Antiviral Therapy 2012; 17: (doi: /IMP2065)

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1 Antiviral Therapy 2012; 17: (doi: /IMP2065) Original article Determinants of low-density lipoprotein particle diameter during antiretroviral therapy including protease inhibitors in HIV-1-infected patients Randa Bittar 1,2 *, Philippe Giral 2,3, Elisabeth Aslangul 4,5, Lambert Assoumou 6,7, Marc Antoine Valantin 6,7,8, Olga Kalmykova 6,7, Valérie Fesel-Fouquier 1, Dominique Costagliola 6,7,8, Dominique Bonnefont Rousselot 1,9, the ANRS 126 study group 1 Unité Fonctionnelle de Biochimie des Maladies Métaboliques, Service de Biochimie Métabolique, Groupe Hospitalier Pitié-Salpêtrière, Assistance Publique-Hôpitaux de Paris, Paris, France 2 UPMC Univ Paris 06, UMR S 939, Paris, France 3 Unité de Prévention Cardiovasculaire, Service d Endocrinologie Métabolisme, Groupe Hospitalier Pitié-Salpêtrière, Assistance Publique- Hôpitaux de Paris, Paris, France 4 Service de Médecine Interne, Hôtel-Dieu, Assistance Publique-Hôpitaux de Paris, Paris, France 5 Université Paris Descartes, rue de l école de Médecine, Paris, France 6 INSERM, U 943, Paris, France 7 UPMC Univ Paris 06, UMR S 943, France 8 Service des Maladies Infectieuses et Tropicales, Groupe Hospitalier Pitié-Salpêtrière, Assistance Publique-Hôpitaux de Paris, Paris, France 9 EA 4466, Département de Biologie Expérimentale, Métabolique et clinique, Faculté des Sciences Pharmaceutiques et Biologiques, Université Paris Descartes, Sorbonne Paris Cité, Paris, France *Corresponding author randa.bittar@psl.aphp.fr Background: Lipid disorders are frequent in HIV-1- infected patients taking combination antiretroviral therapy (cart) that includes protease inhibitors (PIs). The presence of small dense low-density lipoprotein particles might be an important predictive marker of cardiovascular disease in this setting. This cross-sectional substudy of the ANRS 126 trial was designed to identify variables influencing LDL diameter. Methods: We studied 81 stable HIV-1-infected patients with dyslipidaemia (LDL-cholesterol >4.1 mmol/l, triglycerides <8.8 mmol/l) receiving PI-including cart regimens and no lipid-lowering drugs. LDL diameter was assessed by gradient gel electrophoresis. Relationships between LDL diameter and demographic, metabolic and HIV-related variables were identified by using non-parametric univariate tests and multiple linear regression models. Results: In univariate analysis, LDL diameter was related to demographic variables, triglyceride (TG) levels, highdensity lipoprotein cholesterol (HDL-c) levels, and the numbers and duration of exposure to nucleoside reverse transcriptase inhibitors and PIs. In multivariable linear regression analysis, LDL diameter was negatively associated with the TG level (P<0.0001) and positively associated with the HDL-c level (P<0.0001). For each 1-mmol/l increase in TG, LDL diameter fell by nm. Conversely, for each 1-mmol/l increase in HDL-c, LDL diameter rose by nm. Conclusions: Higher TG and lower HDL-c levels are associated with smaller LDL particle diameter. Small-diameter LDL particles could contribute to early atherogenic processes in HIV-1-infected patients on cart. Introduction Cytokine-mediated lipid disorders are a complication of HIV-1 infection [1 3], and can be aggravated by combination antiretroviral therapy (cart) which includes protease inhibitors (PIs) [4,5]. A high prevalence of atherogenic lipoprotein phenotype has been reported in patients on cart [6,7]. The atherogenic lipoprotein phenotype is characterized by elevated triglyceride (TG) levels (>2.28 mmol/l), small 2012 International Medical Press (print) (online) 855

2 R Bittar et al. low-density lipoprotein (LDL) particles, and low highdensity lipoprotein (HDL)-cholesterol levels (HDL-c; <1.040 mmol/l) [8]. Hypertriglyceridaemia has an atherogenic effect by promoting the formation of smaller LDL particles which are TG-enriched, called small dense LDL (sdldl) [7]. The atherogenicity of sdldl is due to their propensity to infiltrate the arterial wall and to be oxidized, and to their lower affinity for LDL receptors [7,9]. High sdldl levels have been shown to increase the risk of cardiovascular disease (CVD) [7,10], and to hasten the progression of coronary artery disease [6,10]. These particles have also been implicated in the elevated risk of CVD observed in cart-treated patients [11]. Until now, the number of HIV-1-infected PI-treated patients enrolled in studies of the LDL particle diameter was too small for multivariable analysis of variables potentially influencing it. Here, we analysed variables related to LDL particle diameter in an adequate number of stable HIV 1- infected patients receiving cart regimens comprising ritonavir-boosted PIs. Methods Study population This cross-sectional substudy of the ANRS 126 trial included 81 HIV-1-infected patients [12]. The aim was to study LDL size distribution and its determinants in dyslipidaemic PI-treated HIV-1 infected patients (LDLcholesterol [LDL-c]>4.1 mmol/l). The institutional review board of Pitié-Salpêtrière Hospital approved the study protocol, and all the patients gave their written informed consent. The ClinicalTrials.gov identifier is NCT [12]. Patients were eligible for this study if they had been receiving a stable cart regimen including a boosted PI for at least 2 months, with no planned treatment modification over the following two months, and plasma viral load 10,000 RNA copies/ml at baseline. In addition, the following fasting biological values were required: LDL-c>4.1 mmol/l, triglycerides <8.8 mmol/l, transaminases 3 the upper limit of normal (ULN; 5 in case of HCV or HBV infection, without cirrhosis), and creatine phosphokinase 5 ULN. Patients were not eligible if they had documented coronary heart disease, muscle disorders, a previous muscular adverse reaction to a statin or fibrate; impaired hepatic function (prothrombin <70%) or renal function (creatinine clearance <30 ml/min); proteinuria exceeding ++, alcohol consumption over 40 g per day, an ongoing opportunistic infection; pregnancy; or statin or fibrate exposure less than 2 months before the first dose of study medication. The clinical section of the CDC classification of HIV-1 disease was used to define AIDS [13]. Lipid parameters (fasting serum levels of total cholesterol, HDL-c, LDL-c and triglycerides, and LDL size) were determined in a central laboratory [12]. Laboratory methods Lipid analysis Fresh venous blood was collected in gel-containing Vacutainer tubes (Becton-Dickinson, Plymouth, UK) after a 12-h fast. Serum lipids were analysed in a central reference laboratory (Unité Fonctionelle de Biochimie des Maladies Métaboliques, GH Pitié-Salpêtrière, Paris, France), using routine enzymatic methods on a Konelab 30i (Thermo Electron Corporation, Cergy-Pontoise, France). Triglycerides were measured with the PAP 1000 enzymatic method (BioMérieux, Marcy-l étoile, France) [14]. Total cholesterol, direct LDL-c, and direct HDL-c were determined as recommended by ARCOL (Comité Français de Coordination des Recherches sur l Athérosclérose et le Cholestérol) using automated enzymatic methods (Konelab Thermo Fisher Scientifics, Cergy-Pontoise, France) [15 17]. When the TG level was >2.28 mmol/l free glycerol was measured by using the Randox colorimetric method (Randox Laboratories Ltd, Crumlin, UK). LDL diameter measurement LDL subclasses were determined in serum by gradient gel electrophoresis previously described by Blanche et al. [18]. A detailed description has been published elsewhere [19 21]. In brief, lipoproteins were isolated from 3 ml of serum, after adjustment with potassium bromide, and after two ultracentrifugations at 90,000 rpm for 4 h at 10 C (NVT rotor Beckman XL 90; Beckman, Roissy Charles de Gaulle, France). LDL size was then determined by electrophoresis in a nondenaturing polyacrylamide gradient gel (Spiragel %; Lara-Spiragel, Couternon, France) using Tris/ boric acid/nan3 buffer (ph 8.3). Markers of known molecular weight (High Molecular Weight Calibration Kit for native electrophoresis; Amersham Biosciences, Courtaboeuf, France), were run simultaneously with lipoproteins isolated from the HIV-1-infected patients and from a normolipidaemic healthy donor whose LDL size had been measured in a reference laboratory, in order to determine reproducibility. Gels were fixed and stained with Coomassie Brillant Blue G250/perchloric acid, and finally scanned with an optical densitometer Hyris2 (Sebia, Evry, France). The usual distribution of LDL particles measured in 63 healthy subjects in our laboratory was as follows: 10 16% LDL-1 ( nm), 30 50% LDL-2 ( nm), 30 50% LDL-3 ( nm) and 6 12% LDL-4 ( nm) [20]. The diameter of the main LDL peak was calculated (25.49 ±0.49 nm) as described elsewhere [22 25]. A diameter of <25.5 nm for the International Medical Press

3 Determinants of LDL diameter in PI-treated HIV-1-infected patients main LDL peak classically identifies an atherogenic phenotype (phenotype B) [10,20,21]. Statistical analyses All continuous variables were described by their median IQR or by their mean ± standard deviation (sd). Associations between LDL diameter and categorical variables were tested with the non-parametric Mann Whitney U test. Associations between LDL diameter and continuous variables were assessed using Spearman s non-parametric correlation coefficient. Backward multivariable linear regression was used to identify factors independently associated with LDL diameter. All variables with P-values below 0.10 were included the multivariable model. Results Characteristics of the patients The baseline demographic and clinical characteristics of the 81 patients are listed in Table 1. Median age was 47 years and 78% of the patients were men. The median (IQR) known duration of ART exposure was 9 years and the patients had received a median of seven different drugs (four nucleoside reverse transcriptase inhibitors, one non-nucleoside reverse transcriptase inhibitors and two PIs). At baseline, 30% of the patients were receiving lopinavir/r (n=25), 28% atazanavir/r (n=23), 23% fos-ampinavir/r (n=19) and 19% (n=16) other PIs (saquinavir/r, indinavir/r or tipranavir/r). The median plasma HIV RNA level was <400 copies/ml and the median CD4 + T-cell count was 482/mm 3. The distribution of cardiovascular risk factors, including gender, smoking, arterial hypertension, diabetes and HDL-c, is shown in Table 1. Only 59% (n=45) of the 81 patients had no risk factors for CVD, while 26% (n=20) had one risk factor, 13% (n=10) had two risk factors and 1% (n=1) had three risk factors. Serum lipid parameters The median (IQR) lipid concentrations were as follows: total cholesterol 7.32 mmol/l ( ), triglycerides 2.30 mmol/l ( ), LDL-c 4.92 mmol/l ( ) and HDL-c 1.33 mmol/l ( ; Table 1). The total cholesterol/hdl-c ratio was 5.40 ( ). An atherogenic phenotype was found in 11% of patients. LDL diameter The mean LDL diameter particle was 25.5 ±0.9 nm. The percentage distribution of LDL diameter subclasses was as follows: LDL1 ( nm): 7.5 ±6.3%, LDL-2 ( nm): 44.2 ±20.1%, LDL3 ( nm) 36.0 ±15.8%, and LDL4 ( nm): 12.3 ±14.5%. Associations of LDL diameter with demographic and clinical variables are shown in Table 2 and Table 3. LDL diameter was associated with gender (female > male; P<0.001), the HIV transmission group (men who have sex with men > other groups; P=0.004), origin (sub-saharan African > other origin; P=0.019) and prior AIDS-defining events (P=0.046). No association was found with smoking. LDL diameter was associated with higher triglyceride levels (P<0.001) and lower HDL-c levels (P<0.001). LDL diameter also correlated negatively with the duration of exposure to nucleoside reverse transcriptase inhibitors (P=0.048) and PIs (P=0.026), and with the number of antiretroviral drugs received (P=0.001). In the backward multivariable linear regression model, LDL diameter was associated negatively with the TG level ( per mmol/l TG; P<0.0001) and positively with the HDL-c level ( per mmol/l Table 1. Baseline characteristics Characteristic Total (n=81) Male sex, n (%) 63 (78) Median age, years (IQR) 47 (42 54) Sub-Saharan African, n (%) 9 (11) Transmission groups Men who have sex with men, n (%) 42 (52) Heterosexual, n (%) 31 (38) Unknown/others, n (%) 8 (10) Median time since HIV diagnosis, years (IQR) 12 (6 16) Median CD4 + T-cell count, per mm 3 (IQR) 482 ( ) Plasma HIV RNA<400 copies/ml, n (%) 74 (91) Prior AIDS event, n (%) 18 (22) HBs-positive, n (%) 3 (4) Median number of previous ARVs (IQR) 7 (5 9) Median time on cart, years (IQR) 9 (5 13) Current smoker or stoppped less than 3 years, n (%) 29 (36) Number of cardiovascular risk factors a (n=76) 0, n (%) 45 (59) 1, n (%) 20 (26) 2, n (%) 10 (13) 3, n (%) 1 (1) Median body mass index, kg/m 2 (n= 77; IQR) 23 (22 25) Median total cholesterol, mmol/l (IQR) 7.32 ( ) Median LDL cholesterol, mmol/l (IQR) 4.92 ( ) Median HDL cholesterol, mmol/l (IQR) 1.33 ( ) Median triglycerides, mmol/l (IQR) 2.30 ( ) Total cholesterol/hdl-cholesterol ratio (IQR) 5.40 ( ) Median glycaemia, mmol/l (n=77; IQR) 5.00 ( ) Median LDL diameter, nm (IQR) 25.7 ( ) LDL subfractions Median LDL1, % (IQR) 6 (3 10) Median LDL2, % (IQR) 49 (23 61) Median LDL3, % (IQR) 36 (24 50) a Men >50 years, women >60 years, current smoker or stopped <3 years previously, high-density lipoprotein (HDL)<1 mmol/l, high blood pressure, diabetes, family history of cardiovascular disease (available for 76 patients). If HDL 1.5 mmol/l, subtract one risk factor. ARVs, antiretrovirals; cart, combination antiretroviral therapy; HBs, hepatitis B surface antigen; LDL, low-density lipoprotein. Antiviral Therapy

4 R Bittar et al. Table 2. Association between low-density lipoprotein diameter, in nm, and categorical variables Yes No Variable n Median (IQR) n Median (IQR) P-value Male sex ( ) ( ) <0.001 Men who have sex with men ( ) ( ) Sub-Saharan African ( ) ( ) Prior AIDS event ( ) ( ) Current smoker or stopped <3 years ( ) ( ) Data analysed by Mann Whitney U test. Bold values represent P<0.05. HDL-c; P<0.0001), these two variables explaining 62% of the variance of LDL diameter. Discussion LDL diameter was positively associated with the HDL-c level (P<0.0001) and negatively associated with the TG level (P<0.0001) in PI-treated HIV-1-infected patients. For each 1-mmol/l increase in HDL-c, mean LDL diameter rose by nm. Conversely, for each 1-mmol/l increase in TG, mean LDL diameter fell by nm. Triglyceride and HDL-c levels explained most of the variance of LDL diameter, as previously reported both in HIV-1-infected patients [6,7], and in HIV-seronegative subjects [10,26]. This is the first study that includes a sufficiently large number of patients receiving a boosted PI (n=81) to authorize multivariable analysis of LDL size determinants in the HIV-1-infected population. Previous studies included only 22 and 10 such patients [6,7]. In our study, we are not able to compare the effects of each PI or RTV dosage used, since groups are too small. Some authors have defined an atherogenic lipoprotein phenotype characterized by an elevated TG level (>2.28 mmol/l), small LDL particles (<25.5 nm) and a low HDL-c level (<1.040 mmol/l) [8,21,27]. This phenotype contributes to the development of CVD and is accepted as an emerging cardiovascular risk factor by the National Cholesterol Education Program Adult Treatment Panel III (NCEP ATP III) [8,10]. This atherogenic phenotype is a complication of PI-containing cart therapy for HIV-1 infection, and is associated with a high risk of CVD in this setting [6,7,28]. In our study, this phenotype was found in 11% of patients receiving a boosted PI. In a study conducted before the cart era, an atherogenic lipoprotein phenotype was found to be 2.5-fold more frequent in untreated HIV- 1-infected patients than in age-matched controls [3]. PI-containing cart improves virological and immunological status but accentuates HIV-1-related dyslipidaemia, with a large increase in TG levels and a reduction in LDL particle diameter [6,7]. Several studies have shown a higher incidence of myocardial infarction in Table 3. Association between LDL diameter, in nm, and continuous variables Variable n Coefficient P-value Age Body mass index Time on cart Time on NRTI Time on PI Time on NNRTI Number of carts Number of NRTIs Number of PIs Number of NNRTIs Total cholesterol LDL cholesterol HDL cholesterol <0.001 Triglycerides <0.001 Data analysed by Spearman correlation. Bold values represent P<0.05. cart, combination antiretroviral therapy; HDL, high-density lipoprotein; LDL, lowdensity lipoprotein; NNRTI, non-nucleoside reverse transcriptase inhibitor; NRTI, nucleoside reverse transcriptase inhibitor; PI, protease inhibitor. HIV-1-infected patients who receive cart regimens that include a boosted PI [5,29]. The incidence of coronary heart disease in these patients is at least two- to threefold that observed in the general population and increases with the duration of cart [11,30 32]. A recent study, in healthy HIV patients receiving cart under PI, observed that switching PI with a new drug (for example, raltegravir) improved the atherogenic phenotype [33]. Similar results as our study, concerning the close association of TG and HDL-c levels with LDL diameter when assessing cardiovascular risk, were observed in the large population of the Women Interagency HIV Study [34]. In this study, performed in a cohort of HIV-uninfected, HIV-infected/HAART-naive, and HIV-infected/on HAART women, the LDL particle size did not remain associated with HIV infection irrespective of HAART, after adjustment for TG level and HDL particle size. The mechanisms underlying the lipid changes associated with PI-containing cart are unclear. Very low International Medical Press

5 Determinants of LDL diameter in PI-treated HIV-1-infected patients density lipoprotein accumulation due to hyper-synthesis and/or delayed clearance by the liver has been reported in HIV-1-infected patients [35 37]. The persistent presence of LDL and of very low density lipoprotein particles rich in TG in the circulation leads to exchange pathways mediated by cholesteryl ester transfer protein, which results in the formation of sdldl, thereby affecting HDL metabolism and rendering HDL particles less protective against atherosclerosis [38,39]. Interestingly, TG and HDL-c levels did not explain all the LDL diameter variability in our study population, suggesting that other factors may be involved. Many studies applied nuclear magnetic resonance [6,34], in addition to gradient gel electrophoresis [10,21] to measure the LDL diameter. These two methods were not totally superimposable in results and interpretation. As measurement of LDL subclasses becomes increasingly important, standardization of methods is required; otherwise, variation among these currently available methods renders them unreliable and limits their clinical usefulness [40]. In conclusion, some of the HIV-1-infected patients receiving ritonavir-boosted PIs have an atherogenic lipoprotein profile, and their triglyceride and HDLcholesterol levels predict their LDL particle size distribution. These findings may help to understand the early atherogenic phenomena observed in this population. Acknowledgements The French National Agency for AIDS and Viral Hepatitis Research (ANRS) sponsored the trial and was involved in the study design. After approving the protocol, the sponsor had no involvement in the collection, analysis, or interpretation of the data, writing of the report or the decision to submit the article for publication. This study was registered with clinicaltrials.gov, number NCT ANRS received a grant from Astra Zeneca for the study. This work was presented at the 79th European Atherosclerosis Society Congress, June 2011, Gothenburg, Sweden, as a poster presentation (Abstract A ). Disclosure statement RB has received travelling grants, payment of registration fees and lecture fees from Gilead Sciences and Solvay. PG has received lecture fees from Pfizer, Astra- Zeneca and Solvay, and travel grants from Merck. EA has received travel grants from Sanofi Aventis, Pfizer and Bristol Myers Squibb. M-AV has received lecture fees, travelling expenses and payment of registration fees from Roche, Tibotec (Johnson&Johnson), Gilead, GlaxoSmithKline, Bristol Myers Squibb, MSD and Boehringer Ingelheim. DC has received travel grants, consultancy fees, honoraria or study grants from various pharmaceutical companies, including Abbott, Boehringer Ingelheim, Bristol Myers Squibb, Gilead Sciences, GlaxoSmithKline, Janssen, Merck and Roche. LA, DB-R, VF-F and OK declare no competing interests. References 1. Grunfeld C, Miyin P, Doerrler W, Shigenaga JK, Jensen P, Feingold PR. Lipids, lipoproteins, triglyceride clearance, and cytokines in human immunodeficiency virus infection and the acquired immunodeficiency syndrome. J Clin Endocrinol Metab 1992; 74: Khovidhunkit W, Kim MS, Memon RA, et al. Effects of infection and inflammation on lipid and lipoprotein metabolism: mechanisms and consequences to the host. J Lipid Res 2004; 45: Feingold KR, Krauss RM, Pang M, et al. The hypertriglyceridemia of acquired immunodeficiency syndrome is associated with an increased prevalence of low density lipoprotein subclass pattern B. J Clin Endocrinol Metab 1993; 76: Samaras K. 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