Effect of orlistat on postprandial lipemia, NMR lipoprotein subclass profiles and particle size

Atherosclerosis 180 (2005) 127 135 Effect of orlistat on postprandial lipemia, NMR lipoprotein subclass profiles and particle size Paolo M. Suter a,, Gabrielle Marmier a, Caroline Veya-Linder b, Edgar Hänseler c, Judith Lentz b, Wilhelm Vetter a, James Otvos d a Department of Internal Medicine, Medical Policlinic, University Hospital, Rämistrasse 100, 8091 Zürich, Switzerland b Roche Pharma, Schweiz, AG, Reinach, Switzerland c Institute of Clinical Chemistry, University Hospital, Zürich, Switzerland d LipoScience Inc., Raleigh, NC, USA Received 23 September 2003; received in revised form 31 October 2004; accepted 15 November 2004 Available online 22 January 2005 Abstract Evidence suggests that metabolic phenomena during postprandial lipemia may be important in the pathogenesis of atherosclerosis. Both lipid concentrations and lipoprotein subclass patterns may be important cardiovascular risk modifiers. The pancreatic lipase inhibitor orlistat reduces fat absorption by 30% and is used for the treatment of overweight and obesity. We evaluated the effect of orlistat on postprandial lipemia and lipoprotein particle distribution after moderate-and high-fat meals in healthy volunteers. In this double-blind, randomized, crossover study, 10 healthy young men received orlistat 120 mg plus a high-fat meal (HFO), orlistat plus a moderate-fat meal (MFO) or placebo plus a high-fat meal (HFP). Plasma triacylglycerol, glucose, insulin, and free fatty acids were measured at baseline (fasting) and postprandially for 8 h. Lipoprotein subclass profile was assessed by nuclear magnetic resonance spectroscopy. The 8 h postprandial mean triacylglycerol area under the curve (AUC) was significantly lower with MFO and HFO (0.79 versus 1.33 mmol/l h) versus HFP (4.33 mmol/l h; p = 0.02). Mean change in large VLDL subclass concentration during the 4 8 h and mean VLDL size after 8 h was significantly lower with HFO and MFO versus HFP (p < 0.001). Small HDL particle concentration decreased significantly with HFP versus MFO or HFO (p < 0.001). There was no significant difference in postprandial concentrations of glucose, insulin or free fatty acids on the different regimens. The lowering of postprandial triacylglycerol AUC, shorter postprandial lipemia, lower concentration of large triacylglycerol-rich particles and decrease of VLDL particle size supports the hypothesis of a less atherogenic postprandial lipoprotein profile following orlistat ingestion. 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Orlistat; Postprandial lipemia; Triacylglycerol; NMR lipoprotein particle subclass profile; Atherosclerosis; Cardiovascular risk; Obesity 1. Introduction More than 20 years ago, Zilversmit postulated that postprandial metabolic phenomena may play a role in the pathogenesis of atherosclerosis [1]. This concept has subsequently gained considerable support from various studies [2 6]. Dur- The triacylglycerol data have been presented as an abstract at the NAASO Annual Meeting (Quebec City, Canada, November 2001) and Nutrition Week (San Diego, USA, February 2002). Corresponding author. Tel.: +41 1 255 11 11; fax: +41 1 255 44 26. E-mail address: paolo.suter@usz.ch (P.M. Suter). ing the postprandial period, lipid changes, mainly resulting from increased concentrations of triacylglycerol (TG)-rich particles such as chylomicrons, chylomicron remnants and very low-density lipoproteins (VLDL), may play a causal role in the development and progression of atherosclerosis. Different factors, such as a low-fat diet, a pre-or postprandial exercise session [7,8] and alcohol intake, have been reported to modulate postprandial TG levels [9,10]. The major determinants of postprandial lipemia are the amount and type of fat absorbed and its metabolic clearance. Over the last few years, evidence has accumulated suggesting that lipoprotein subclass distributions may be important 0021-9150/$ see front matter 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2004.11.023

128 P.M. Suter et al. / Atherosclerosis 180 (2005) 127 135 modifiers of atherogenic risk [11 16]. Individuals with a high concentration of small, dense low-density lipoprotein (LDL) particles and their metabolic precursors, the large VLDL particles, have a higher cardiovascular risk than those with predominantly large LDL particles and small VLDL particles [17]. Orlistat (tetrahydrolipstatin) is a selective, nonsystemically acting inhibitor of gastrointestinal lipase activity which has been developed for the treatment of obesity [18]. When taken with meals, orlistat prevents the absorption of 30% of dietary fat [18,19]. Moreover, when combined with a mildly hypocaloric diet (30% of calories from fat), orlistat induced an average weight loss of 10% of the initial weight after 1 year of treatment [20]. In view of the pharmacological effects of orlistat (reducing fat absorption), favorable effects on postprandial lipemia, as well as on lipoprotein particle distribution, may be expected following acute and/or long-term therapy. Accordingly, we evaluated the effect of orlistat on postprandial lipemia and lipoprotein particle distribution in response to moderate-and high-fat meals in a population of healthy young men. 2. Subjects and methods 2.1. Subjects To be included in the study, young adult male subjects had to have a body weight that was stable for the last 3 months, and to have a normal physical examination and medical history, and not take any medications and/or recreational drugs. All were non-smokers, except one subject who reported to be an occasional smoker (one to two times per month), but who refrained from smoking during the study period. 2.2. Study design The protocol of this randomized, double-blind crossover study was approved by the Ethics Committee of the Medical Faculty of the University Hospital Zurich, Switzerland. Participation was voluntary and each subject gave written consent before entering the study. During the study period, subjects had to pursue their usual lifestyle and in particular their usual patterns of physical activity and eating. All subjects were studied on three separate test days. Subjects refrained from any strenuous physical activity and/or exercise and alcohol intake for 3 days before each test day and were instructed to consume a low-fat meal (i.e. pasta) on the evenings before each test day. On each occasion, after an overnight fast of at least 12 h, subjects were studied for an 8 h postprandial period. On each test day, subjects were admitted to the University Hospital at 07:00 h, placed in a semirecumbent position on a bed and had a catheter inserted into the antecubital vein. After a resting period of 20 30 min, a first sample of blood was drawn immediately before ingestion of the test meal (baseline value). Each test meal contained either 1 g (high-fat) or 0.67 g (moderate-fat) of fat per kg body weight. Subjects were randomized, in a double-blind manner, to receive one of three treatment regimens: (a) orlistat 120 mg plus a high-fat meal (HFO), (b) orlistat 120 mg plus a moderate-fat meal (MFO), or (c) placebo plus a high-fat meal (HFP) (Fig. 1). Subjects then received the two remaining treatments on the two remaining test days sequentially in a random order, with each treatment separated by 11 ± 2 days. Fig. 1. The experimental design regarding procedures on the single study days (A) and regarding the composition of the meal as well as drug (B).

P.M. Suter et al. / Atherosclerosis 180 (2005) 127 135 129 The energsy content of the high-fat meal was 1003 ± 62 kcal and of the moderate-fat meal was 788 ± 49 kcal. The high-fat meal (HFO and HFP regimens) comprised 4, 31, and 65% of protein, carbohydrates and lipids, respectively. In the moderate-fat meal (MFO regimen), the ratio of these substrates was 4.5%:40%:55%, respectively. Fat was given as butter and carbohydrates as marmalade and Zwieback (rusk) (all products from Migros Genossenschaftsbund, Zurich, Switzerland). Each meal was accompanied by 200 ml of tap water and all had to be ingested within a 10 min period. The drug (i.e. orlistat or placebo) was swallowed exactly halfway through the meal (i.e. after 5 min). The first two postprandial blood samples were drawn at 30 and 60 min after ingestion of the meal, and hourly thereafter (Fig. 1). EDTA plasma was immediately analyzed for lipid and glucose concentrations. During the test, subjects were seated in a semi-recumbent position on a bed and were allowed to read, study, sleep or watch video tapes. Walking or any other type of physical activity was not allowed. On each test day, 200 ml of tap water was given to subjects hourly to maintain adequate hydration. 2.3. Biochemical tests Plasma concentrations of TG, free fatty acid (FFA), insulin and glucose were determined immediately by chemical analysis at the Institute of Clinical Chemistry of the University Hospital Zurich using standard techniques. The latter analysis were done immediately without placing the blood samples on ice. Plasma samples frozen at 70 C for less than 1 month after termination of the study were analyzed by nuclear magnetic resonance (NMR) spectroscopy for lipoprotein subclass levels and mean VLDL, LDL, and high-density lipoprotein (HDL) particle diameters at LipoScience Inc. (Raleigh, NC, USA), as previously described [21 24]. In brief, the NMR method uses the characteristic signals broadcast by lipoprotein subclasses of different size as the basis of their quantification. Each measurement shows the concentration of seven subclasses of TG-rich particles (including chylomicrons, chylomicron remnants and VLDL), intermediatedensity lipoprotein, three subclasses of LDL, and five subclasses of HDL. The subclass levels are used to calculate weighted-average VLDL, LDL and HDL particle sizes (nm diameter). To simplify data analysis, we grouped subclasses to give a total of nine subclass categories: large VLDL (>60 nm; chylomicrons are included in this category), intermediate VLDL (35 60 nm), small VLDL (27 35 nm), large LDL (21.3 23 nm), intermediate LDL (19.8 21.2 nm), small LDL (18.3 19.7 nm), large HDL (8.8 13.0 nm), intermediate HDL (8.2 8.8 nm), and small HDL (7.3 8.2 nm). Since NMR distinguishes lipoprotein subclasses on the basis of particle size alone and is unable to differentiate intestinally-derived chylomicrons and chylomicron remnants from liver-derived VLDL particles [22], what we refer to as large VLDL includes chylomicrons in postprandial samples. VLDL subclass levels are reported in TG mass concentration units (mmol/l) and LDL and HDL subclass levels in units of mmol/l cholesterol. Lipoprotein subclass distributions determined by NMR and gradient gel electrophoresis are highly correlated [22,23]. However, LDL subclass diameters, which are referenced to those determined by electron microscopy [21], are uniformly 5 nm smaller than those estimated by gradient gel electrophoresis. NMR analyses of fresh and frozen ( 70 C) plasma specimens produce closely equivalent results, as confirmed in this study by the observed high correlation (r = 0.96; p < 0.0001) between chemically-determined TG concentration (immediately after the blood was drawn) and that estimated from the NMR subclass measurements on frozen samples. 2.4. Statistical analysis All values are expressed as mean ± S.E.M. unless stated otherwise. Postprandial changes in concentrations at specific time points are reported as concentration values, calculated by subtracting the baseline values from the postprandial values. The postprandial responses of the different parameters were evaluated as absolute values, incremental change at each time point, and as area under the curve (AUC) for the 8 h postprandial period, following the trapezoidal rule. In calculating the incremental postprandial variations in the concentration of certain parameters (e.g. TG) compared with baseline, negative concentration values were obtained. Since these negative values correspond to actual changes in the absolute concentration, these were used in the calculations. For selected biochemical and NMR measurements, the mean ± S.E.M. of the concentration changes during the first 4 h (time points 0 240 min) and the second 4 h (time points 240 480 min) postprandially, were computed. To evaluate the repeated measurements linear mixed models with period (day of intervention) and treatment (intervention) as fixed factors and subject as random factor have been applied to the primary variables, i.e. TG AUC, maximum change of TG concentrations, time to reach the maximum and time to return to baseline. Carry over effects have been not expected because of sufficiently long washout period between the interventions, and statistical tests of treatment sequence effects or period by treatment interactions lead to non-significant results. Finally the main effects model y ijk = µ + s k + π i + τ j(i) + ε ijk has been used, where y ijk denotes the response of subject k to the treatment j applied in period i, µ the general mean, s k the random subject effect, π i the period effect, τ j(i) the treatment effect and ε ijk is the random deviation. First the three interventions have been compared applying the ANOVA F-test (global test). In case of a significant result all pair of interventions have been tested using the corresponding contrasts. This procedure follows the closure principle of Marcus et al. [25], i.e. no α-adjustment is required.

130 P.M. Suter et al. / Atherosclerosis 180 (2005) 127 135 The same level of significance (α = 0.05) has been used for all tests. All statistical tests were performed with SAS 8.02 statistical software programs for PC (SAS Institute, Cary, NC, USA). 3. Results 3.1. Study population characteristics Ten young healthy men were studied. Their mean age (±S.D.) was 25 ± 3 years, mean body weight 70 ± 4 kg, and mean body mass index 21.5 ± 1.2 kg/m 2. All subjects were normoglycemic and normolipidemic with respect to TG and cholesterol. Their mean fasting cholesterol level was 4.5 ± 0.7 mmol/l (range 3.7 5.8) and fasting plasma TG concentration was 0.98 ± 0.23 mmol/l (range 0.73 1.30). All subjects were students and reported to be in good health. The test meal and orlistat/placebo were well tolerated by all subjects. 3.2. Postprandial lipemia Table 1 summarizes the concentrations of selected variables for the different regimens at the beginning and end of the test as well as the maximal concentrations observed during the postprandial period. There was no significant difference in the postprandial concentrations of glucose, insulin or free fatty acids on the different regimens. The time courses of the change in TG concentration are shown in Fig. 2. The maximal changes in postprandial TG concentration (Fig. 2) were not significantly different between the different treatment regimens (HFO: 0.64 ± 0.22 mmol/l; MFO: 0.38 ± 0.13 mmol/l; HFP: regimen 1.17 ± 0.52 mmol/l). There was a positive correlation between the baseline and the maximal TG levels which reached the level of significance only for the MFO regimen (r = 0.89, p = 0.008). The maximal postprandial TG concentration (using the individual maximal concentration values) occurred earlier with the HFO (150 ± 13 min after the meal, p = 0.02 versus HFP) and MFO (171 ± 25 min, p = 0.15 versus HFP) regimens compared with the HFP regimen (225 ± 27 min). The time to reach maximal TG concentration was not significantly different between HFO and MFO regimens. The time point at which postprandial lipemia returned to baseline (i.e. fasting) levels (using the individual first time point with a TG concentration below the baseline concentration) was significantly later with the HFP regimen (456 ± 24 min) than with the HFO (360 ± 23 min; p = 0.03 versus HFP) and MFO (348 ± 40 min; p = 0.01 versus HFP) regimens (no significant difference between HFO and MFO) (Fig. 2). The mean standardized TG concentration during the first four postprandial hours had a tendency to be higher than baseline with the HFP regimen (0.63 mmol/l) than with the HFO (0.37 mmol/l, p = 0.07 versus HFP) and MFO (0.23 mmol/l, p = 0.01 versus HFP) regimens (difference between MFO and HFO was not significant) (Table 2). During the 4 8 postprandial hour, TG concentration was higher than baseline by 0.33 mmol/l with the HFP regimen compared with almost identical lower concentration as compared to baseline by 0.14 mmol/l with the HFO regimen and 0.16 mmol/l with the MFO regimen (ANOVA, p < 0.001) (Table 2). The AUC (mean ± S.E.M.) for the increase in TG concentration during the 8 h postprandial period was significantly lower with the HFO (1.33 ± 0.52 mmol/l h) and Table 1 The fasting concentration, maximal postprandial concentration (maximal pp), and the concentration after 8 h postprandially (after 8 h pp) for selected variables on the three test days (n = 10, all values mean ± S.E.M.) Fasting Maximal pp After 8 h pp High-fat-orlistat (HFO) Triacylglycerol (mmol/l) 1.03 ± 0.13 1.50 ± 0.16 ** 0.83 ± 0.09 * Free fatty acids (mmol/l) a 401 ± 52 735 ± 69 ** Glucose (mmol/l) 5.0 ± 0.1 6.3 ± 0.3 ** 4.8 ± 0.1 Insulin (pmol/l) 56 ± 7 250 ± 27 ** 47 ± 5 Moderate-fat-orlistat (MFO) Triacylglycerol (mmol/l) 0.99 ± 0.11 1.34 ± 0.21 * 0.82 ± 0.19 Free fatty acids (mmol/l) a 434 ± 73 802 ± 86 ** Glucose (mmol/l) 4.9 ± 0.1 6.3 ± 0.5 ** 4.8 ± 0.1 Insulin (pmol/l) 54 ± 6 289 ± 42 ** 44 ± 4 ** High-fat-placebo (HFP) Triacylglycerol (mmol/l) 0.76 ± 0.13 1.77 ± 0.42 ** 0.83 ± 0.14 Free fatty acids (mmol/l) a 494 ± 78 611 ± 85 ** Glucose (mmol/l) 5.14 ± 0.10 6.14 ± 0.22 ** 4.80 ± 0.1 Insulin (pmol/l) 60 ± 2 285 ± 30 ** 49 ± 4 * a The maximal free fatty acid concentration corresponds to the concentration at the end of the test. p < 0.05 vs. baseline. p < 0.01 vs. baseline.

P.M. Suter et al. / Atherosclerosis 180 (2005) 127 135 131 Fig. 2. Postprandial time-courses of the concentration changes of triacylglycerol (A), and triacylglycerol-rich subclasses: large VLDL (B), intermediate VLDL (C), small VLDL (D), in mean VLDL particle size (E) on the high-fat-orlistat [HFO] ( ), high-fat-placebo [HFP] ( ) and moderate-fat-orlistat [MFO] ( ) regimens (mean ± S.E.M.). MFO (0.79 ± 0.56 mmol/l h) regimens than with HFP (4.33 ± 1.37 mmol/l h; ANOVA global p = 0.009, HFO-HFP p = 0.01, MFO-HFP p = 0.005) (Fig. 2). TG AUC values for HFO versus MFO regimens were not significantly different (p = 0.70). The postprandial time course of the plasma concentrations of glucose, FFA, insulin, total cholesterol, and apolipoproteins A-1 and B, were similar with the different treatment regimens. The concentration time course of glucose, FFA and insulin is shown in Fig. 3 (there was no significant difference in the time course for all three parameters on the different intervention days). 3.3. NMR lipoprotein analysis Table 2 summarizes the mean changes in NMR-measured variables during the first and second 4 h postprandial periods with the different regimens. The concentrations of the

Table 2 Mean ± S.E.M. changes in plasma triacylglycerol and NMR-measured lipoprotein concentrations during the 1 4 and 4 8 postprandial hour on the different dietary regimens Parameter Baseline Postprandial h 1 4 Postprandial h 4 8 HFP MFO HFO HFP MFO HFO HFP MFO HFO Triacylglycerol (TG) (mmol/l) 0.63 ± 0.15 0.23 ± 0.05 0.33 ± 0.07 0.33 ± 0.08 0.16 ± 0.04 0.14 ± 0.04 VLDL (mmol/l) Large 0.20 ± 0.06 0.40 ± 0.22 0.34 ± 0.12 0.51 ± 0.15 0.17 ± 0.04 = 0.28 ± 0.07 0.36 ± 0.07 ***, 0.11 ± 0.05 === 0.05 ± 0.04 === Intermediate 0.26 ± 0.10 0.22 ± 0.05 0.37 ± 0.08 0.07 ± 0.02 0.08 ± 0.02 0.04 ± 0.02 0.08 ± 0.04 0.01 ± 0.03 ** 0.14 ± 0.03 Small 0.11 ± 0.02 0.19 ± 0.03 0.14 ± 0.02 0.05 ± 0.01 0.03 ± 0.01, * 0.03 ± 0.02 0.03 ± 0.02 0.05 ± 0.01 *** 0.05 ± 0.02 LDL (mmol/l) Large 1.67 ± 0.24 1.75 ± 0.34 1.81 ± 0.32 0.09 ± 0.06 0.09 ± 0.04 0.01 ± 0.06 0.14 ± 0.06 0.08 ± 0.05 0.10 ± 0.07 Intermediate 0.44 ± 0.11 0.36 ± 0.12 0.30 ± 0.09 0.13 ± 0.04 0.10 ± 0.03 0.06 ± 0.03 0.09 ± 0.04 0.02 ± 0.05 0.003 ± 0.05 Small 0.07 ± 0.05 0.18 ± 0.09 0.22 ± 0.15 0.10 ± 0.03 * 0.07 ± 0.03 0.01 ± 0.03 0.08 ± 0.02 0.06 ± 0.05 0.03 ± 0.04 HDL (mmol/l) Large 0.58 ± 0.09 0.56 ± 0.10 0.59 ± 0.10 0.00 ± 0.01 0.00 ± 0.01 0.02 ± 0.01 0.06 ± 0.01 ** 0.04 ± 0.01 0.02 ± 0.01 == Intermediate 0.17 ± 0.03 0.22 ± 0.05 0.21 ± 0.06 0.04 ± 0.01 0.01 ± 0.01 = 0.04 ± 0.01 0.15 ± 0.01, *** 0.04 ± 0.01 === 0.06 ± 0.02 Small 0.72 ± 0.05 0.65 ± 0.06 0.70 ± 0.07 0.08 ± 0.02 0.01 ± 0.01 * 0.05 ± 0.02 0.25 ± 0.02 *** 0.001 ± 0.03 * 0.09 ± 0.02 ===, VLDL size (nm) 55.37 ± 2.85 53.47 ± 5.03 53.86 ± 2.52 39.11 ± 5.36 27.98 ± 2.96 33.48 ± 4.08 49.03 ± 3.7 *** 11.1 ± 3.1 9.3 ± 2.5 === LDL size (nm) 21.54 ± 0.13 21.43 ± 0.22 21.49 ± 0.23 0.05 ± 0.05 0.03 ± 0.03 0.07 ± 0.05 0.04 ± 0.04 0.007 ± 0.03 0.08 ± 0.05 HDL size (nm) 9.06 ± 0.14 9.07 ± 0.13 9.09 ± 0.13 0.02 ± 0.02 0.04 ± 0.01 0.02 ± 0.01 0.12 ± 0.02 ***, 0.03 ± 0.02 == 0.03 ± 0.02 === HFO: high-fat-orlistat; MFO: moderate-fat-orlistat; HFP: high-fat-placebo. *p < 0.05, **p < 0.01, ***p < 0.001 vs. HFO; p < 0.05, p < 0.01, p < 0.001 vs. MFO; = p < 0.05, == p < 0.01, === p < 0.001 vs. HFP. 132 P.M. Suter et al. / Atherosclerosis 180 (2005) 127 135

P.M. Suter et al. / Atherosclerosis 180 (2005) 127 135 133 Fig. 3. Postprandial time course of glucose, insulin and FFA. Fig. 4. Postprandial time-courses of the concentration changes of the HDL subclasses: large HDL (A), intermediate HDL (B), small HDL (C) on the high-fat-orlistat [HFO] ( ), high-fat-placebo [HFP] ( ) and moderate-fatorlistat [MFO] ( ) regimens (mean ± S.E.M.). different lipoprotein subclasses assessed by NMR, and the mean lipoprotein particle sizes, at baseline and at the end of the 8 h postprandial period are given in Table 2. The intermediate VLDL subclass concentration was significantly lower than baseline at the end of the 8 h period with the HFP and HFO regimens (Table 2). After 8 h, there were no significant differences in the mean concentrations of the LDL subclasses for any regimen as compared to baseline. The mean concentration change of large HDL particles were significantly higher during the 4 8 postprandial hour compared with the 1 4 postprandial hour for all regimens (p < 0.01). Likewise, the mean concentration change of inter- mediate HDL particles was higher during the 4 8 postprandial hour compared with the 1 4 postprandial hour for all regimens, but reached significance only on the HFP regimen (p < 0.001). The change in the standardized HDL size was greater with the HFP regimen than with the other regimens (Table 2). The concentration of the small HDL subclass declined significantly with the HFP regimen (Table 2, Fig. 4) and the mean concentration change of the small HDL subclass was significantly lower during the 4 8 postprandial hour on the HFP and the HFO regimen (p < 0.001 for both) as compared with the MFO regimen. The concentration of the small HDL subclass during the postprandial period and at the end

134 P.M. Suter et al. / Atherosclerosis 180 (2005) 127 135 of the 8 h period was significantly higher on the MFO and HFO regimens (Table 2, Fig. 4). VLDL size increased significantly with the HFP regimen and was significantly higher with this regimen than with the HFO or MFO regimens during the second 4 h postprandial period (Table 2 and Fig. 2)(p < 0.0001). The increase in VLDL particle size is explained by the postprandial time course of large and intermediate VLDL subclass concentration changes shown in Fig. 2. At the end of the 8 h period, the concentration of large VLDL was higher than baseline for HFP while that of the intermediate VLDL subclass was lower. With HFO and MFO, the concentration of large VLDL particles during the second 4 h period was significantly lower than with HFP (Fig. 2). The concentration of small VLDL particles during the second 4 h was significantly lower on the MFO regimen compared with the HFO or HFP regimens (p < 0.0001) (Table 2 and Fig. 2). There were no significant changes in the LDL subclass profile with the different regimens; LDL particle size changes during the postprandial phase were not significantly different between the three regimens (Table 2). 4. Discussion Accumulating evidence indicates that an increased concentration of TG-rich lipoproteins, especially during the postprandial period, may promote the development and progression of atherosclerosis [1 5]. Our study shows that ingestion of the gastrointestinal lipase inhibitor orlistat with a meal led to a significantly shorter postprandial TG response as well as a significant reduction in the TG AUC over an 8 h postprandial period. Postprandial lipemia, as assessed by the AUC, was not significantly different on the HFO and MFO regimens. This is not surprising, since there is a close relationship between postprandial lipemia and the absolute amount of fat ingested [26]. With the MFO regimen, subjects were already receiving a relatively moderate fat load (i.e. 0.6 g/kg body weight) and so the amount of non-absorbed fat resulting from orlistat therapy was smaller in these subjects than in those with the high-fat diet (i.e. 1 g/kg body weight). With HFO and MFO regimens, postprandial lipemia returned to fasting levels much sooner than with the HFP regimen (see Fig. 2). If the hypothesis that atherosclerosis represents a postprandial phenomenon is correct [1,4,6], the observed effects of orlistat on postprandial lipemia in the HFO and MFO regimens would, with long-term therapy, result in a reduction of the risk of atherosclerosis independent from the classical cardiovascular risk factors. Our data are in agreement with that of Reitsma et al. [27] where 8 weeks of orlistat treatment in hyperlipidemic subjects with a body mass index of 19 28 kg/m 2 led to a reduction in the postprandial TG AUC. Although another study failed to show a favorable effect of orlistat on postprandial lipemia [28], this can be explained by the comparatively small fat load (28 g fat) that was given [26]. In view of the pharmacological effects of orlistat [18], we hypothesized that, due to the inhibition of fat absorption and consecutively smaller amounts of fat entering the systemic circulation, the absolute concentration of plasma TG as well as the lipoprotein particle subclass distribution may be influenced. Large VLDL particles [13], small dense LDL [29,30] and small HDL particles [31] may be associated with a higher cardiovascular risk. We found that the postprandial concentration changes of large VLDL particles (including chylomicrons and remnants) were significantly more pronounced with the HFP regimen. Accordingly, the VLDL size during the second 4 h postprandial phase was significantly larger with HFP than with MFO or HFO regimens. Large VLDL particles are preferentially metabolized to small dense LDL particles which may have an increased atherosclerotic potential [17,32]. Consequently, these findings suggest that ingestion of a single dose of orlistat with a meal may have a favorable effect on VLDL particle size and thus on atherosclerosis risk. Based on our NMR data, we cannot discriminate between TG-rich lipoproteins of intestinal (chylomicrons) and hepatic (VLDL) origin. However, in view of the pharmacological effect of orlistat at the level of fat absorption, it might be assumed that the observed changes in the particle concentration are mainly caused by changes in the concentration of lipoproteins of intestinal origin. In the present study, we did not observe any significant postprandial changes in LDL subclass concentrations nor LDL particle size with any of the regimens. There was a significantly greater decrease in small HDL particle concentration (which was significantly lower at the end of the experimental period) and a greater increase in intermediate HDL with HFP compared with HFO and MFO regimens (Fig. 4). It is not certain what significance, if any, these differences may have in terms of atherosclerotic risk. While promising, the data from this single-dose study do not allow us to draw firm conclusions about the effects of long-term treatment with orlistat on lipoprotein subclass pattern. It is known that lipid-lowering drugs may favorably affect postprandial lipemia and lipoprotein subclass profile [33]. Therefore, one can speculate that if the effect of orlistat was maintained over longer periods of time, the inhibition of fat absorption, with consequently lower postprandial and fasting TG concentrations and alterations in postprandial lipid exchange between lipoprotein particles, might be associated with a lower atherogenic risk. In conclusion, current cardiovascular risk reduction measures (both non-pharmacological and pharmacological) remain suboptimal and future preventive strategies will have to focus on novel factors [34], including postprandial lipid metabolism and lipoprotein particle size. Our data suggest that inhibition of gastrointestinal lipase may represent another relatively unexplored pharmacological approach to cardiovascular risk modulation. In modern society, most individuals spend >50% of their day in the postprandial state. In addition, many cardiovascular events occur in the postprandial period which might be explained by postprandial

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