Atherosclerosis 234 (2014) 200e205

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Bariatric surgery in morbidly obese patients improves the atherogenic qualitative properties of the plasma lipoproteins Josep Julve f, g, h,1, Eva Pardina a,1, Montserrat Pérez-Cuéllar f, Roser Ferrer b, Joana Rossell a, Juan Antonio Baena-Fustegueras e, José Manuel Fort b, Albert Lecube c, d, g, h, Francisco Blanco-Vaca f, g, h, José Luis Sánchez-Quesada f, Julia Peinado-Onsurbe a, * a

Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain Departament de Bioquímica i Unitat de Cirurgia, Institut de Recerca Hospital Universitari Vall d’Hebron, Barcelona, Spain Departament d’Endocrinologia i Nutrició, Hospital Universitari Arnau de Vilanova, Universitat de Lleida, Lleida, Spain d Unitat de Recerca en Diabetes i Metabolisme, Institut de Recerca Hospital Universitari Vall d’Hebron, Barcelona, Spain e Unitat de Cirurgia, Hospital Arnau de Vilanova, Lleida, Spain f Institut d’Investigació Biomèdica de l’Hospital de la Santa Creu i Sant Pau, IIB-Sant Pau, Barcelona, Spain g CIBER de Diabetes y Enfermedades Metabólicas Asociadas, CIBERDEM, Barcelona, Spain h Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Barcelona, Spain b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 26 November 2013 Received in revised form 14 February 2014 Accepted 17 February 2014 Available online 14 March 2014

Objective: The purpose of this study was to evaluate the effect of weight loss induced in morbidly obese subjects by Roux-en-Y gastric bypass bariatric surgery on the atherogenic features of their plasma lipoproteins. Methods: Twenty-one morbidly obese subjects undergoing bariatric surgery were followed up for up to 1 year after surgery. Plasma and lipoproteins were assayed for chemical composition and lipoproteinassociated phospholipase A2 (Lp-PLA2) activity. Lipoprotein size was assessed by non-denaturing polyacrylamide gradient gel electrophoresis, and oxidised LDL by ELISA. Liver samples were assayed for mRNA abundance of oxidative markers. Results: Lipid profile analysis revealed a reduction in the plasma concentrations of cholesterol and triglycerides, which were mainly associated with a significant reduction in the plasma concentration of circulating apoB-containing lipoproteins rather than with changes in their relative chemical composition. All patients displayed a pattern A phenotype of LDL subfractions and a relative increase in the antiatherogenic plasma HDL-2 subfraction (>2-fold; P < 0.001). The switch towards predominantly larger HDL particles was due to an increase in their relative cholesteryl ester content. Excess weight loss also led to a significant decrease in the plasma concentration of oxidised LDL (w
25%; P < 0.01) and in the total Lp-PLA2 activity. Interestingly, the decrease in plasma Lp-PLA2 was mainly attributed to a decrease in the apoB-containing lipoprotein-bound Lp-PLA2. Conclusion: Our data indicate that the weight loss induced by bariatric surgery ameliorates the atherogenicity of plasma lipoproteins by reducing the apoB-containing Lp-PLA2 activity and oxidised LDL, as well as increasing the HDL-2 subfraction. Ó 2014 Elsevier Ireland Ltd. All rights reserved.

Keywords: Bariatric surgery Obesity HDL Oxidised LDL Lipoprotein-associated phospholipase A2 (Lp-PLA2) Weight loss

Abbreviations: apo, apolipoprotein; HDL, high-density lipoprotein; HDL-C, HDL cholesterol; LDL, low-density lipoprotein; LDL-C, LDL cholesterol; Lp-PLA2, lipoprotein-associated phospholipase A2; MSR1, scavenger receptor A (SRA) gene; nonHDL-C, non-HDL cholesterol; OLR1, oxidised (lectin-like) LDL receptor-1 gene; oxLDL, oxidised LDL; PLA2G7, group VII phospholipase A2 gene; PON1, plasma paraoxonase-(PON)1 gene; RYGBP, Roux-en-Y gastric bypass; SCARB1, scavenger receptor B type I (SR-BI) gene; VLDL, very-low-density lipoprotein. * Corresponding author. Departament de Bioquímica. Facultat de Biologia. Universitat de Barcelona. Av. Diagonal 643, 08028 Barcelona, Spain. Tel.: þ34 93 4021542; fax: þ34 93 4021559. E-mail address: [email protected] (J. Peinado-Onsurbe). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.atherosclerosis.2014.02.034 0021-9150/Ó 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Obesity is a major health problem in westernised societies [1]. Bariatric surgery is the only treatment that can effectively achieve a stable weight reduction and improve dyslipidaemia and cardiovascular disease risk in the morbidly obese [2e6]. Although a direct association between weight gain and total plasma cholesterol, triglycerides and lipoprotein levels, especially low-density lipoprotein (LDL), has been reported in different

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human studies [7,8], the relationship between plasma LDLcholesterol (LDL-C) concentration and obesity still remains controversial. For instance, the plasma concentration of LDL-C does not correlate with body mass index [1] and frequently remains unchanged in severe morbidly obese subjects compared to healthy, non-obese subjects [9]. Conversely, a favourable reduction in the plasma concentration of LDL-C has been reported in the obese undergoing bariatric surgery in recent systematic reviews and meta-analyses [10,11] and most clinical studies [12e20]. Oxidative stress is increased in obesity [5,6]. In this condition, an elevated plasma concentration of oxidised LDL (oxLDL) particles is frequently found in obese individuals [21] and exhibits a much stronger association with the degree of obesity than the LDL concentration itself [21]. Accordingly, weight loss induced by bariatric surgery has been reported to improve the oxidative stress by reducing the plasma concentration of oxLDL [22]. Oxidative stress can also lead to HDL dysfunction [23]. HDL are complex and functionally heterogeneous [24]. The ability of HDL to inhibit LDL oxidation is considered one of its antiatherogenic actions [25]. The HDL-mediated protective effect depends on the presence of apolipoproteins and enzymes that possess antioxidant and antiinflammatory properties. In this regard, the HDL-associated antioxidant enzymes, the plasma paraoxonase (PON)-1 and lipoproteinassociated phospholipase A2 (Lp-PLA2), are the main contributors to the antioxidant and anti-inflammatory properties of HDL [26]. LpPLA2 also associates with LDL in plasma where, in contrast with its antiatherogenic role in HDL, it exhibits a proatherogenic function [27]. For this reason, the relative distribution of Lp-PLA2 in HDL and LDL has been suggested to influence the progression of atherosclerosis [27]. A low level of circulating PON-1 and high plasma Lp-PLA2 activity have been associated with obesity, metabolic syndrome and coronary artery disease in most prospective studies to date [27]. Recent data suggest that excess weight loss improves the oxidative status of morbidly obese patients, as shown by decreases in different plasma markers, including the plasma concentration of oxLDL, and an increase in the plasma PON-1 activity [22]. However, no favourable changes have been found in the Lp-PLA2 mass in a previous study [28]. The impact of weight loss on the detailed composition of circulating lipoprotein species, the activity and distribution of LpPLA2 among lipoproteins, and the relationship, if any, of these systemic changes to the hepatic-related expression of lipoproteinassociated enzymes (ie., PON1 and PLA2G7) and receptors involved in the clearance of modified lipoproteins (ie.,SCARB1, MSR1 and CD36) have not been previously analysed in obese subjects after bariatric surgery. In this context, we tested the hypothesis that significant weight loss might improve both the quantitative (ie., concentration and chemical composition) and qualitative (ie., size and content in Lp-PLA2 activity) properties of circulating lipoproteins. Therefore, the purposes of this study were to deeply evaluate the effect of RYGBP on [i] the changes in the plasma circulating lipoprotein mass, composition, and size; [ii] the oxidative status by analysing the plasma concentration of oxLDL and lipoprotein distribution of Lp-PLA2 activity; and [iii] the liver-related gene expression of target proteins directly involved in the antiatherogenic properties of HDL and clearance of modified LDL in morbidly obese subjects, as a baseline, and up to 12 months after bariatric surgery-induced weight loss. 2. Materials and methods 2.1. Patients A group of 21 morbidly obese subjects (15 women and 6 men), between 20 and 60 years of age, who underwent RYGBP surgery

201

were enrolled and followed up at the Hospital de la Vall d’Hebron in Barcelona, as described elsewhere [27,29e33]. The subjects presented the necessary indications for bariatric surgery: BMI >40 kg/ m2 or greater than 35 kg/m2 with at least one comorbidity, including hypertension, DM2, dyslipidaemia, obstructive sleep apnoea or weight-induced rheumatological disease. The diagnostic criteria used for DM2, hypertension, and metabolic syndrome are detailed in the National Cholesterol Education Program [34]. All subjects were free of inflammatory and infectious diseases, and none of the subjects were receiving anti-obesity or antiinflammatory drugs at the time of the study. Patients were excluded if they had neoplastic, renal, or active systemic diseases, hypothyroidism, or an endocrine disease other than diabetes. All patients reported that their weight had been stable during the previous three months. None of the diabetic patients were being treated with insulin. All patients were required to complete a 2week preoperative low carbohydrate, high-protein liquid commercially formulation of 800 kilocalories per day or less replacing all usual food intakes. This diet was administered with the adequate support of a nutritional expertise and was supplemented with a multivitamin complex in order to provide the daily allowances of all essential nutrition requirements. After surgery, patients were discharged if they were able to tolerate liquids and/or puréed diet by mouth, and incrementally advanced to full feeding. Standard discharge medication included a daily multivitamin, omeprazole 20 mg daily during the first month, and intramuscular vitamin B(12) administered monthly. The study protocol was reviewed and accepted by the hospital ethics committee, and all subjects gave their written informed consent to participate. Blood sample collection and tissue biopsies were performed for each subject at baseline. Six out of 21 participants volunteered to undergo liver biopsy at 12 mo after surgery. Anthropometric parameters, including BMI, height, and waist and hip circumferences, were assessed before surgery and at 1 mo, 3 mo, 6 mo, and 12 mo after surgery. Blood samples were collected using EDTA-containing Vacutainer tubes after an overnight fast of 12 h at the same time points. 2.2. Biochemical analyses The lipid profile, oxidised LDL, lipoprotein composition and LpPLA2 activity distribution were determined from the plasma. The lipid profile included total cholesterol (Roche), triglycerides (Roche), LDL-C (LDL-C Plus, Roche) and HDL-C (HDL-C Plus, Roche). VLDL-cholesterol (VLDL-C) was calculated by subtracting the amount of LDL-C and HDL-C from the total cholesterol. Free cholesterol and phospholipids were determined using reagents from Wako Diagnostics. Human apoA-I and apoB were quantified using commercially available turbidimetric assays (RAL). All determinations were performed using commercial kits that are routinely used in the chemistry laboratory at the Hospital. The bicinchoninic acid assay reagent (Pierce) was used for total protein quantification. LDL size was determined by non-denaturing polyacrylamide gradient (2e16%) gel electrophoresis (GGE) with Sudan black prestained samples, as previously described [35]. The LDL subfraction phenotype B was defined by a predominant LDL diameter lower than 25.5 nm, whereas phenotype A subjects had an LDL diameter higher than 25.5 nm, as previously described [36]. The relative distribution of HDL-2 and HDL-3 was determined using densitometry in the same gels. The composition analysis of circulating lipoproteins was carried out in the 6 out of 21 obese subjects who volunteered to undergo liver biopsy at 12 mo after surgery. The plasma lipid and apolipolipoprotein profile and its evolution after surgery shown by this

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subset of patients did not differ from that displayed by the whole group. Lipoprotein fractions were isolated by sequential ultracentrifugation at 100,000 g for 24 h [37]. Lipoprotein subfraction composition was analysed for lipids, including total cholesterol, free cholesterol, phospholipids, and triglycerides, and proteins (total protein content) at the indicated times using commercial methods. OxLDL was measured using a commercially available specific ELISA assay (Mercodia AB). Plasma Lp-PLA2 activity was measured using 2-thio-PAF (Cayman) as a substrate, according to the manufacturer’s instructions. To determine the distribution of Lp-PLA2 between the lipoprotein fractions, apoB-containing lipoproteins were precipitated from the plasma using dextran sulphate, as previously described [35]. Briefly, 100 mL of plasma was mixed with 25 mL of dextran sulphate reagent, incubated at room temperature for 5 min, and centrifuged at 10,000 g for 10 min. The supernatant, which was depleted of apoB-containing lipoproteins, was collected and assayed for HDL-associated Lp-PLA2 activity. 2.3. Quantitative real-time RT-PCR analyses The liver-related gene expression analysis was performed for 5 out of 21 of the obese subjects who voluntarily underwent biopsy using real-time RT-PCR. RNA was isolated from 10 to 25 mg of human liver biopsy using the TripureÒ RNA isolation method (Roche). The integrities of the total RNA samples were determined using a Bioanalyser. Total RNA (40 ng) was reverse-transcribed with random primers using a TaqMan high-capacity cDNA reverse transcription kit (Applied Biosystems) to generate cDNA. Predesigned, validated primers (Assays-on-Demand; Applied Biosystems) were used with the TaqManÒ probes. Real-time PCR assays were performed on a C1000 Thermal Cycler coupled to a CFX96 Real-Time System (Bio-Rad Laboratories SA, Life Science Group). All analyses were performed in duplicate, and relative RNA levels were determined using beta-actin (BACT) as an internal control. 2.4. Statistical methods The data are presented as the median and the 25th and 75th percentiles. Statistical analyses were performed using the GraphPad Prism software (GPAD, version 5.0, San Diego, CA, USA). The effects of weight loss after surgery on the plasma chemical parameters or the expression of target genes were determined using a non-parametric repeated-measure Friedman test followed by a Dunn multiple comparison test or using a Wilcoxon test, when indicated. Differences between groups were considered statistically significant when the P value was 2-fold; P < 0.001) in the antiatherogenic plasma HDL-2 subfraction relative to the total HDL at 6 months (38.0 (25.6; 59.4) %) and 12 months (47.6 (41.0; 58.3) %) after surgery compared to the baseline values (20.1 (16.3; 32.8) %) (Fig. 1; panel C). The weight loss induced switch towards a higher proportion of larger HDL particles in the plasma was consistently associated with a decrease in the protein-to-lipid ratio (Supplemental Fig. 2; panel C), mainly due to an increased cholesteryl ester content (w1.7-fold; P < 0.05) in combination with a significant decrease in the protein content (w
0.7-fold; P < 0.05) in these particles (Fig. 1; Supplemental Table 2). Effect of RYGBP on the plasma concentration of oxLDL and LpPLA2 activity and liver-related gene expression of antioxidant lipoprotein-associated enzymatic activities and receptors involved in the clearance of oxLDL. Bariatric surgery also caused a positive impact on the oxidative status of the plasma (Fig. 2; panel A). A significant reduction (w
25%; P < 0.05) in the plasma concentration of oxidised LDL was observed both at 6 (34.5 (23.8; 45.3) U/L) and 12 months (35.3 (25.3; 40.0) U/L) after surgery compared to the baseline values (46.1 (40.5; 54.9) U/L) (Fig. 2; panel A). Interestingly, the reduction in the plasma concentration of oxLDL was further associated with mild (w
10%), but significant, changes (P < 0.01) in the total plasma levels of Lp-PLA2 activity (Fig. 2; panel B). Total plasma Lp-PLA2 activities were lower in obese patients both at 6 (14.1 (11.3; 16.7) mmol mL
1 min
1) and 12 months (15.1 (12.7; 17.6) mmol mL
1 min
1) after surgery compared to the obese baseline values (17.0 (14.8; 20.7) mmol mL
1 min
1) (Fig. 2; panel B). Because no significant differences were observed in the HDL-associated LpPLA2 activity, the reduction in total Lp-PLA2 was mainly attributed to a decrease in the apoB-containing lipoprotein-associated LpPLA2 activity (Fig. 2; panel B) and could not be explained by significant alterations in the relative expression of the PLA2G7 gene in the liver (Supplemental Fig. 3; panel A), despite a slight decrease in its mRNA synthesis (
2-fold; P ¼ 0.19) at 12 months after surgery (0.5 (0.4; 1.4)) compared to the baseline values (1.0 (0.8; 1.6)). In addition to the liver expression of the PLA2G7 gene, PON1 gene expression was also analysed (Supplemental Fig. 3; panel B). Our data showed that although it tended to increase (wþ20%; P ¼ 0.12), the expression of the PON1 gene remained unchanged in

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Fig. 1. Size, distribution, and relative chemical composition of plasma lipoproteins after bariatric-induced weight loss in morbidly obese patients. A. Representative native gradient gel electrophoresis of the plasma lipoproteins of patients at the indicated times after surgery compared to their baseline values. B. LDL size [nm] calculated from the native gradient gel electrophoresis of plasma lipoproteins of patients at the indicated times compared to their baseline values. C. Per cent content of the HDL-2 subfraction vs. the total HDL calculated from the native gradient gel electrophoresis of plasma lipoproteins of patients at the indicated times after surgery compared to their baseline values. D. Chemical composition of VLDL. E. Chemical composition of LDL. F. Chemical composition of HDL. For the chemical composition analysis of plasma lipoproteins (panels D, E and F), the lipoproteins were isolated by ultracentrifugation from the fasting plasma of obese subjects before and after surgery at the indicated times. In panels B and C, the data are expressed as the median (25th; 75th percentiles) from 21 subjects, whereas in panels D, E and F, the data are expressed as the median (25th; 75th percentiles) from a cohort group of 6 subjects. * P < 0.05, **P < 0.01, ***P < 0.001 vs. baseline, yP < 0.01 vs. 6 mo.

the patients’ livers after surgery (1.7 (1.4; 1.9)) compared to the baseline expression (1.3 (0.9; 1.5)). The relative expression of genes encoding different scavenger receptors involved in the plasma clearance of modified lipoproteins, including CD36, MSR1 and SCARB1, and the gene encoding the oxLDL (lectin-like) receptor-1, OLR1, were also assessed in the liver before and a year after intervention (Supplemental Fig. 3; panels Ce F). Although the relative gene expression of CD36 and MSR1 tended to decrease after surgery (CD36: 0.4 (0.4; 0.9); MSR1: 0.7 (0.5; 1.0)), this change was not significant compared to the baseline expression values (CD36: 1.0 (0.7; 1.3); MSR1: 1.1 (1.0; 1.1)) (Supplemental Fig. 3; panels DeE). The hepatic expression of OLR1 was almost undetectable at baseline but, notably, its expression tended to show even lower levels after surgery (Supplemental Fig. 3; panel F). 4. Discussion Herein, we report the effects of RYGBP on the atherogenic lipoprotein profile in the plasma, including favourable changes in the concentration and the qualitative characteristics of circulating lipoproteins. For the first time, we describe the association between RYGBP and the changes in the distribution of Lp-PLA2 in the plasma lipoproteins and the gene expression of proteins involved in lipoprotein metabolism and oxidative stress in the liver. Favourable changes in the fasting lipid and apolipoprotein profiles in the plasma occurred over 1 year after RYGBP bariatric intervention. A commensurate positive evolution of the lipid or

apolipoprotein ratios further confirmed a weight loss induced amelioration of the atherogenic lipoprotein profile and insulin resistance shown by our obese patients [29]. As previously reported [11,12,14,15,18,20,38], a significant reduction in the plasma concentrations of total cholesterol and triglycerides was mainly due to a reduction in the plasma concentration of apoB-containing lipoproteins, particularly LDL. Moreover, the percent chemical composition and size of these lipoproteins did not vary along with the weight loss, which is likely due to the mild extent of hypertriglyceridemia and hypercholesterolemia present in the morbid obese individuals analysed in the present study. Consistent with previous reports [12,14,16,18], the plasma concentration of HDL-C was not significantly modified after surgery when compared to the baseline value. The chemical analysis of HDL revealed a significant reduction in the protein content and enrichment in the cholesteryl ester moiety 12 months after surgery, and these changes were associated with an enlargement of the particle core and size. This finding was further consistent with the increase of predominantly larger HDL particles (HDL-2 subfraction) observed in the plasma at the indicated times after surgery. A similar switch towards larger HDL particles in the plasma of the morbidly obese undergoing gastric bariatric surgery has been recently reported in the absence of significant changes in the plasma HDL-C concentration [12,14,16]. LCAT and CETP are thought to be involved in the intravascular remodelling of plasma HDL. Although these two plasma enzyme activities were not directly determined in this study, an increase in the LCAT activity [39] in

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oxLDL [U/L]

150

A

100

*** 50

Lp-PLA2 activity [μmol·mL-1·min-1]

0

30 20

Baseline

6 mo.

B

***

12 mo. Baseline 6 mo. 12 mo.

* * * *

10 0 Total Lp-PLA2

Non-HDL Lp-PLA2

HDL Lp-PLA2

Fig. 2. Plasma concentration of oxLDL and Lp-PLA2 enzyme activity in morbidly obese subjects before and 6 and 12 months after gastric surgery. A. Plasma oxLDL concentration determined in the plasma lipoproteins of patients 6 and 12 months after surgery compared to their baseline values. B. Total plasma Lp-PLA2 (Total Lp-PLA2) and Lp-PLA2 distribution in the lipoprotein classes (apoB-containing lipoproteinassociated PLA2: Non-HDL Lp-PLA2; HDL-associated PLA2: HDL Lp-PLA2) in the patients at the indicated times after surgery compared to their baseline values. The data are expressed as the median (25th; 75th percentiles) from 21 subjects. *P < 0.05, *** P < 0.001 vs. baseline.

combination with a decrease in CETP activity [12e14,16] reported in obese patients after surgery might lead to an increase in HDLcholesteryl ester content and, consequently, contribute to the enlargement of the particle size observed in our patients after surgery. Oxidative stress plays a key role in the onset and pathogenesis of obesity-related comorbidities [40]. Increased adiposity and insulin resistance in the morbidly obese contribute to global oxidative stress by increasing the production of reactive oxygen species [6]. Conversely, minimising obesity induces a reduction in oxidative stress [6,41,42]. As previously described [21,22], the bariatric surgery-induced weight reduction leads to a decrease in the plasma concentration of oxLDL. Our data revealed that the liver-related gene expression pattern of two of the scavenger receptors (i.e., MSR1 and CD36), tended to decrease in response to the weight loss induced by the bariatric surgery, thereby suggesting that the liver, which is central in the clearance of oxLDL [21], would be sensitive to the amelioration of systemic oxidative indicators, including oxLDL. OxLDL are proatherogenic lipoproteins that are thought to result from the oxidation of small, dense LDL particles [21]. However, because our patient cohort displayed a phenotype A LDL pattern, factors other than the LDL size could account for these differences. First, oxidative stress, which is known to be increased in the morbidly obese [5], might contribute to LDL oxidation. Second, the total plasma LDL concentration significantly correlates with plasma concentration of oxLDL [43]. In this regard, the elevated concentration in plasma of LDL shown by our obese patients at baseline might be also contributing to the increased plasma levels of oxLDL. A third factor would be the HDL antioxidant activity, which protects

LDL from oxidation [44]. The extent of antioxidant activity exhibited by the HDL is mainly attributed to the content of PON-1 and LpPLA2 [26]. Serum PON-1 activity level has been shown to negatively correlate with BMI and is increased in obese patients undergoing gastric surgery, compared to the baseline value [22]. Consistently after surgery, PON-1 gene expression tended to increase in the liver, which is a main site for PON-1 synthesis in the body. In our study, a significant reduction in the total plasma Lp-PLA2 activity, mainly due to a drop in the non-HDL-associated Lp-PLA2 activity, was observed after surgery. Our data contrast with a previous study, which found that the total Lp-PLA2 mass was unchanged after gastric surgery in morbidly obese subjects [28]. However, in the latter work the Lp-PLA2 activity was not directly determined. It is noteworthy to mention that Lp-PLA2 can be dysfunctional under inflammatory conditions [44]. Moreover, misleading associations between Lp-PLA2 activity and mass and the risk of cardiovascular disease have been previously reported in other pathologies (e.g., DM2) with increased oxidative stress [45]; thus, the differential distribution of Lp-PLA2 among the lipoprotein classes is a possible explanation for this still unsolved controversy. Our data further showed that the decrease in total Lp-PLA2 after surgery was mainly due to the decrease of its apoB-containing lipoprotein content, which is strongly dependent on the amount of circulating LDL and VLDL in the plasma. In contrast, the absence of changes in the HDLassociated Lp-PLA2 activity before and after surgery would suggest that the HDL-associated Lp-PLA2 is independent of the HDL remodelling induced by the weight loss. Furthermore, the switch in the distribution of Lp-PLA2, which results in lower activity of the apoB-containing lipoproteins, suggests a protective effect of the bariatric surgery. Taken together, our data indicate that the weight loss induced by bariatric surgery ameliorates the plasma proatherogenic lipoprotein profile by decreasing both the plasma concentration of circulating apoB-containing lipoproteins, particularly LDL, and the plasma oxidative markers, including oxLDL and Lp-PLA2 activity, as well as increasing the HDL-2 subfraction. The study’s main flaw includes the relatively small number of patients, particularly in the lipoprotein and gene expression analyses. This, especially in the case of the gene expression analysis, may limit the interpretation of the observations and should therefore be considered with caution. Thus, the expression of target genes should be confirmed in future studies with a larger number of patients. Another limitation was the restricted availability of liver biopsies from the control, healthy donors. Besides, given that there was no matched control group that did not undergo RYGBP, this study should be categorized as an uncontrolled case series. Lastly, even though we assessed the LDL size and plasma concentration of oxLDL, the susceptibility of LDL to oxidation and the protection provided by the HDL against LDL oxidation was not explored in the present study. Hence, further investigation is warranted. Conflict of interest statement The authors declare no conflicts of interest. Acknowledgements This work was funded by Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III, FIS Grants PI10/00277 (to J.J.), PI11/ 01076 (to F.B.-V.), PI11/01159 (to J.P-.O.) and the Quality Research Group 2009-SGR-1205, Generalitat de Catalunya. CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM) is a project of the Instituto de Salud Carlos III. The English grammar and language was corrected by American Journal Experts (www.aje.com).

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Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.atherosclerosis.2014.02.034

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