Received: 21 November 2019 | Revised: 16 January 2020 | Accepted: 4 February 2020 DOI: 10.1002/jcla.23282 RESEARCH ARTICLE Precipitated sdLDL: An easy method to estimate LDL particle size Bárbara Fernández-Cidón1,2 | Beatriz Candás-Estébanez1 | Josep Ribalta3 | Edmond Rock4 | Montserrat Guardiola-Guionnet3 | Núria Amigó3 | Ariadna Padró-Miquel1 | Pedro Alía-Ramos1 | Xavier Pintó-Sala1 1 Hospital Universitari de Bellvitge, L'Hospitalet de Llobregat, Spain Abstract 2 Department of Biochemistry, Molecular Background: LDL-C lowering is the main measure in cardiovascular disease preven- Biology and Biomedicine, Autonomous tion but a residual risk of ischemic events still remains. Alterations of lipoproteins, University of Barcelona (UAB), Barcelona, Spain specially, increase in small dense LDL (sdLDL) particles are related to this risk. Objective: To investigate the potential use of sdLDL cholesterol concentration 3 Institut d'Investigació Sanitària Pere Virgili, Reus, Spain 4 (sdLDL-C) isolated by an easy precipitation method and to assess the impact of a set French National Institute for Agricultural Research, Paris, France of clinical and biochemical variables determined by NMR on sdLDL concentration. Methods: sdLDL-C and NMR lipid profile were performed in 85 men samples. Correspondence Beatriz Candás-Estébanez, Hospital Association among them was evaluated using Pearson coefficients (r xy). A multivari- Universitari de Bellvitge, L'Hospitalet de ate regression was performed to identify the influence of NMR variables on sdLDL-C. Llobregat, Catalunya, Spain. Email: beatrizcandas@gmail.com Results: A strong association between sdLDL-C and LDLLDL-P (r xy = 0.687) and with LDL-Z (r xy = −0.603) was found. The multivariate regression explained a 56.8% in Funding information This study was funded by Hospital sdLDL-C variation (P = 8.77.10-12). BMI, ApoB, triglycerides, FFA, and LDL-Z showed Universitari de Bellvitge, part of the Institut a significant contribution. The most important ones were ApoB and LDL-Z; a 1nm Català de la Salut (ICS) and Department of Biochemistry, Molecular Biology and increase (LDL-Z) leads to decrease 126 nmol/L in sdLDL-C. Biomedicine, Autonomous University of Conclusion: The association between sdLDL-C, LDL-Z, and LDL-P is clear. From a large Barcelona (UAB), Barcelona, Spain. number of variables, especially LDL-Z and apoB influence on sdLDL-C. Results show that the smaller the LDL size, the higher their cholesterol concentration. Therefore, sdLDL-C determination by using this easy method would be useful to risk stratifica- tion and to uncover cardiovascular residual risk. KEYWORDS atherosclerosis, lipoprotein precipitation, nuclear magnetic resonance lipid profile, residual cardiovascular risk, small dense LDL Abbreviations: ALT, alanine aminotransferase; Apo, CIII apolipoprotein CIII; ApoA-I, apolipoprotein A; ApoB, apolipoprotein B; ApoE, apolipoprotein E; AST, aspartate aminotransferase; BMI, body mass index; CRP, creatinine, c-reactive protein; CVD, Cardiovascular diseases; CVRFs, cardiovascular risk factors; FFAs, free fatty acids; Fib, fibulin; GPT, glutamate-pyruvate transaminase; HDL, high-density cholesterol; IDL-C, intermediate-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; LDL-P, LDL particles; LDL-Z, LDL size; LPA, lipoprotein A; MUFAs, monounsaturated fatty acids; NMR, nuclear magnetic resonance; PUFAs, polyunsaturated fatty acids; r xy, Pearson coefficients; sdLDL, small dense LDL particles; sdLDL-C, small dense LDL particles cholesterol; Tg, triglycerides; Vit A, vitamin A; Vit E, vitamin E; VLDL-C, very low-density lipoprotein cholesterol; ρ, Spearman correlation coefficients. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. © 2020 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals, Inc. J Clin Lab Anal. 2020;34:e23282.  wileyonlinelibrary.com/journal/jcla | 1 of 9 https://doi.org/10.1002/jcla.23282 2 of 9 | FERNÁNDEZ-CIDÓN et al. 1 | I NTRO D U C TI O N They concluded that the combination of 150 U/mL Heparin-Na and 90 mmol/L MgCl2 allow to precipitate selectively lipoproteins with Cardiovascular diseases (CVD) are the leading cause of mortality in density <1.044 g/mL. the occidental countries being subclinical atherosclerosis the trig- However, until 2017 there were not reference values established gering factor for most of these events.1 Premature atherosclerotic by this method.17 disease commonly occurs in individuals with atherogenic dyslipid- Nuclear magnetic resonance (NMR) can be used to estimate the emia who share a phenotype characterized by centripetal obesity, number and diameter of lipoprotein particles from different sub- insulin resistance, and physical inactivity. In addition, they also have classes.18,19 This method is consolidated to measure LDL particles a lipid profile characterized by an increase in triglycerides (Tg) and number (LDL-P) and their diameters (LDL-Z). However, discrepancies small dense lipoproteins (sdLDL) concentrations. Also, a decrease are often observed between LDL-P and the concentration of choles- in concentration of high-density cholesterol (HDL) and normal to terol derived from these particles (LDL-C). 20-22 LDL-P is more linked moderately elevated low-density lipoprotein cholesterol (LDL-C) are to atherothrombosis and is a better predictor of coronary events generally observed in these patients. 2 than LDL-C alone in some cases.13,23 NMR lipid profile provides bet- Reducing the incidence of cardiovascular events requires the ter risk stratification for subclinical atherosclerosis than traditional early detection of cardiovascular risk factors (CVRFs), such as lipid profile. 24 New consensus guides for cardiovascular risk assess- LDL-C concentration, nowadays the most important therapeutic ment recommend that lipid-lowering therapies should be guided by target.3 However, it has been demonstrated that lowering LDL-C LDL-P and not by LDL-C. 25 concentration is not enough to prevent all ischemic events even in Taking into account the literature, LDL-P has been established patients without CVRFs.4 People with LDL-C concentrations lower as a predictor of cardiovascular events. However, the relationship than 3.4 mmol/L suffer from subclinical atherosclerosis. In fact, it between LDL-P, LDL-Z, and LDL-C is still unclear. has been shown that in 20% of patients whose treatment results in The aim of this study is to investigate the relationship between reductions in LDL-C to 1.81 mmol/L or less continue to show ath- sdLDL-C obtained by precipitation and characteristics established erosclerotic plaque progression.5 Therefore, it seems that measuring by NMR in order to know whether performing the sdLDL-C concen- LDL-C alone does not provide enough information for CVD early de- tration measurement could be useful as a complementary tool in car- tection, and treatment strategy based on lowering LDL-C concen- diovascular risk assessment. tration would be not enough for preventing all premature ischemic events. Nowadays, in some American scientific societies the LDL-C therapeutic threshold value is simply being lowered.6 Nevertheless, 2 | M ATE R I A L S A N D M E TH O DS the latest data suggest that LDL-C, by itself, cannot explain all as- pects of atherosclerotic disease,7,8 summarizing that LDL-C is not a 2.1 | Subjects good predictor of cardiovascular events in all cases.9 The number of particles, their size, and cholesterol concentra- Eighty-five patients from a population selected in Reus (Spain) and tion in different subclasses of lipoproteins are three factors that Clermont-Ferrand (France) were included in this study. All 85 partici- could be useful to estimate the residual cardiovascular risk unex- pants were 19- to 75-year-old male non-smokers who were enrolled plained by LDL-C concentrations alone.8-10 It is well known that in the VITAGE study. The exclusion criteria were as follows: enolism sdLDL particles are more atherogenic. Due to their small size and and history of familial hypercholesterolemia and chronic diseases, high density, these particles can easily infiltrate the vascular endo- such as diabetes, cancer, heart failure, and arterial hypertension. 11 thelium and promote atheroma formation. The concentration of Demographic and dietary variables were collected using a question- cholesterol derived from sdLDL (sdLDL-C) particles is related to the naire. These variables included: age, BMI, systolic and diastolic blood thickening of the intima media7,12 and to a higher risk of cardiovascu- pressure, alcohol consumption (g/d), and diet (kcal/d). Informed con- lar disease.13,14 Despite sdLDL particles are considered a new CVRF sent was obtained from all participants, after which blood samples and its measurement is recommended by guidelines, sdLDL analyses were drawn. Samples were collected in tubes containing the anti- are not being easily implemented in the clinical routine.15 The princi- coagulant EDTA-K4 and were centrifuged immediately for 15 min- pal reason is that quantifying sdLDL concentration is expensive and utes at 1500 g and at 4°C. The plasma was separated and stored at time consuming. An alternative method using Magnesium Heparin −80°C until analyzed. In order to guarantee the study reliability, all as surfactant was developed to precipitate lipoproteins in a way that measurements, which differ from the results obtained before sam- facilitates the enrichment of sdLDL particles.16 ples freezing more than 3% for Cholesterol and 4% for HDL, were It was well known that divalent cations and polyanions precipitate excluded of the study. apolipoprotein B containing lipoproteins. Basing in this fact, Hirano The research protocol was approved by the ethics committees of et al tested different combinations of polyanion and divalent cations both participating hospitals. and compared the results with the reference method, the ultracen- Each patient had their biochemical profiles assessed. This in- trifugation. They achieved to find the combination which showed cluded plasma concentrations of alanine aminotransferase (ALT), as- the best correlation with results obtained by the ultracentrifugation. partate aminotransferase (AST), glutamate-pyruvate transaminase FERNÁNDEZ-CIDÓN et al. | 3 of 9 (GPT), creatinine, c-reactive protein (CRP), albumin, transthyre- TA B L E 1 Demographic variables and lipid and biochemical tin, glucose, Free Fatty Acids (FFAs), polyunsaturated fatty acids profile (PUFAs), monounsaturated fatty acids (MUFAs), fibulin (Fib), vi- Variable Units Average (range) tamin A (Vit A), and vitamin E (Vit E). A complete lipid profile for Age years 45.8 (19-75) each patient was also attained including plasma concentrations of 2 BMI kg/cm 25.1 (19.3-30) cholesterol, very low-density lipoprotein cholesterol (VLDL-C), in- Systolic pressure mm Hg 124 (100-140) termediate-density lipoprotein cholesterol (IDL-C), triglycerides (Tg), Diastolic pressure mm Hg 78 (60-96) low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein Alcohol g/d 13 (0-100) cholesterol (HDL-C), apolipoprotein A (ApoA-I), apolipoprotein B (ApoB), apolipoprotein E (ApoE), apolipoprotein CIII (ApoCIII), and Diet kcal/d 2240 (999-4347) lipoprotein A (LPA). In addition, atherogenic indices were calculated ALT µkat/L 0.40 (0.16-1.55) using the following formula: IA = LDL-C/HDL-C. LDL-Z and LDL-P AST µkat/L 0.39 (0.25-0.80) and sdLDL-C were also measured (Table 1). GPT µkat/L 0.35 (0.1-1.3) Creatine µmol/L 91.2 (57-120) CRP mg/L 6.2 (6-28.3) 2.2 | Determining sdLDL-C Albumin mg/L 44 (34-49) TTR g/L 0.28 (0.19-0.37) As a first step, sdLDL particles were isolated. This was carried out Glucose mmol/L 5.3 (4.2-6.8) using a previously reported precipitation method adapted for use Fib ng/L 0.48 (0-2) in our laboratory to process samples containing elevated concen- Vit A µmol/L 1.81 (1.15-2.81) trations of triglycerides. This method has been validated comparing Vit E µmol/L 30.12 (18.14-48.42) the results obtained with the reference method, the ultracentrifu- gation.16,17 Interassay imprecision of the precipitation method was Cholesterol mmol/L 1.67 (1.13-2.63) calculated measuring 10 aliquots of 2 samples, one of them with Tg mmol/L 0.47 (0.52-2.76) pathological values of sdLDL-c and the other one with values within LDL-C mmol/L 2.65 (1.40-4.28) the reference interval for sdLDL-c. The study has been carried out HDL-C mmol/L 1.27 (0.76-2.39) during 2 weeks, and the aliquots were stored at −80C°. Interassay IDL-C mmol/L 0.19 (0.01-0.56) imprecision estimated by the variation coefficient (CV %) was 9%, 2%. VLDL-C mmol/L 0.32 (0.06-1.62) The surfactant magnesium heparin was prepared as a solution Apo A mg/dL 129.8 (95-195) of 150 U/mL of Heparin-Na+ (Sigma Aldrich H3393) and 90 mmol/L Apo B mg/dL 72.2 (37-122) of MgCl2. 300 μL of Magnesium Heparin was added to each 300 μL Apo E mg/dL 3.49 (1.6-5.8) of plasma and incubated for 10 minutes at 37°C. The mixture was Apo CIII mg/dL 16.03 (9.4-22.8) then kept at 0°C for 15 minutes and centrifuged in at 21 913 g for Lpa mg/L 25.22 (2-144) 15 minutes at 4°C (6K15 SIGMA centrifuge). At this point, lipopro- FFAs ng/mL 10 802 (6929-21506) teins with a density <1.044 g/mL were precipitated. The supernatant PUFAs ng/mL 16.08 (6.19-52.49) contained HDL and sdLDL particles that had densities of between 1.044 and 1.063 g/mL. Supernatant HDL-C and total cholesterol MUFAs ng/mL 49.90 (22.62-80.32) were measured using a Cobas 8000 modular analyzer (Roche ® AI n.a. 2.20 (1.04-4.80) Diagnostics by homogeneous assays. The linearity range for cho- LDL-Z nm 21.13 (19.1-22.7) lesterol (Cholesterol gen.2) is 0.08‑20.7 mmol/L and for HDL (HDL- LDL-P nmol/L 684 (0-2432) Cholesterol plus 3rd generation) is 0.08‑3.12 mmol/L. sdLDL-C nmol/L 350 (0-1260) Cholesterol concentration is determined enzymatically using Abbreviations: AI = LDL-C/HDL-C, atherogenic index; ALT, alanine cholesterol esterase and cholesterol oxidase in Cobas 701, Roche amino transferase; ApoA-I, apolipoprotein A; ApoB, apolipoprotein B; Diagnostics®. HDL is also determined by a homogeneous enzymatic ApoCIII, apolipoprotein CIII; ApoE, apolipoprotein E; AST, aspartate assay in Cobas 701, which involves the same enzymes modified with aminotransferase; BMI, body mass index; CRP, c-reactive protein; FFA, free fatty acids; Fib, fibulin; GPT, glutamate-pyruvate transaminase; polyethyleneglycol (HDL-Cholesterol plus 3rd generation). In these con- HDL-C, high-density lipoprotein cholesterol; IDL-C, intermediate- ditions, hydrogen peroxide is liberated and oxidizes 4‑aminoantipyrine density lipoprotein cholesterol; LDL-C, low-density lipoprotein to a quinoneimine, which is directly proportional to HDL concentration. cholesterol; LDL-P, particle concentration; LDL-Z, LDL diameter; Since supernatant only contained HDL and sdLDL particles, the Lpa, lipoprotein A; MUFAs, monounsaturated fatty acids; PUFAs, polyunsaturated fatty acids; sdLDL-C, small, dense LDL cholesterol sdLDL-C was calculated by subtracting the HDL-C from the total concentration; Tg, triglycerides; TTR, transthyretin; Vit A, vitamin A; Vit cholesterol concentration. E, vitamin E; VLDL-C, very low-density lipoprotein. 4 of 9 | FERNÁNDEZ-CIDÓN et al. 2.3 | NMR characterization of LDL particles 3 | R E S U LT S The NMR analyses were carried out with the Vantera® analyzer 3.1 | Subjects (LipoScience. Inc). This equipment is approved for in vitro diagno- ses by the US Food and Drug Administration (FDA). This technique All 85 participants included in the study completed a questionnaire. was used to determine the LDL diameters and estimate particle Demographic, biochemical, and lipid variables are listed in Table 1. concentrations (LDL-Z and LDL-P). NMR spectra were acquired by The average age was 45.8 (19-75) years. 400 MHz 1H NMR spectrometers included in the Vantera Clinical Analyzer. The methyl signal envelope appearing between 0.718 and 0.914 ppm was analyzed using the LipoProfile-3 algorithm. 3.2 | Validation of precipitation technique to The contribution of each subcomponent was determined by lin- measure sdLDL-C ear least squares Results were expressed as LDL-P in nmol/L and LDL-Z in nm. 26 Only three outliers were discarded. The correlation study showed that there was a direct linear association between sdLDL-C and LDL-P (r xy = 0.687 [0.555-0.785]; P = 2.1 × 10−7, (Figure 1). In addi- 2.4 | Statistical analyses tion, there was an inverse association between sdLDL-C and LDL-Z (r xy = −0.603 [−0.723-0.447]; P = 1.6 × 10−12; (Figure 2). Both asso- 2.4.1 | Validation of precipitation technique to ciations were statistically significant. measure sdLDL-C First of all, a correlation study was carried out in order to assess the 3.3 | Impact of a set of clinical and biochemical association between LDL-Z, LDL-p, and sdLDL-C by precipitation. variables on sdLDL concentration The NMR lipid profile data are use as the gold standard. Outliers were identified using the Bland-Altman concordance analysis of To evaluate the impact between a set of clinical and biochemical paired data. The association of sdLDL-C with LDL-Z and LDL-P variables on sdLDL, a multivariate analysis was carried out. Results was determined by calculating the Pearson correlation coefficient of the univariate analysis are laid out in Table 2. Variables found to be (r xy) with a confidence interval of 95% [95% CI] as well as levels of significant in the univariate analyses were BMI, ALT, Vit E, Tg, LDL- significance. C, HDL-C, VLDL-C, IDL-C, ApoA-I, ApoB, ApoE, ApoCIII, FFAs, and LDL-Z. These variables were included in the multivariable model. By convention, age was also included. 2.4.2 | Assessing the impact of a set of clinical and Three pairs of variables were found to be collinear. These were biochemical variables on sdLDL concentration as follows: LDL-C and ApoB; VLDL-C and Tg; and HDL-C and ApoA-I. LDL-C, VLDL-C, and ApoA-I contribute less to sdLDL-C concentra- To study the relation between LDL-Z and sdLDL-C, a multivariate tion variations and were excluded in later analyses. regression analysis adjusted for control variables was performed. The multivariate regression model was found to be significant Here, the dependent variable (Y) was the sdLDL-C, and the inde- (P = 8.77 × 10 −12; R 2 = 0.568). The model explains 56.8% of sdLDL-C pendent variable (X) was LDL-Z. variation. Regression coefficients were calculated for each variable Those variables which contribute significantly to the sdLDL-C included in the multivariate analysis (see Table 3). B values were concentration in univariate analysis were included in the multivariate significant for BMI, ApoB, Tg, FFAs, and LDL-Z. ApoB and sdLDL-C model. Significance levels were set to 0.05. concentrations were the two variables, which contributed most to By using correlation matrices and calculating Spearman correla- the model (β = 0.415) and LDL-Z (β = −0.394). The way of LDL-Z sig- tion coefficients (ρ), collinearities between variables were evaluated. nificantly influence on sdLDL-C concentration is by a 1nm increase Those variables with a ρ > 0.5 were discarded. A multivariate re- in LDL-Z leads to a 126 nmol/L reduction in sdLDL-C concentration. gression model was constructed using the selected variables. The adjusted coefficient of determination (adjusted R 2) was calculated to determine the proportion of variation in sdLDL-C concentration that 4 | D I S CU S S I O N could be explained by the adjusted model. B and β coefficients were used to assess the contribution of each variable to the multivariate In this article, we investigated the relationship between the choles- regression model. A higher β coefficient was indicative of a greater terol derived from precipitated sdLDL particles (sdLDL-C) and the contribution. characteristics of LDL lipoproteins, measured by NMR. From a co- The analysis was adjusted for the control variables. The SPSS hort of 85 frozen samples belonging to men enrolled in the VITAGE v.17.0 software package was used in all statistical analyses. Results study, 27 the sdLDL-C concentration was measured and its associa- with a P-value < .05 were considered statistically significant. tion with all variables collected was investigated. FERNÁNDEZ-CIDÓN et al. | 5 of 9 3000 rxy =0.687 [0.555-.785] 2500 2000 1160, 1941 500, 1855 480, 1814 640, 1736 760, 1705 LDL-P (nmol/L) 440, 1634 660, 1648 1140, 1667 480, 1604 580, 1572 1500 260, 1444 660, 1469 140, 1413 440, 1367 1260, 1366 0, 1295 340, 1321 480, 1316540, 1313 80, 1277 240, 1252 280, 1246340, 360, 360, 1267 440,460, 1288 1245 1293 1268 480, 1247 1040, 1250 260,280, 220, 1191 11981193 600, 1226 120, 1169 1178 320, 1152 180, 1090 320, 1123 480, 1095 560, 1114 680, 1117 160, 1076220, 1085280, 1074 240, 1041 320, 1052 1000 220,240, 9831003 300, 1025 280, 1012340, 1010 420, 1019 360,380, 1008 956967 440, 953 964 962 620, 943 20, 92560, 895 0, 924 140,160, 904 180,200, 907 884898 180, 860 320, 853 240,260, 200, 791 805778 784 300, 810 360, 830 300, 779 0, 729 140, 705 200, 721 60, 671100,120, 663694 140, 599 100, 598 340, 616 40, 556 500 0 0 200 400 600 800 1000 1200 1400 sdLDL-C (nmol/L) FIGURE 1 Association between sdLDL-C and LDL-P 23 260, 22.7 100, 22.6 140, 22.6 22.5 240, 22.5 200, 22.3 340, 22.3 0, 22.2 60, 22.2 280, 22.2 160, 22.1 440, 22.1 rxy =0.687 [0.555-0.785] 22 180, 22 320, 22 360, 22 0, 21.8 360, 21.8 240, 21.8 320, 21.8 180, 21.7 300, 320, 21.7 340, 21.7 420, 21.7 21.7 20, 21.6 460, 21.6 21.5 60, 21.5 100, 21.5 360, 21.5 120, 21.4 300, 21.4 500, 21.4 600, 21.4 140, 21.3200, 21.3 240, 21.3 280, 21.3 360, 380, 21.3 21.3 620, 21.3 80, 21.2 480, 21.2 LDL-Z (nm) 0, 21.140, 21.1 200, 21.1260, 160, 21.1 21.1 280, 21.1340, 21.1 680, 21.1 21 140, 21 300, 21 480, 21 120, 20.9180, 20.9 220, 20.9 360, 20.9420, 20.9 220, 20.8280, 20.8 360, 20.8 240, 20.7 260, 20.7 480, 20.7 440, 20.7 220, 20.6 560, 20.6 20.5 140, 20.5 440, 20.5 280, 20.4 580, 20.4640, 20.4 260, 20.3 480, 20.3540, 20.3 660, 20.3 1260, 20.2 480, 20.1 20 660, 20 320, 19.8 440, 19.7 760, 19.6 19.5 1040, 19.4 1140, 19.3 1160, 19.1 19 18.5 0 200 400 600 800 1000 1200 1400 sdLDL-C (nmol/L) FIGURE 2 Association between sdLDL-C and LDL-Z In Tables 1 and 2, some characteristic of population studied are 2, there was a direct linear association between sdLDL-C and LDL-P shown. In table 1, means and ranges of each variable included in (r xy = 0.687 [0.555-0.785]; P = 2.1 × 10−7). Besides, there was an VITAGE study can be seen. Means and ranges from each variable are inverse association between sdLDL-C and LDL-Z (r xy = −0.603 representative of general population. [−0.723-0.447]; P = 1.6 × 10−12). Both were statistically significant With these data, the principal aim of this study was to investigate associations. the relationship between all these characteristics and the concentra- The strong positive association between sdLDL-C and LDL-P tion of sdLDL-C obtained by precipitation. proves that the cholesterol isolated by the precipitation method Firstly, a correlation study was carried out as a tool to assess represents a great part of the cholesterol contained in sdLDL the validity of the precipitation method As shown in Figures 1 and particles as determined by NMR. Therefore, it could be that by 6 of 9 | FERNÁNDEZ-CIDÓN et al. TA B L E 2 Univariate regression analyses results TA B L E 3 Regression coefficients for the variables included in the multivariate model Variable B P Variable B [95% CI] β P Age (y) 0.18 .174 BMI (kg/cm2) 23.30 .04 Age (y) 0.169 [−2.627 a 2.966] 0.011 .904 2 Systolic pressure (mm Hg) −1.350 .755 BMI (kg/cm ) 0.016 [0.032 a 0.114] 0.175 .048 Diastolic pressure (mm Hg) 2.859 .571 ALT (µkat/L) 78.172 [−95.288 a 251.631] 0.068 .372 Alcohol (g/d) 0.62 .8 Vit E (µmol/L) −8.780 [−20.717 a 3.158] -0.230 .147 Diet (kcal/d) 0.073 .925 Tg (mmol/L) 0.182 [0.391 a 0.325] 0.352 .013 ALT (µkat/L) 323.063 .021 HDL-C (mmol/L) -10.388 [−248.315 a 227.539] -0.013 .931 AST (µkat/L) 414.234 .087 IDL-C (mmol/L) 245.684 [−769.679 a 278.312] 0.080 .353 GPT (µkat/L) 210.727 .167 Apo B (mg/dL) 0.071 [0.025 a 0.116] 0.415 .003 Creatinine (µmol/L) 2.248 .500 Apo E (mg/dL) 3.997 [−70.137 a 62.142] −0.011 .904 CRP (mg/L) 1.167 .648 Apo CIII (mg/dL) 10.638 [−27.980 a 6.702] 0.132 .225 Albumin (mg/L) 1.163 .942 FFAs (ng/mL) 0.001 [−0.004 a 0.061] 0.264 .084 TTR (g/L) 1.614 .070 LDL-Z (nm) −126 [−212 a −396] -0.394 .005 Glucose (mmol/L) 4.585 .487 Abbreviations: ALT, alanine aminotransferase; ApoB, apolipoprotein B; ApoCIII, apolipoprotein CIII; ApoE, apolipoprotein E; B, regression Fib (ng/L) 23.233 .063 coefficient at a 95% confidence interval or CI 95%; BMI, body mass Vit A (µmol/L) −21.507 .062 index; c-HDL, high-density lipoprotein cholesterol; c-IDL, intermediate- Vit E (µmol/L) −15.525 .001 density lipoprotein; FFAs, free fatty acids; LDL-Z, LDL diameter; Tg, triglycerides; Vit E, vitamin E; β, standardized regression coefficient. Cholesterol (mmol/L) 113.001 .059 Tg (mmol/L) 374.575 5.43 × 10−13 LDL-C (mmol/L) 135.64 .01 more cholesterol transported by them. To summarize, these first HDL-C (mmol/L) −445.004 .06 × 10 −6 results confirm that there is an association between LDL charac- IDL-C (mmol/L) 414.234 5.63 × 10 −5 teristics obtained by NMR and cholesterol measured after isolat- VLDL-C (mmol/L) 688.12 1.02 × 10 −9 ing sdLDL by precipitation. Taking into account these results, it would be interesting to study Apo A (mg/dL) −5.036 0.002 whether it is possible to estimate the presence of particles of small Apo B (mg/dL) 10.55 2.83 × 10 −8 size and its number by measuring sdLDL-C. Apo E (mg/dL) 77.65 .04 According to recent articles, it seems that the presence of Apo CIII (mg/dL) 28.064 .001 an elevated number of sdLDL particles is a clear risk. In fact, the Lpa (mg/L) 1.538 .376 23 Hortega-Liposcale Follow-up study found an association be- FFAs (ng/mL) 0.077 6.26 × 10 -9 tween the presence of lipoproteins with small and medium size PUFAs (ng/mL) 2.498 .988 and the increased cardiovascular risk, especially of coronary heart MUFAs (ng/mL) 0.598 .476 disease. IA 240.718 .078 This is particularly important due to the fact that discordance LDL-Z (nm) −185.137 1.02 × 10 -9 between LDL-C and LDL-P has been described in some cases. 20,21 The discordance is due to the fact that cholesterol concentration Abbreviations: AI = c-LDL/c-HDL, atherogenic index; ALT, alanine amino transferase; ApoA-I, apolipoprotein A; ApoB, apolipoprotein B; in these particles varies depending on the size of the different LDL ApoCIII, apolipoprotein CIII; ApoE, apolipoprotein E; AST, aspartate subclasses. 20-22 Especially relevant is the MESA study. 22 This study aminotransferase; B, regression coefficient; BMI, body mass index; has demonstrated the discordance between LDL-C and LDL-P and its c-HDL, high-density lipoprotein cholesterol; c-IDL, intermediate-density relationship with the development of atherosclerosis and coronary lipoprotein cholesterol; c-LDL, low-density lipoprotein cholesterol; CRP, c-reactive protein; c-VLDL, very low-density lipoprotein; FFA, events. free fatty acids; Fib, fibulin; GPT, glutamate-pyruvate transaminase; In another study (clinical trial), it has been found that in women LDL cholesterolTg, triglycerides; LDL-P, particle concentration; LDL-Z, with discordant LDL-related measures, coronary risk could be over- LDL diameter; Lpa, lipoprotein A; MUFAs, monounsaturated fatty estimated or underestimated when LDL-C concentration was con- acids; PUFAs, polyunsaturated fatty acids; sdLDL-C, small dense; TTR, transthyretin; Vit A, vitamin A; Vit E, vitamin E. sidered alone. 20 Recently, Tsai MY et al have demonstrated that sdLDL-C concentration measured by Denka Seiken automated assay is associated with CHD events independent of LDL-C concentration. measuring sdLDL-C, LDL-P can be estimated. On the other hand, Clinically, they found that sdLDL-C assessment would be more ben- the negative association between in sdLDL-C concentration and eficial in patients with intermediate CHD risk, since they could be LDL size indicates that people that have smaller particles have benefit from intensive treatments. 28 FERNÁNDEZ-CIDÓN et al. | 7 of 9 On account of these studies, LDL-C concentration is not these are linked to atherogenic dyslipidemia, which is well-related to enough to estimate the cardiovascular risk in patients in which the presence of sdLDL particles. Individuals with a high BMI have an LDL-C goals are achieved but elevated concentration of sdLDL excess of FFA, which in turn leads to a high concentration of Tg. An particles is found. Keeping in mind this fact, it would be really elevated concentration of Tg leads to increased formation of sdLDL interesting to know this information through a simple, fast, and particles. Consequently, the concentration of Tg influences sdLDL-C inexpensive method. In a recent study, it was demonstrated that more that it does LDL-C; reviewing the most relevant articles, we in patients with elevated LDL-P the risk of a CHD event increased, could deduce that the LDL-C reducing therapies should not be the and patients undergoing LDL-P measurements were more likely only therapeutic strategy for preventing atherothrombosis events. to receive more aggressive lipid-lowering therapy and had a Other factors should be considered, such as Tg concentration, which lower cardiovascular risk than a cohort in which just LDL-C was implies sdLDL particles formation. measured. 28,29 Only significant variables from the univariate analyses were used Considering the results from this first study, a regression anal- to construct the multivariate model. ysis was performed. The main objective of this second part is to Vitamin E concentration is relevant even though it was not in- assess the impact of a set of clinical and biochemical variables on cluded in the multivariate model. However, Vit E contribution to sdLDL concentrations. In Table 2, all univariate regressions between variations in sdLDL-C was more significant than other lipid variables sdLDL-C concentration and each variable are shown. Only those sig- directly related to the formation of sdLDL particles such as IDL-C. nificant variables (P < .05) were included in the multivariate analysis: Vit E is an antioxidant primarily transported by LDL particles. The age, BMI, ALT, vitamin E, and concentrations of HDL, IDL, ApoB, Apo oxidation resistance of these particles will depend on their carrying E, Apo CIII, FFAs, and LDL-Z. Once the variables were included in the antioxidants such as Vit E. 30 We have verified that there is an inverse multivariate analysis, only TG, LDL-Z, FFA and ApoB concentration relationship between Vit E and sdLDL-C. However, a study should be continued to be significant (see Table 3). carried out to determine whether a Vit E deficiency favors the for- The final multivariate model is summarized in Table 3. It ex- mation of sdLDL particles. In addition, we should find out whether plains a 57% of sdLDL-C variation. The major contributors in the Vit E deficiency is the reason why sdLDL particles have a greater model are ApoB and LDL-Z. This is a coherent result because sdLDL tendency to undergo oxidation and give rise to more unstable ather- particles are formed by cholesterol and a single ApoB molecule. omas, which increase the risk of ischemic events.31 However, LDL-C was not finally included in the last model due to To summarize, it is important to emphasize the fact that the num- its collinearity with apoB and minor significance. Extrapolating ber of articles that show the lack of concordance between the LDL-C to clinical practice, these results agree with literature, since they and the number of sdLDL particles is increasing, and it must be took imply that there are individuals with a normal LDL-C and a high into account to explore residual cardiovascular risk. In cases when 20 sdLDL-C and vice versa. The discordance phenomenon could be NMR is not available, to determine the sdLDL-C concentration could present in people with low LDL-C concentration, even in people be a good strategy to carry out. who achieve LDL-C goals. 20,21 Regarding the relation between sdLDL-C concentration varia- tion and LDL-Z, we found that an increase in the diameter of LDL 5 | CO N C LU S I O N particles implies a decrease in sdLDL-C concentration. Importantly, taking into account the multivariate regression, an increment of This study demonstrates that the cholesterol contained in sdLDL 1 nm in LDL size leads to a 126 nmol/L reduction in sdLDL-C con- measured by an easy and inexpensive method is related to the parti- centration. As a consequence, smaller LDL particles contain a higher cle size determined by NMR independently of a great number of vari- concentration of cholesterol. Due to its composition, smaller LDL ables. Taking into account the necessity of improving risk stratification particles would support the formation and progression of the ath- in cases when LDL-C determination is not enough, when NMR is not eroma plaques in higher degree than larger ones. Considering the available, to quantify the cholesterol concentration in sdLDL particles relationship between sdLDL particles and the development of ath- can be a reliable alternative to estimate residual cardiovascular risk. erosclerosis, sdLDL-C should be thoroughly investigated as a novel predictor of premature ischemic events. Therefore, when NMR is AC K N OW L E D G M E N T S not available, measuring sdLDL-C concentration could be a reliable The authors thank CERCA Program/Generalitat de Catalunya for tool for diagnosis risk stratification instead of LDL-C measurement. providing institutional support. This study was funded by Hospital Implementing this method in clinical laboratories is easy since it Universitari de Bellvitge, part of the Institut Català de la Salut (ICS) does not require the acquisition of new equipment nor does it re- and Department of Biochemistry, Molecular Biology and Biomedicine, quire specialized training. It could be especially important in cases as Autonomous University of Barcelona (UAB), Barcelona, Spain. premature cardiovascular disease, or in patients under lipid-lowering therapy with high cardiovascular CVR who achieve LDL-C goals. AU T H O R C O N T R I B U T I O N S Finally, it is also important to consider the contribution of signif- BFC and BCE involved in design of the study, experimental part, icant variables as Tg, BMI, and FFAs in the multivariate model. All of and writing and revising full article; JR and ER contributed to select 8 of 9 | FERNÁNDEZ-CIDÓN et al. population, experimental part, and revising full article; MGG, APM, 13. Mora S, Otvos JD, Rifai N, Rosenson RS, Buring JE, Ridker PM. Lipoprotein particle profiles by nuclear magnetic resonance com- and PA involved in revising full article; NA involved in experimental pared with standard lipids and apolipoproteins in predicting incident part and revising full article; XPS involved in writing and revising full cardiovascular disease in women. Circulation. 2009;119:931-939. article. 14. Nishikura T, Koba S, Yokota Y, et al. Elevated small dense low-den- sity lipoprotein cholesterol as a predictor for future cardiovascular events in patients with stable coronary artery disease. J Atheroscler ORCID Thromb. 2014;21(8):755-767. Beatriz Candás-Estébanez https://orcid. 15. National Cholesterol Education Program (NCEP) Expert Panel on org/0000-0001-9154-5193 Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III). 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