C and sdLDL-apoB concentrations. Accordingly, carriers of the LDL B phenotype had higher values of both % sdLDL-C and % sdLDL-apoB (Table 2). 3.3. Associations of sdLDL-C and sdLDL-apoB with serum lipids and LDL size Our results (Table 3) showed significant correlations between LDL size and HDL-C, TG and apoAI concentrations. No significant associations were found between LDL size, TC, LDL-C and apoB. In contrast, sdLDL-C and sdLDL-apoB concentrations correlated with all the examined serum lipid parameters. Similar results were found for % sdLDL-C and % sdLDL-apoB, the exception being the relationships between % sdLDL-apoB and serum TC and LDL-C concentrations. We also examined associations between different features of LDL particles (Fig. 1). The strongest positive association was found between sdLDL-C and sdLDL-apoB concentrations and both parameters inversely correlated with LDL size. Variables identified by the multiple linear regression analysis as significant and independent determinants of sdLDL-C and sdLDLapoB are shown in Table 4. When age, gender and all examined lipid parameters were entered in the regression analysis the significant predictors of sdLDL-C concentration were sdLDL-apoB, serum apoB and TG concentrations, as well as LDL particle size. The variations in sdLDL-apoB concentrations were significantly associated with
Table 3 Spearman’s correlations between LDL size, sdLDL-C and sdLDL-apoB concentrations and serum lipid and lipoprotein parameters. LDL size (nm) TC (mmol/L) HDL-C (mmol/L) LDL-C (mmol/L) TG (mmol/L) ApoAI (g/L) ApoB (g/L) * ** ***
P < 0.05. P < 0.01. P < 0.001.
−0.155 0.353** −0.143 −0.400*** 0.369** −0.152
sdLDL-C (mmol/L) ***
0.686 −0.362** 0.688*** 0.649*** −0.295* 0.650***
sdLDL-C (%) *
0.256 −0.333** – 0.549*** −0.320** 0.265*
sdLDL-apoB (g/L) ***
0.570 −0.477*** 0.507*** 0.748*** −0.414*** 0.468***
sdLDL-apoB (%) 0.069 −0.360*** 0.101 0.487*** −0.391** –
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Fig. 1. Correlation between LDL size and sdLDL-C (A), LDL size and sdLDL-apoB (B) and sdLDL-C and sdLDL-apoB concentrations (C).
age, male gender and changes in sdLDL-C, LDL size and serum TG concentration. 4. Discussion Although the accumulation of sdLDL plays a pivotal role in the pathogenesis of atherosclerosis, the use of sdLDL in clinical practice remains a matter of debate [5,13]. Hirano et al. introduced a new precipitation method for the quantification of cholesterol and apoB in sdLDL particles and so far, it has been shown that elevated concentrations of sdLDL-C may contribute to the increased CVD Table 4 Stepwise multiple linear regression analysis for sdLDL-C and sdLDL-apoB concentrationsa . Variable
Predictors
sdLDL-C (mmol/L)
sdLDL-apoB (g/L) apoB (g/L) LDL size (nm) TG (mmol/L)
sdLDL-apoB (g/L)
sdLDL-C (mmol/L) TG (mmol/L) Gender (w/m) LDL size (nm) Age (years)
 (SE )
P
Adjusted R2 for model
0.791 (0.149)
<0.001
0.795
0.441 (0.155) −0.206 (0.022) −0.153 (0.057)
<0.001 <0.01 <0.05
0.599 (0.031)
<0.001
0.266 (0.023) 0.222 (0.048) −0.168 (0.011) 0.166 (0.002)
<0.001 <0.001 <0.01 <0.05
0.839
a Variables included in the models were: age, gender (0—woman, 1—man), TC, HDL-C, LDL-C, TG, apoAI, apoB, LDL size and sdLDL-apoB or sdLDL-C with respect to the analysed dependent variable. Values are given as  (SE ).
risk [11]. Moreover, high sdLDL-C, rather than smaller LDL particle size, has been found to be closely and independently associated with the severity of CVD [14]. The same authors also investigated the influence of circadian rhythm, fat and glucose intake on sdLDL-C concentration [15,16]. It has been suggested that sdLDLapoB determination provides no additional information beyond that of sdLDL-C [10,11]. Despite the increasing body of evidence that sdLDL-C determination is superior to that of LDL size with respect to CVD risk prediction, such conclusions have arisen exclusively from studies conducted in Japanese population. Whether these conclusions can be extrapolated to Caucasians remains to be clarified. Our present study has evaluated the relationship between LDL size and the concentrations of sdLDL-C and sdLDL-apoB in a sample of healthy subjects from a European population. 4.1. Effects of gender and menopausal status on sdLDL-C and sdLDL-apoB concentrations We found that non-modifiable cardiovascular risk factors, such as gender and menopausal status, influenced both sdLDL-C and sdLDL-apoB concentrations (Table 1). It is recognized that CVD risk is generally lower in women than in men, but increases progressively in women after menopause [17]. Accordingly, our results demonstrated that men and menopausal women had comparable sdLDL-C concentrations, both significantly higher compared with premenopausal women. Several studies have consistently shown more favourable lipoprotein profiles among premenopausal women than among men due to estrogen-related protective mechanisms. At menopause the LDL particle size distribution shifts toward sdLDL particles [18]. According to Carr et al. [19] such redistri-
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bution in favour of sdLDL occurs prior to the increase in LDL-C concentration. In our study menopausal women had both higher LDL-C and reduced LDL size, but also significantly increased sdLDLC and sdLDL-apoB concentrations than premenopausal women. Alterations in LDL size and composition during menopause are mainly attributable to increased hepatic lipase activity caused by reduced levels of circulating estrogens [20]. They could also be associated with ageing and age-related changes in lifestyle that have a significant impact on lipoprotein metabolism. The observed influence of gender and menopausal status on sdLDL-C and sdLDL-apoB concentrations persisted after taking into account the differences in TG, total LDL-C and apoB concentration and even in LDL size, a finding that supports the importance of advanced lipid testing in clinical practice. However, it is still unknown to what extent an elevated sdLDL-C contributes to increased CVD risk in men and menopausal women. In our present study these two groups were characterised by a cluster of unfavourable LDL features such are smaller LDL size and elevated sdLDL-C and sdLDL-apoB concentrations. Bearing in mind that sdLDL particles are more readily oxidised than larger LDLs [7], we can assume that higher levels of sdLDL-C and sdLDL-apoB could represent increased concentrations of substrates available for oxidation. We recently reported that individuals with smaller LDL particles exhibited evidence of increased oxidative stress [21]. Thus, the abundance of cholesterol combined with increased oxidative stress may lead to acceleration of the atherosclerotic process. 4.2. Effect of LDL phenotype on sdLDL-C and sdLDL-apoB concentrations We found that sdLDL-C was significantly higher in subjects with the LDL B phenotype (Table 2). According to Capell et al. [22], LDL particles lack cholesterol in subjects with the LDL B phenotype. We confirmed such findings by calculating the indexes of cholesterol contents per sdLDL and lbLDL. On the other side, we found that subjects possessing the LDL B phenotype had significantly higher sdLDL-apoB concentrations. Furthermore, the evaluation of % sdLDL-C and % sdLDL-apoB indicated a considerably higher proportion of sdLDL particles in carriers of the LDL B phenotype. Based on these findings, the above-mentioned difference in sdLDLC between the two LDL phenotype groups cannot be interpreted as increased cholesterol concentration per sdLDL particle, but rather as an overload of sdLDL particles in phenotype B subjects. Therefore, our results suggest that simply measuring sdLDL-C might be misleading if one neglects a proportion of sdLDLs in the total LDL mass. In a recent study, Shoji et al. [23] found strong positive association between sdLDL-C and carotid artery intima-media thickness. However, the authors postulated that the association depended on the number of sdLDL particles, rather than on the cholesterol content of sdLDL. They also concluded that the measurement of sdLDL-C offered indirect information on the number of sdLDL, but it did not provide direct evidence for the overproduction of apoB [23]. Therefore, measuring sdLDL-apoB could be as important as measuring sdLDL-C to characterise an individual’s LDL profile. 4.3. Relationships between LDL particle characteristics and serum lipids and lipoproteins A strong association was found between smaller LDL size, higher sdLDL-C and sdLDL-apoB levels and increased TG concentration (Table 3), underlining the crucial role of TG in LDL particle remodelling. It is generally admitted that the combined effects of lipases activities and lipid transfer between TG-rich lipoproteins and LDL enhance the conversion of larger LDL particles into smaller ones. Furthermore, sdLDL particles are cleared less effectively from the circulation in hypertriglyceridemia leading to an
additional increase in their concentration [4]. To gain further insight we performed multiple regression analysis and found strong independent association between reduced sdLDL-C and elevated TG concentration (Table 4). This finding completely fits into conclusion that hypertriglyceridemia generates cholesterol-poor LDL particles. Namely, although LDL particles serve as the acceptors of cholesterol esters from HDL in the reaction mediated by cholesterol ester transfer protein, in the case of hypertriglyceridemia cholesterol esters are preferentially transferred to TG-rich lipoproteins rather than to LDL [24]. 4.4. Study limitations The cross-sectional nature of the present study did not allow us to investigate a causal relationship between elevated sdLDL-C and sdLDL-apoB and the formation of atherosclerotic plaques. Secondly, age at commencement of menopause was self-reported and might have been subjected to recall bias. Even though a measurement of estrogen level is a more reliable approach for confirmation of menopausal status, self-reported ages at menopause are most commonly used in epidemiologic studies. 5. Conclusions The results of our study indicate that gender and menopausal status have significant impact on sdLDL-C and sdLDL-apoB concentrations. Furthermore, our findings confirmed that the protein/lipid composition of LDL particles is strongly influenced by other metabolic alterations, primarily by changes in TG concentration. Finally, we emphasise the importance of controlling for particle concentration in evaluating sdLDL-C, a task that can be accomplished by measuring sdLDL-apoB. Further longitudinal studies are needed to evaluate the relationship between elevated sdLDL-C and sdLDL-apoB levels and the development of atherosclerosis in later life. Conflict of interest statement None declared. Acknowledgements This work was supported by a grant from the Ministry of Science and Technological Development, Republic of Serbia (Project No. 145036B). The authors would also like to thank Dr. David R. Jones for the help in editing the manuscript. References [1] St-Pierre AC, Ruel IL, Cantin B, et al. Comparison of various electrophoretic characteristics of LDL particles and their relationship to the risk of ischemic heart disease. Circulation 2001;104:2295–9. [2] Rizzo M, Berneis K. Low-density-lipoproteins size and cardiovascular risk assessment. QJM-Int J Med 2006;99:1–14. [3] Krauss RM, Burke DJ. Identification of multiple subclasses of plasma low density lipoproteins in normal humans. J Lipid Res 1982;23:97–140. [4] Berneis KK, Krauss RM. Metabolic origin and clinical significance of LDL heterogeneity. J Lipid Res 2002;43:1363–79. [5] Sacks FM, Campos H. Low-density lipoprotein size and cardiovascular disease: a reappraisal. J Clin Endocrinol Metab 2003;88:4525–32. [6] Galeano NF, Al-Haideri M, Keyserman F, Rumsey SC, Deckelbaum RJ. Small dense low density lipoprotein has increased affinity for LDL receptor-independent cell surface binding sites: a potential mechanism for increased atherogenicity. J Lipid Res 1998;39:1263–73. [7] Tribble DL, Rizzo M, Chait A, et al. Enhanced oxidative susceptibility and reduced antioxidant content of metabolic precursors of small, dense lowdensity lipoproteins. Am J Med 2001;110:103–10. [8] Zeljkovic A, Spasojevic-Kalimanovska V, Vekic J, et al. Does simultaneous determination of LDL and HDL particle size improve prediction of coronary artery disease risk? Clin Exp Med 2008;8:109–16.
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