Journal of Clinical Medicine Article Changes in the Composition and Function of Lipoproteins after Bariatric Surgery in Patients with Severe Obesity Idoia Genua 1,2,† , Núria Puig 2,3,† , Inka Miñambres 1,2,4,† , Sonia Benítez 3 , Pedro Gil 1,2 , Margarida Grau-Agramunt 3 , Andrea Rivas-Urbina 2,3 , Carme Balagué 2,5 , Sonia Fernández-Alanin 5 , Álvaro García-Osuna 3 , Antonio Pérez 1,2,4, * and José Luis Sánchez-Quesada 3,4, * 1 Endocrinology and Nutrition Department, Hospital de la Santa Creu i Sant Pau, 08041 Barcelona, Spain; igenua@santpau.cat (I.G.); iminambres@santpau.cat (I.M.); pgimll@icloud.com (P.G.) 2 Bellaterra Campus, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, 08041 Barcelona, Spain; npuigg@santpau.cat (N.P.); arivas@santpau.cat (A.R.-U.); cbalague@santpau.cat (C.B.) 3 Cardiovascular Biochemistry Group, Research Institute of the Hospital de la Santa Creu i Sant Pau (IIB Sant Pau), 08041 Barcelona, Spain; sbenitez@santpau.cat (S.B.); mgrauag@santpau.cat (M.G.-A.); agarciao@santpau.cat (Á.G.-O.) 4 CIBER of Diabetes and Metabolic Diseases (CIBERDEM), Instituto de Salud Carlos III (ISCIII), 28029 Madrid, Spain 5 General and Digestive Surgery Department, Metabolic & Bariatric Surgery Unit, Hospital de la Santa Creu i Sant Pau, 08041 Barcelona, Spain; sfernandeza@santpau.cat * Correspondence: aperez@santpau.cat (A.P.); jsanchezq@santpau.cat (J.L.S.-Q.); Tel.: +34-3553-5657 (A.P.);  +34-3553-7588 (J.L.S.-Q.)  † The authors contributed equally to this work. Citation: Genua, I.; Puig, N.; Miñambres, I.; Benítez, S.; Gil, P.; Abstract: The effect of bariatric surgery on lipid profile and the qualitative characteristics of lipopro- Grau-Agramunt, M.; teins was analyzed in morbidly obese subjects. Thirteen obese patients underwent bariatric surgery. Rivas-Urbina, A.; Balagué, C.; Plasma samples were obtained before surgery and at 6 and 12 months after the intervention. Thirteen Fernández-Alanin, S.; healthy subjects comprised the control group. Lipid profile, hsCRP, and the composition and func- García-Osuna, Á.; et al. Changes in tional characteristics of VLDL, LDL, and HDL were assessed. At baseline, plasma from subjects with the Composition and Function of obesity had more triglycerides, VLDLc, and hsCRP, and less HDLc than the control group. These Lipoproteins after Bariatric Surgery in Patients with Severe Obesity. J. Clin. levels progressively normalized after surgery, although triglyceride and hsCRP levels remained Med. 2021, 10, 1716. https://doi.org/ higher than those in the controls. The main differences in lipoprotein composition between the 10.3390/jcm10081716 obese subjects and the controls were increased apoE in VLDL, and decreased cholesterol and apoJ and increased apoC-III content in HDL. The pro-/anti-atherogenic properties of LDL and HDL Academic Editor: Vanessa Bianconi were altered in the subjects with obesity at baseline compared with the controls, presenting smaller LDL particles that are more susceptible to modification and smaller HDL particles with decreased Received: 9 April 2021 antioxidant capacity. Bariatric surgery normalized the composition of lipoproteins and improved Accepted: 14 April 2021 the qualitative characteristics of LDL and HDL. In summary, patients with obesity present multiple Published: 16 April 2021 alterations in the qualitative properties of lipoproteins compared with healthy subjects. Bariatric surgery reverted most of these alterations. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in Keywords: obesity; bariatric surgery; lipoproteins; lipoprotein function; LDL; HDL; apolipoproteins published maps and institutional affil- iations. 1. Introduction Obesity is a major public health issue that has become increasingly prevalent world- Copyright: © 2021 by the authors. wide [1]. Patients with obesity have an increased cardiovascular (CV) risk [2], mostly Licensee MDPI, Basel, Switzerland. This article is an open access article because of the presence of comorbidities, such as diabetes, hypertension, or dyslipidemia. distributed under the terms and The chronic inflammation and increased oxidative stress present in obesity may also con- conditions of the Creative Commons tribute to the increased CV risk [3]. One of these obesity-related comorbidities is the Attribution (CC BY) license (https:// alteration in lipid profile. Atherogenic dyslipidemia in patients with obesity is charac- creativecommons.org/licenses/by/ terized by an increased serum concentration of triglycerides and decreased high-density 4.0/). lipoprotein cholesterol (HDLc) levels [4]. In addition to the quantitative alterations found J. Clin. Med. 2021, 10, 1716. https://doi.org/10.3390/jcm10081716 https://www.mdpi.com/journal/jcm J. Clin. Med. 2021, 10, 1716 2 of 15 in obese subjects, these patients have alterations in the characteristics of lipoproteins, dis- playing small LDL and HDL particles, and large VLDL particles [5]. These qualitative changes in lipoproteins may alter their functionality, contributing to the increased CV risk [6]. Bariatric surgery lowers CV mortality [7], and the improvement of atherogenic dyslipi- demia has been proposed to explain the benefits on CV outcomes after bariatric surgery [8]. The improvements in lipid profile after surgery include a decrease in triglycerides and an increase in HDLc [8,9], as well as qualitative and functional modifications to lipoprotein particles [10]. The potential benefits on lipoprotein composition, size, and functionality after bariatric surgery might contribute to lowering the risk of CV disease, and, conse- quently, the focus has shifted from studying the levels of lipoproteins to studying the quality of lipoproteins. However, information about the effect of weight loss on the composition and function- ality of lipoproteins is limited. Different studies have reported the alteration of different properties of both HDL and LDL. Most of these studies focused on the cholesterol efflux capacity of HDL, and an improvement in cholesterol efflux after bariatric surgery was reported by most [11–14], but not all [15], authors. The distribution of HDL subspecies has also been extensively studied in obese patients, and there is a consensus regarding the shift of HDL subfractions from small to large HDL particles after bariatric surgery [10,16]. However, other properties of HDL in obese subjects, such as its antioxidant capacity, have only been analyzed in one study [15], and there is scarce information regarding changes in HDL composition after bariatric surgery. Regarding LDL, few studies have analyzed the qualitative properties of this lipopro- tein, and they focused mainly on LDL size and plasma levels of oxidized LDL (oxLDL). There are contradictory reports on the effect of bariatric surgery on LDL size, with some studies indicating increased size after surgery [17,18], and others reporting no change [16,19]. Some studies described an increased concentration of oxLDL in obesity, which decreases after bariatric surgery [16,19–21]. However, other characteristics of LDL, such as lipid com- position, the content of minor apolipoproteins, susceptibility to oxidation or aggregation, and the proportion of modified LDL particles in blood, have not been studied in obese subjects after bariatric surgery. The scarcity of studies on LDL is even more evident in the case of VLDL, beyond its known larger size in obese individuals with dyslipidemia [22]. Most previous studies focused on a specific lipoprotein fraction, so determining how alterations in one lipoprotein can affect other lipoproteins has not been possible. This aspect is relevant, as the metabolism of the different lipoproteins is closely interconnected, determining the characteristics and catabolism of VLDL and the properties of LDL and HDL. Furthermore, there is a close interaction between LDL and HDL in blood, and the exchange of lipids and apolipoproteins between LDL and HDL generates particles with greater or lesser atherogenic potential [23,24]. For this reason, carrying out a complete analysis of all lipoprotein fractions is necessary to uncover the mechanisms by which bariatric surgery modulates the pro- or anti-atherogenic properties of lipoproteins. The aim of this study was to evaluate the lipid profile, conduct an exhaustive analysis of the composition and function of the different lipoprotein fractions in patients with morbid obesity before and after bariatric surgery, and compare the data with those of a normal- weight healthy control group. 2. Materials and Methods 2.1. Study Population Thirteen morbidly obese patients were included in the study. The inclusion criteria were patients with severe morbid obesity who underwent bariatric surgery at Hospital de Sant Pau. All patients met the criteria used in usual clinical practice for bariatric surgery in our clinic: body mass index (BMI) > 40 Kg/m2 or BMI > 35 Kg/m2 with comorbidities, in- cluding hypertension, diabetes, and hyperlipidemia. All subjects were free of inflammatory and infectious diseases, and none of them were receiving anti-obesity or anti-inflammatory J. Clin. Med. 2021, 10, 1716 3 of 15 drugs. The exclusion criteria were undergoing treatment with anti-inflammatory drugs or a prior diagnosis of a disease affecting lipid metabolism. Obese patients underwent bariatric surgery (sleeve gastrectomy or gastric bypass according to routine clinical practice), and blood samples were obtained one week before the intervention (baseline) and at 6 and 12 months after. Blood was collected in EDTA-containing Vacutainer tubes after an overnight fast of 12 h. Plasma was obtained by centrifugation at 1500 g for 15 min at 4 ◦ C, and samples were frozen at −80 ◦ C until analysis. Thirteen healthy subjects were included in the control group. The controls were of normal weight, normolipidemic, and normoglycemic, and had no antecedents of inflam- matory, infectious, or coronary disease or other diseases known to affect lipid metabolism. The clinical and anthropometric data of all groups are shown in Table 1. All subjects gave written informed consent before participating in the study, and the protocol was approved by the ethical committee of the Hospital de la Santa Creu i Sant Pau. The study was performed in accordance with the Helsinki Declaration. Table 1. Anthropometric and clinical characteristics, lipid profile, and inflammatory markers. Obese Control Group Parameters (n = 13) (n = 13) Age (years) 51.3 ± 8.1 42.5 ± 11.6 Gender (M/F) 8/5 6/7 Baseline 6 months 12 months BMI (kg/m2 ) 42.7 ± 3.9 34.1 ± 3.0 a 30.0 ± 3.6 a,b 22.6 ± 2.1 a,b,c % TWL 20.02 ± 6.42 28.87 ± 8.99 b Fasting glucose (mmol/L) 6.68 ± 2.04 5.61 ± 1.30 a 5.32 ± 0.97 a 4.74 ± 0.47 a,b HbA1c (%) 5.91 ± 0.80 5.58 ± 0.59 5.51 ± 0.73 a 5.13 ± 0.27 a,b Cholesterol (mmol/L) 5.05 ± 0.85 4.34 ± 0.80 a 5.08 ± 1.00 b 5.22 ± 0.78 b Triglycerides (mmol/L) 1.45 ± 0.51 1.12 ± 0.35 a 1.13 ± 0.36 a 0.86 ± 0.28 a,b,c VLDL-c (mmol/L) 0.67 ± 0.24 0.51 ± 0.16 a 0.51 ± 0.17 a 0.40 ± 0.13 a LDL-c (mmol/L) 3.23 ± 0.76 2.67 ± 0.70 3.33 ± 0.83 b 3.28 ± 0.68 b HDL-c (mmol/L) 1.16 ± 0.19 1.15 ± 0.19 1.38 ± 0.22 a,b 1.54 ± 0.29 a,b ApoB (g/L) 1.06 ± 0.23 0.86 ± 0.17 a 1.00 ± 0.16 b 0.92 ± 0.15 a,b NEFA (mmol/L) 0.56 ± 0.28 0.66 ± 0.23 0.62 ± 0.20 0.49 ± 0.18 hsCRP (mg/L) 9.96 ± 6.71 5.28 ± 4.83 a 2.88 ± 2.21 a 0.84 ± 0.15 a,b,c a p < 0.05 vs. baseline. b p < 0.05 vs. 6 months. c p < 0.05 vs. 12 months. BMI: body mass index; TWL: total weight loss. 2.2. Plasma Determinations Lipid profile, C-reactive protein (CRP), total apoJ, Lp-PLA2 activity, LDL size, and HDL subfraction proportion were determined in plasma obtained in EDTA-containing Vacutainer tubes. The lipid profile included total cholesterol (Roche), triglycerides (Roche), apoB (Roche), and VLDL, LDL, and HDL cholesterol. Cholesterol of lipoprotein fractions was quantified using a direct HDL-cholesterol method (HDL-C plus, Roche) or by ultra- centrifugation when the TG concentration was higher than 3 mmol/L, according to the National Cholesterol Education Program [25]. Non-esterified fatty acids (NEFA) were quantified using a commercial kit (Wako Pure Chemical, Osaka, Japan). All these deter- minations and CRP (Roche Diagnostics, Basel, Switzerland) were performed in a Cobas 6000/c501 autoanalyzer. ApoJ plasma levels were measured with commercial ELISA kits (Mabtech, Stockholm, Sweden). LDL size and HDL subfraction proportions were evaluated from plasma by non-denaturing polyacrylamide gradient (2.5%–16%) gel electrophoresis, as described previously [26]. Lp-PLA2 activity was determined using 2-thio-PAF (Cayman Chemicals, Ann Arbor, MI, USA) as a substrate [27] according to the manufacturer’s in- structions. The distribution of Lp-PLA2 between lipoprotein fractions was measured by precipitating apoB-containing lipoproteins from plasma with dextran sulfate [28]. J. Clin. Med. 2021, 10, 1716 4 of 15 2.3. Lipoprotein Isolation and Composition Lipoproteins were isolated from thawed plasma by flotation sequential ultracentrifu- gation, according to density: VLDL (1.006–1.019 g/mL), LDL (1.019–1.063 g/mL), and HDL (1.063–1.210 g/mL). Their lipid and apolipoprotein composition was determined by measuring the content of cholesterol, triglycerides, apoB, apoA-I (Roche Diagnostics, Basel, Switzerland), phospholipids, free cholesterol (Wako Pure Chemical, Osaka, Japan), apoA-II, apoE, and apoC-III (Kamiya Biomedicals, Seattle, WA, USA) in the autoanalyzer. ApoJ was evaluated using commercial ELISA (Mabtech). 2.4. Lipoprotein Functional Assays Electronegative LDL (LDL(-)). The proportion of LDL(-) was quantified from the total LDL using stepwise anion exchange chromatography in a MonoQ 5/50 GL column (GE Healthcare, Chicago, IL, USA), as described previously [29]. LDL susceptibility to aggregation. LDL was dialyzed against phosphate-buffered saline (PBS) pH 7.4 using gel filtration chromatography in a PD10 column (Sephadex G-25, GE Healthcare). Aggregation of LDL was induced by SMase lipolysis. LDL (0.6 mmol/L cholesterol) was incubated at 37 ◦ C for 1 h with SMase from Bacillus cereus sp. (Sigma Diagnostics, Livonia, MI, USA) with a final concentration of 50 mU/mL in the presence of 2 mM CaCl2 and 2 mM MgCl2 . Size-exclusion chromatography was performed for monitoring aggregation using a Superose 6 Increase 5/150 GL column in an AKTA-FPLC system (GE Healthcare), as described [30]. LDL and HDL susceptibility to oxidation. LDL and HDL were dialyzed against PBS pH 7.4 using gel filtration chromatography in a PD10 column (Sephadex G-25, GE Healthcare). Susceptibility to oxidation was evaluated by monitoring the formation of conjugated diene at 234 nm in a Synergic HT spectrophotometer (BioTek). LDL or HDL at 0.15 mM of cholesterol was incubated with 5 µM of CuSO4 , and the lag phase time of the oxidation kinetics was determined, as described [31]. Antioxidant capacity of HDL. HDL at 0.15 mM of cholesterol was incubated with a standard LDL (obtained from a pool of normolipidemic plasma and stored with 10% sucrose at −80 ◦ C), and oxidation was induced by adding 5 µM of CuSO4 . Conjugated diene formation was monitored, as explained in the LDL section. The results were expressed as the capacity of HDL to prolong the lag phase time of the standard LDL alone, as described previously [32]. See Supplementary Materials for details. 2.5. Statistical Analysis Statistical analysis was performed using GraphPad Prism 6.0 software. Unpaired data (obese versus the control group) were compared using the nonparametric Mann– Whitney test. Paired data (obese patients before and after surgery) were analyzed using the nonparametric Wilcoxon test. Correlation analysis was conducted using the Spearman test for nonparametric variables. Data are expressed as mean ± SD or mean ± SEM. p < 0.05 was considered significant. 3. Results 3.1. Lipid Profile and Inflammation Markers Table 1 shows the anthropometric and clinical characteristics, lipid profile, and in- flammatory markers of the control subjects and the obese patients at baseline and at 6 and 12 months after bariatric surgery. Within the obese group, nine (69%) patients underwent sleeve gastrectomy, and four (31%) patients underwent gastric bypass. At baseline, obese patients presented similar levels of total cholesterol, LDLc, and apoB as the control group, but their triglycerides and VLDLc were higher, and their HDLc levels were lower than those in the control group. Bariatric surgery decreased TG and VLDLc at 6 months after surgery, and these levels remained stable, but were still higher compared with those of the control group. By contrast, the effect of surgery on HDLc levels was deferred, and the increase was only statistically significant at 12 months after surgery. Total cholesterol, The inflammatory biomarker hsCRP suggested increased systemi the obese patients at baseline, with high hsCRP levels compared with Surgery progressively decreased these levels, albeit without re J. Clin. Med. 2021, 10, 1716 concentration as that in the control group. 5 of 15 3.2. Changes in VLDL Composition LDLc, and apoB slightly decreased at 6 months but showed a rebound effect, with an The between increment analysis6 andof VLDLafter 12 months composition showed surgery. No difference thatconcentration in NEFA the VLDL fro was observedmore contained amongtriglycerides the groups. and less apoB than the VLDL from the con The inflammatory biomarker hsCRP suggested increased systemic inflammation S1), which in the is suggestive obese patients ofwith at baseline, thehigh larger VLDL hsCRP levels particles in the compared with obese grou the control apoE group.content was decreased Surgery progressively in these decreased the levels, obesealbeit patients withoutat baseline. reaching The only the same concentration as that in the control group. surgery was an increase in the apoE content in VLDL, reaching the subjects. 3.2. Changes in VLDL Composition The analysis of VLDL composition showed that the VLDL from obese patients con- tained more triglycerides and less apoB than the VLDL from the control group (Figure S1), 3.3. Changes which in LDL is suggestive of the Composition and in larger VLDL particles Function the obese group. In addition, the apoE content was decreased in the obese patients at baseline. The only effect of bariatric surgery The LDL composition is shown in Figure S2. No difference was an increase in the apoE content in VLDL, reaching the levels of control subjects. b patients at baseline and the control group was observed, and m 3.3. Changes in LDL Composition and Function components of LDL remained unchanged. The only significant mo The LDL composition is shown in Figure S2. No difference between the obese patients increase in the at baseline and free cholesterol, control whichandwas group was observed, almost most of the main significant components of LDLat 6 mo statistical difference remained unchanged. at 12 The only months. significant Accordingly, modification when was an increase thecholesterol, in free rate of choles which was almost significant at 6 months and reached statistical difference at 12 months. was assessed by measuring the ratio of esterified cholesterol/free chol Accordingly, when the rate of cholesterol esterification was assessed by measuring the LDL, ratio ofaesterified decrease in such ratio cholesterol/free was(EC/FC) cholesterol observed in LDL,ata 6decrease months after in such ratiosurgery was (F observed at 6 months after surgery (Figure 1). Figure 1. Ratio of esterified cholesterol (EC) versus free cholesterol (FC) in LDL. Data were calculated Figure from the1. Ratio % of ofeach mass of esterified component,cholesterol as described in (EC) versus the Methods, andfree cholesterol are expressed as mean(FC) ± SD.in LDL calculated Bars indicate from P. Blue the %indicate values of mass of each statistically component, significant as described in the Methods differences. as mean ± SD. Bars indicate P. Blue values indicate statistically significant diffe Despite this, the only change detected after surgery in the main components of LDL was in the EC/FC ratio. The LDL from obese patients presented important alterations in their atherogenic properties compared with those from the control group (Figure 2). Thus, the LDL from the obese group was more susceptible to oxidation than the LDL from the controls. In addition, the proportion of the inflammatory plasma subfraction electronegative LDL (LDL(-)) was increased, and the susceptibility to in vitro induced aggregation was higher in the obese patients than in the controls. Interestingly, bariatric surgery reverted all these abnormalities in LDL to values similar to those shown by the control group. controls. In addition, the proportion of the inflammatory pla electronegative LDL (LDL(-)) was increased, and the susceptibility to aggregation was higher in the obese patients than in the controls. Inter J. Clin. Med. 2021, 10, 1716 surgery reverted all these abnormalities in LDL to values similar 6 of 15 to th control group. Figure Figure 2. Qualitative characteristics 2. Qualitative of LDL. (a) Proportion characteristics of LDL. of(a) electronegative ProportionLDL of (LDL(-)) deter- electronegative L mined by anion-exchange chromatography. (b) Susceptibility to aggregation. The proportion of determined by anion-exchange chromatography. (b) Susceptibility to aggregati aggregated LDL was determined by gel filtration chromatography after in vitro lipolysis with SMase. of Susceptibility (c) aggregated to LDL was oxidation determined induced by gel filtration by CuSO4 incubation, expressed chromatography as lag phase time. Data after are in vi SMase. as expressed (c)mean Susceptibility to oxidation ± SD. Bars indicate induced P. Blue values by CuSO indicate statistically 4 incubation, significant expressed differences. Data are expressed as mean ± SD. Bars indicate P. Blue values indicate statistica On the other hand, the size of LDL was smaller in the obese patients than in the differences. controls (Figure 3a). Bariatric surgery increased the LDL size to normal levels at 6 months, and this remained stable for 12 months. The small size of LDL in the obese subjects suggests a higher On prevalence the otherof subjects hand, presenting the size the atherogenic of LDL was LDL subfraction smaller phenotype in the obeseB pa than would be expected from the relatively near-normal values of plasma triglycerides (all controls patients (Figure were below 2.43a). Bariatric mmol/L). surgery Phenotype increased B indicates the LDLofsize the predominance smallto LDLnormal l and this particles remained (diameter below 25.5stable nm), andfor 12 months. is considered The small as the atherogenic size of phenotype LDL in t of LDL. Specifically, seven obese patients presented phenotype B. Six months after surgery, only suggests a higher prevalence of subjects presenting the atherogenic three patients displayed phenotype B, and after one year of surgery, only one patient had phenotype phenotype B than B (Figure would 3b). Figure be expected S3 shows from a representative GGEthethat relatively near-normal includes samples from one obese patient at the three time points and one control triglycerides (all patients were below 2.4 mmol/L). Phenotype subject. predominance of small LDL particles (diameter below 25.5 nm), and is atherogenic phenotype of LDL. Specifically, seven obese patients presen Six months after surgery, only three patients displayed phenotype B, a of surgery, only one patient had phenotype B (Figure 3b). Fig representative GGE that includes samples from one obese patient at the and one control subject. J. J.Clin. Clin.Med. Med.2021, 10,x1716 2021,10, FOR PEER REVIEW 7 of 7 15of 15 J. Clin. Med. 2021, 10, x FOR PEER REVIEW 7 of 15 Figure 3. LDL Figure LDLsize. size.(a)(a) LDLLDLsizesize determined determined byby non-denaturing non-denaturing gradient gradient gelgel electrophoresis. electrophoresis. (b) Individual changes in LDL size after bariatric surgery. The dashed red line indicates 25.5 nm, the (b) Individual changes in LDL size after bariatric surgery. The dashed red line indicates 25.5 nm, Figure 3. LDL size. (a) LDL size determined by non-denaturing gradient gel electrophoresis. (b) threshold the threshold Individual between changes in LDL between LDLLDL subfraction sizesubfraction phenotypes phenotypes after bariatric AAand surgery. The andB,B,red dashed and and numbers indicatesin numbers line in parentheses parentheses 25.5 nm, the indicate the indicate number the number threshold of patients LDLwith of patients between withthethe atherogenic subfraction atherogenic phenotypes phenotype and B, andB.numbers Aphenotype Data B. Dataare expressed expressedas areparentheses in asmean mean indicate ±±SD. the SD.Bars Bars indicate indicate P.patients number ofP. Bluevalues Blue values with theindicate statistically atherogenic indicate phenotype statistically significant B. Data are significant differences. expressed as mean ± SD. Bars differences. indicate P. Blue values indicate statistically significant differences. 3.4. Changes 3.4. Changes in inHDL HDLComposition Compositionand andFunction Function 3.4. Changes in HDL Composition and Function The HDL TheHDL HDL from from the theobese obese patients patients had had less free andandesterified cholesterol and and moremore The apoC-III from compared the withobese that patients from the freeless hadcontrols less andfree esterified (Figure esterified cholesterol S4). Bariatric cholesterol and more surgery normalized apoC-III compared apoC-III compared withwith that that fromfrom the controls (Figure the controls S4).surgery Bariatric surgery normalized cholesterol and apoC-III content in HDL. (Figure Surgery S4). Bariatric also induced normalized a decrease in apoA-II cholesterol cholesterol andand apoC-III apoC-III content content in HDL.in HDL. SurgerySurgery also ainduced also induced decrease aindecrease apoA-II in apoA-II content at 6 months, which redounded in a decrease in total protein. Regarding the content atat6 6months, content months, which whichredounded redoundedin a decrease in total protein. in a decrease totalRegarding the qualitative qualitative characteristics characteristics of of HDL, HDL, obese obese patients patients had a had low lowin apredominance protein. predominance of large of HDL Regarding large HDL the qualitative particles characteristics (HDL2) compared ofwith HDL, obese patients had a low 4a).predominance of large theHDL particles (HDL2) compared with the the control control group group (Figure (Figure 4a). SurgerySurgery normalized normalized the particles relative (HDL2) proportion compared with the control group (Figure 4a). Surgery normalized GGE the relative proportion ofof HDL2/HDL3 HDL2/HDL3 at 6 months. at 6 months. FigureFigure S3 showsS3 shows a representative a representative GGE relative that proportion includesone that includes oneobese of obese HDL2/HDL3 patient patient at the at the at threethree6 months. time time points Figure points and oneand S3 shows one control control a representative subject.subject. GGE that includes one obese patient at the three time points and one control subject. Figure 4.4.Qualitative Figure characteristics Qualitative of HDL. characteristics (a) Proportion of HDL. of HDL2of (a) Proportion (large HDL2HDL) determined (large by HDL) determined by non-denaturing gradient gel electrophoresis. (b) Susceptibility to oxidation induced by non-denaturing gradient gel electrophoresis. (b) Susceptibility to oxidation induced by CuSO4 CuSO 4 incubation, expressed as lag phase time. (c) Antioxidant capacity of HDL to prevent the oxidation incubation, expressed as lag phase time. (c) Antioxidant capacity of HDL to prevent the oxidation of LDL, expressed as an increment of the lag phase time. Data are expressed as mean ± SD. Bars of LDL, expressed indicate P. Blue valuesasindicate an increment of the statistically lag phase significant time. Data are expressed as mean ± SD. Bars differences. indicate P. Blue values indicate statistically significant Figure 4. Qualitative characteristics of HDL. (a) Proportion differences. of HDL2 (large HDL) determined by non-denaturing gradient gel electrophoresis. (b) Susceptibility to oxidation induced by CuSO4 incubation, expressed as lag phase time. (c) Antioxidant capacity of HDL to prevent the oxidation of LDL, expressed as an increment of the lag phase time. Data are expressed as mean ± SD. Bars indicate P. Blue values indicate statistically significant differences. J. Clin. Med. 2021, 10, x FOR PEER REVIEW 8 of J. Clin. Med. 2021, 10, 1716 8 of 15 Regarding oxidative properties, the HDL from the obese patients was mo susceptible to oxidation than the control HDL was (Figure 4b), and its capacity to inhib the oxidation of LDL (prolongation of the lag phase) was also decreased (Figure 4c Regarding oxidative properties, the HDL from the obese patients was more susceptible Bariatric surgery improved the resistance to oxidation of HDL at 12 months and i to oxidation than the control HDL was (Figure 4b), and its capacity to inhibit the oxidation capacity to prevent the oxidation of LDL at 6 months. Figure S5 shows representativ of LDL (prolongation of the lag phase) was also decreased (Figure 4c). Bariatric surgery kinetics of improved the the antioxidant resistance capacity to oxidation of the of HDL at HDL fromand 12 months oneitsobese patient capacity at the to prevent thethree tim points and oxidation of one LDLcontrol subject. at 6 months. Figure S5 shows representative kinetics of the antioxidant capacity of the HDL from one obese patient at the three time points and one control subject. 3.5. Distribution of Lp-PLA2 Activity in Plasma 3.5. Distribution of Lp-PLA2 Activity in Plasma The total Lp-PLA2 activity was increased in the obese group at baseline compare The total Lp-PLA2 activity was increased in the obese group at baseline compared with the control group (Figure 5a), and it decreased 6 months after bariatric surgery. The with the control group (Figure 5a), and it decreased 6 months after bariatric surgery. These differences weremainly differences were mainly attributed attributed to increased to increased activity activity in apoB-containing in apoB-containing lipoprotein lipoproteins, but notininHDL but not HDL(Figure (Figure 5b,c). 5b,c). Figure Figure 5.5. Total Total Lp-PLA Lp-PLA22 activity activity and and distribution in lipoproteins. distribution in lipoproteins. (a) Total plasma activity. Total plasma activity. (b) Lp PLA2 activity (b) Lp-PLA 2 associated with HDL. (c) Lp-PLA2 activity activity associated with HDL. (c) Lp-PLA 2 associated with apoB-containing activity associated with apoB-containing lipoproteins. Dataare lipoproteins. Data areexpressed expressed as as mean ± SD. mean ± SD. Bars Bars indicate indicate P. Blue P. Blue values values indicate indicate statistically statistically significant differences. significant differences. 3.6. Distribution of Apolipoprotein J (apoJ) in Plasma 3.6. Distribution of Apolipoprotein J (apoJ) in Plasma The total plasma concentration of apoJ was increased in the obese patients compared Thecontrols, with the total plasma and noconcentration of apoJ effect of bariatric surgerywas increased was observedin the obese (Figure 6a). Itpatients has beencompare with the that reported controls, and most of noineffect apoJ plasma of bariatric is present surgery wasnot in free form, observed bound to (Figure 6a). It has bee lipoproteins, reported that most of apoJ in plasma is present in free form, not bound to The and only a proportion ranging from 5–30% is transported associated to lipoproteins. lipoprotein proportion and only aofproportion apoJ not bound to lipoproteins ranging from 5–30%(free form) was higherassociated is transported in the obeseto group than lipoproteins. Th in the control group (Figure 6b). Since the apoJ carried by lipoproteins is bound proportion of apoJ not bound to lipoproteins (free form) was higher in the obese grou mainly to HDL, this difference was mainly due to the decreased content of apoJ associated with HDL, than in the control group (Figure 6b). Since the apoJ carried by lipoproteins is boun whereas no difference was observed in the apoJ content in LDL and VLDL (Figure 6c–e). mainly Bariatricto HDL,increased surgery this difference the apoJwas mainly content due to theatdecreased in lipoproteins 6 months, content of apoJofassociate mainly because with HDL, whereas no difference an increase in HDL (Figure 6b,e). was observed in the apoJ content in LDL and VLD (Figure 6c–e). Bariatric surgery increased the apoJ content in lipoproteins at 6 month mainly because of an increase in HDL (Figure 6b,e). FOR PEER REVIEW J. Clin. Med. 2021, 10, 1716 9 of 15 9 of 15 Figure 6. Plasma apoJ concentration and distribution in lipoproteins. (a) Total plasma apoJ. (b) Proportion of apoJ not Figure 6. Plasma apoJ concentration and distribution in lipoproteins. (a) Total plasma apoJ. (b) bound to lipoproteins. (c) Proportion of apoJ bound to VLDL. (d) Proportion of apoJ bound to LDL. (e) Proportion of apoJ Proportion of apoJ not bound to lipoproteins. (c) Proportion of apoJ bound to VLDL. (d) bound to HDL. Data are expressed as mean ± SD. Bars indicate P. Blue values indicate statistically significant differences. Proportion of apoJ bound to LDL. (e) Proportion of apoJ bound to HDL. Data are expressed as mean ± SD. Bars indicate 3.7. P. Blue values Correlation indicate statistically significant differences. Analysis To confirm the robustness of the observed changes and define the parameters they 3.7. Correlation Analysis depend on, a correlation analysis was performed using the total number of samples To confirm the analyzed. robustness TableofS1the shows the statistical observed correlations changes andbetween definelipoprotein profile, lipoprotein the parameters they function, Lp-PLA2 activity, and apoJ-related parameters. The main findings are as follows: depend on, a correlation analysis was plasma triglycerides and performed VLDLc correlated using the total negatively withnumber of samples LDL size and susceptibility analyzed. Table S1to oxidation, shows the statistical correlations between lipoprotein whereas HDLc was positively associated with LDL size and negatively profile,with lipoprotein function,LDL(-) Lp-PLA 2 activity, proportion andsusceptibility and LDL apoJ-related parameters.Interestingly, to aggregation. The mainthe findings parameters of LDL function did not show associations with total cholesterol or LDLc; instead, they are as follows: plasma triglycerides and VLDLc correlated negatively with LDL size and were mainly associated with TG and VLDLc. CRP, a marker of systemic inflammation, susceptibility to oxidation, is strongly whereas associatedHDLc wasparameters with most positively associated of LDL and HDLwith LDLThis function. size and association negatively with LDL(-) proportion is particularly strongand withLDL susceptibility LDL(-), an inflammatory to aggregation. subfraction of LDL Interestingly, present in blood. Lp-PLA the parameters of LDL function 2 activity did not show associations with total cholesterolofor correlated negatively with the susceptibility to oxidation HDL, LDLc;and this activity associated to apoB also correlated negatively with the susceptibility to oxidation of instead, they were mainly associated with TG and VLDLc. CRP, a marker of systemic HDL and with its antioxidant capacity. Finally, apoJ plasma concentration was negatively inflammation, is strongly associated associated with most parameters with the susceptibility to oxidation ofof LDL LDL, and HDL pointing function. to a protective This role of apoJ. association is particularly strong with Associations LDL(-), between an inflammatory the lipoprotein subfraction function and of LDL its composition werepresent also tested. Table S2 shows in blood. Lp-PLA2 activity the correlations correlated in LDL.with negatively The only the function of LDL clearly susceptibility associated with to oxidation of its composition was the LDL susceptibility to oxidation, which was dependent on the degree HDL, and this activity associated to apoB also correlated negatively with the susceptibility of cholesterol esterification (the higher the free cholesterol content, the higher the resistance to oxidation of HDLtoand with its oxidation). antioxidant Weak associationscapacity. Finally,between were also observed apoJ plasma concentration the proportion of LDL(-) and was negatively associated with the the phospholipid susceptibility content, and between LDL to oxidation size and freeof LDL, pointing cholesterol content. to a protective role of apoJ. Regarding HDL (Table S3), the susceptibility to oxidation correlated positively with the content of triglycerides and apoJ, and negatively with apoA-I. The antioxidant capacity Associations between the lipoprotein function and its composition were also tested. of HDL correlated positively with cholesterol, triglycerides, and apoJ, and negatively Table S2 shows the correlations with total protein in LDL. content.The Theonly function proportion of HDL2of LDL clearly correlated associated positively with with cholesterol, its composition wastriglycerides, the LDL susceptibility apoA-I, and apoJ,to andoxidation, which negatively with totalwas proteindependent content on the degree of cholesterol esterification (the higher the free cholesterol content, the higher the resistance to oxidation). Weak associations were also observed between the proportion of LDL(-) and the phospholipid content, and between LDL size and free cholesterol content. Regarding HDL (Table S3), the susceptibility to oxidation correlated positively with J. Clin. Med. 2021, 10, 1716 10 of 15 Lipoprotein function parameters are strongly intertwined with one another (Table S4), with a higher proportion of LDL(-) being statistically associated with higher LDL suscepti- bility to aggregation, smaller LDL and HDL size, increased susceptibility to the oxidation of LDL and HDL, and decreased HDL antioxidant capacity. The parameters of HDL function positively correlated with the content of apoJ in HDL, supporting the role of apoJ in the maintenance of the antiatherogenic properties of HDL. 4. Discussion This study reveals numerous functional alterations in lipoproteins isolated from morbidly obese subjects. The main findings are: (1) VLDL from obese patients at base- line presented only moderate alterations in composition, suggestive of a larger size, and bariatric surgery increased apoE content; (2) despite the similar LDL composition in both groups, the LDL from obese patients showed numerous abnormalities in their function, which improved after bariatric surgery; (3) the protective properties of HDL from obese patients were compromised when compared with the HDL from control subjects, but these characteristics improved after surgery; (4) the total and apoB-associated Lp-PLA2 activities were high in the obese patients and decreased 6 months after surgery; (5) plasma apoJ levels were increased in the obese patients compared with the control group, but the amount of apoJ transported by HDL was lower in the obese patients. Bariatric surgery normalized the cargo of apoJ in HDL. VLDL particles from obese subjects contained more triglycerides and less apoB, indi- cating their larger size. The finding that bariatric surgery promoted a progressive increase in the content of apo E in VLDL, reaching the values of the control group, suggests that VLDL clearance from blood could be faster after surgery, in accordance with the role of apoE as a ligand for lipoprotein receptors [33,34]. This accelerated VLDL catabolism could add to the decreased availability of NEFA in the liver because of loss of visceral adipose tissue, as major mechanisms behind the decrease in VLDLc levels. To our knowledge, this is the first report of increased apoE content in the VLDL of patients with obesity after bariatric surgery. LDL presented several potential atherogenic traits when isolated from obese patients, including a decreased size, increased susceptibility to oxidation and aggregation, and a high proportion of LDL(-). Bariatric surgery succeeded in reverting these alterations. Interestingly, the improvement in the qualitative characteristics of LDL occurred without changes in the plasma levels of LDLc and in the major components of LDL. Only a minor change in the free cholesterol content and the EC/FC ratio was observed after bariatric surgery. This change could underlie the improvement of LDL susceptibility to oxidation, as suggested by the correlation analysis, which showed the association of LDL susceptibility to oxidation with the content of free cholesterol, which was negative with the degree of cholesterol esterification, in accordance with previous reports [35]. Another factor strongly related to the improvement in LDL susceptibility to oxidation is the enlargement of LDL particles after bariatric surgery, as small LDL particles are highly oxidizable [35,36]. LDL(-) is a pool of all the modified forms of LDL present in blood with inflammatory and apoptotic properties [37,38]. The LDL(-) proportion was increased in adult obese patients, which agrees with previous findings of increased LDL(-) in obese adolescents [39]. In addition, we demonstrate for the first time that bariatric surgery decreased LDL(-) pro- portion. LDL(-) correlated with susceptibility to aggregation, which is explained by LDL(-) being highly susceptible to aggregation [40]. LDL aggregation in the subendothelial space is an early event in atherogenesis [41], and the susceptibility of LDL to aggregation has been related to an accelerated development of atherosclerosis [42]. LDL(-) was associated with all functional parameters of HDL, its being proportion higher when the size, susceptibility to oxidation, or antioxidant capacity of HDL was low. This observation emphasizes the role of HDL in the prevention of LDL modification [43]. Our results indicate that in obese patients at baseline, the HDL particles are smaller and more susceptible to oxidation, and they have partially lost their capacity to prevent J. Clin. Med. 2021, 10, 1716 11 of 15 LDL oxidation. Bariatric surgery, not only increased plasma HDLc levels, but also reverted these functional abnormalities. An abnormal size distribution of HDL in obesity has been reported previously, and interventions directed to weight loss, including diet and surgery, increase the size of HDL, shifting its distribution to larger particles [10–16,19,21]. Our results suggest that cholesterol esterification in HDL after surgery is the key element favoring the formation of large HDL particles and the improvement in their antioxidative properties. Indeed, correlation analysis showed a positive association of the antioxidant capacity of HDL with the content of esterified cholesterol and the esterified/free cholesterol ratio. We also found negative associations between the susceptibility to oxidation and the apoA-I content (the longer the lag phase, the lower the apoA-I content), and between the antioxidant capacity and the protein content in HDL (the higher the increment of the lag phase, the lower the protein content). In both cases, the explanation is related to the size of HDL, since HDL2, which contains less protein and apoA-I, is positively associated with the antioxidant capacity of HDL. Another relevant finding is the alteration in the content of some apolipoproteins in HDL. The HDL from the obese patients contained more apoC-III and less apoJ than the HDL from the controls, and bariatric surgery normalized the cargo of both apolipoproteins in HDL. This finding is relevant because similar alterations in apoC-III and apoJ content were observed in HDL from coronary patients by Riwanto et al. [44]. Regarding apoJ, our results demonstrate that compared with the control group, the obese subjects presented increased plasma concentration of apoJ, but a decreased pro- portion bound to HDL. Bariatric surgery reverted both alterations. These findings agree with previous reports on the associations between apoJ, insulin resistance [45], and adipos- ity [46], and indicate an altered distribution of apoJ among lipoproteins in obesity [47,48]. ApoJ prevents the aggregation of LDL [49], inhibits the apoptosis induced by modified LDL [50], and stabilizes several proteins in HDL with antioxidant properties, including apoA-I and paraoxonase 1 [51]. This concurs with our observation that the content of apoJ in HDL positively correlates with the protective properties of HDL, including size, susceptibility to oxidation, and antioxidant capacity. Therefore, apoJ appears to be a key factor contributing to the preservation of the antiatherogenic properties of HDL. From these findings, a scheme can be drawn (Figure 7) in which the decrease in central adiposity after bariatric surgery shifts the altered qualitative characteristics of LDL and HDL to a non-atherogenic phenotype. The driving force could be a decrease in the available free fatty acids in the liver because of the loss of adipose tissue. This decreases hepatic VLDL secretion, which adds to the increased apoE content in VLDL, favoring its receptor- mediated clearance. Consequently, VLDL particles are smaller, and their metabolism in blood generates large LDL particles, which are less susceptible to oxidation. In parallel, the size and composition of HDL particles becomes closer to the composition of healthy subjects. As a result, the capacity of HDL to prevent modification improves, and less modified LDL particles are found in circulation. As a whole, the functions of LDL and HDL in obese patients are normalized after bariatric surgery. Of note, the improvement of the lipoprotein function after surgery occurs in some cases without a complete normalization of the lipoprotein composition and size, suggesting a threshold beyond which lipoprotein function becomes normal. The main limitation of our study is the small sample size in each group. Nevertheless, as a whole, the alterations observed in the composition and function of each lipoprotein fraction are coherent and metabolically connected, and they support the consistency of the achieved results. Another limitation is that a group of obese subjects displaying dyslipidemia were not compared with the obese subjects included in the present study, which would have made it possible to estimate the specific effect of hyperlipemia on alterations in lipoprotein function. Finally, no measures of atherosclerosis, either clinical or subclinical were assessed, therefore, the impact of the findings affecting the function of lipoproteins on developing arteriosclerosis requires further studies, analyzing this particular aspect. Clin. Med.J.2021, 10, x2021, Clin. Med. FOR 10,PEER 1716 REVIEW 12 of 15 12 o Effect7.ofEffect Figure 7. Figure of bariatric surgery on the qualitative properties of lipoproteins. The loss of visceral adipose tissue after bariatric surgery on the qualitative properties of lipoproteins. The loss of visceral adipose tissue after surgery decreases the availability of NEFA in the liver and promotes lower VLDL secretion. This VLDL contains more surgery decreases the availability of NEFA in the liver and promotes lower VLDL secretion. This VLDL contains more apoE, which favors VLDL clearance in plasma, and less triglycerides, indicative of smaller VLDL particles. Therefore, apoE, which favors VLDL clearance in plasma, and less triglycerides, indicative of smaller VLDL particles. Therefore, the the catabolism of smaller VLDL particles produces larger LDL and HDL particles with altered contents of some major catabolism of smaller VLDL particles produces larger LDL and HDL particles with altered contents of some major components. Both large LDL and HDL are more resistant to oxidative modifications, and HDL has an increased capacity components. Both further to prevent large LDL and HDL modifications are more in LDL, both inresistant to in plasma and oxidative modifications, the arterial and the wall. Consequently, HDL has anofincreased deposition LDL in capacity to preventarteries furthershould decline after bariatric surgery. Blue text: events occurring after bariatric surgery. Black text: changes in the of LDL in modifications in LDL, both in plasma and in the arterial wall. Consequently, the deposition arteries should decline after bariatric properties of lipoproteins. Brown surgery. Blue text: changes text: eventsAOX: in composition. occurring after bariatric surgery. Black text: changes in the antioxidant. properties of lipoproteins. Brown text: changes in composition. AOX: antioxidant. 5. Conclusions TheInmain limitation summary, of our our study showsstudy is thequalitative multiple small sample size in in modifications each group. Neverthel the lipoproteins as a from whole,obese thesubjects, which observed alterations are reverted,ininthe part,composition by the weight loss andinduced functionafterofbariatric each lipoprot surgery. A decreased hepatic secretion of VLDL after surgery elicits a cascade of metabolic fraction are coherent and metabolically connected, and they support the consistency changes in LDL and HDL that improve their functional properties. Our study also reveals the achieved results. the intertwined Another relationship amonglimitation the main is that a group components of obese of the three subjects display main lipoprotein dyslipidemia fractions inwere obese not compared patients. Further with studiesthe areobese subjects warranted (1) to included confirm ourinfindings the present in stu which would have made it possible to estimate the specific effect of hyperlipemia larger cohorts of patients with severe obesity, and (2) to relate the alterations of lipoprotein functionin alterations with the development lipoprotein of atherosclerosis function. Finally, noormeasures the emergence of clinical events. either clin of atherosclerosis, or subclinical were assessed, therefore, the impact of the findings affecting the function Supplementary Materials: The following are available online at https://www.mdpi.com/article/ lipoproteins on developing 10.3390/jcm10081716/s1. arteriosclerosis Figure S1: VLDL composition. requires Figure S2: further studies, LDL composition. analyzing Figure S3: particular aspect. Representative gradient gel electrophoresis (GGE). Figure S4: HDL composition. Figure S5: Sus- ceptibility to oxidation of LDL and HDL, and antioxidant capacity of HDL. Table S1: Correlation analysis between lipid profile and total plasma parameters, and the lipoprotein function variables. 5. Conclusions Table S2: Correlation analysis between LDL composition, and LDL function. Table S3: Correlation analysis between HDL In summary, ourcomposition, study shows and HDL function. multiple Table S4: Correlation qualitative analysis between modifications in thethe lipoprote parameters of lipoprotein function. from obese subjects, which are reverted, in part, by the weight loss induced after baria Author surgery. AContributions: Conceptualization, decreased hepatic secretionI.M., A.P. and of VLDL J.L.S.-Q.; after methodology, surgery elicits aI.G., N.P. and cascade of metab A.R.-U., Á.G.-O. and M.G.-A.; investigation, I.G., I.M., P.G., C.B. and S.F.-A.; resources, S.B., Á.G.-O., changes in LDLA.P. C.B., S.F.-A., andandHDL thatwriting—original J.L.S.-Q.; improve their functional draft preparation,properties. Our study A.P. and J.L.S.-Q.; writing— also reve the intertwined relationship among the main components of the three main review and editing, all authors; supervision, I.M., A.P. and J.L.S.-Q.; project administration, A.P. lipopro fractions in obese patients. Further studies are warranted (1) to confirm our finding larger cohorts of patients with severe obesity, and (2) to relate the alterations of lipoprot function with the development of atherosclerosis or the emergence of clinical events. Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1. Figure J. Clin. Med. 2021, 10, 1716 13 of 15 and J.L.S.-Q.; funding acquisition, S.B., A.P. and J.L.S.-Q. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by grants FIS PI13/00364 and PI16/00471 from the Instituto de Salud Carlos III, Spanish Ministry of Health (co-financed by the European Regional Development Fund). CIBERDEM (CB07/08/0016) is an Instituto de Salud Carlos III Project. N.P., S.B., A.R.-U., and J.L.S.-Q. are members of the Quality Research Group 2017-SGR-1149 from Generalitat de Catalunya. N.P., S.B., A.R.-U. and J.L.S.-Q. are members of the Group of Vascular Biology of the Spanish Society of Atherosclerosis. Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of the Hospital de la Santa Creu I Sant Pau (protocol code IIBSP-APO-2013-105, date of approval 01-21-2014). Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: The data presented in this study are available on reasonable request from the corresponding author. Conflicts of Interest: The authors declare no conflict of interest. References 1. Chooi, Y.C.; Ding, C.; Magkos, F. The epidemiology of obesity. Metabolism 2019, 92, 6–10. [CrossRef] 2. Adams, K.F.; Schatzkin, A.; Harris, T.B.; Kipnis, V.; Mouw, T.; Ballard-Barbash, R.; Hollenbeck, A.; Leitzmann, M.F. 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