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Selective uptake of high density lipoproteins cholesteryl ester in the dog, a species lacking in cholesteryl ester transfer protein activity
Comparative Biochemistry and Physiology, Part B 138 (2004) 339 – 345 www.elsevier.com/locate/cbpb
Selective uptake of high density lipoproteins cholesteryl ester in the dog, a species lacking in cholesteryl ester transfer protein activity An in vivo approach using stable isotopes Khadija Ouguerram a,*, Patrick Nguyen b, Michel Krempf a, Etienne Pouteau b, Francßois Briand a,b, Edwige Bailhache a,b, Thierry Magot a a
Centre de Recherche en Nutrition Humaine, INSERM U539, CHU Nantes, 1 place Alexis Ricordeau, 44093 Nantes Cedex 01, France b USC INRA de Nutrition et Endocrinologie, Ecole nationale ve´te´rinaire de Nantes, France Received 16 December 2003; received in revised form 9 April 2004; accepted 13 April 2004
Abstract Amongst the processes involved in the reverse cholesterol transport (RCT) from organs to liver, including high density lipoproteinsapolipoprotein AI (HDL-apoAI) dependent tissue uptake and cholesteryl ester transfer protein (CETP)-mediated transfers, the selective uptake of cholesteryl ester (CE) is of increasing interest through its antiatherogenic role. The purpose of this report is to develop a simple protocol allowing study of this process in an animal model with easier quantification of CE selective uptake. The dog was chosen essentially because this animal has a low CETP activity and an appropriate size to conduce a kinetic study. Tracer kinetics were performed to estimate in vivo the contributions of the pathways involved in HDL-CE turnover in dogs. Stable isotopes, 13C-acetate and D3-leucine as labeled precursors of CE and apoAI, were infused to fasting dogs. Isotopic enrichments were monitored in plasma unesterified cholesterol and in HDL-CE and apoAI by mass spectrometry. Kinetics were analyzed using compartmental modeling. Results concerned the measurement of the activity of cholesterol esterification (0.13 F 0.032 h 1), rate of HDL-apoAI catabolism (0.024 F 0.012 h 1), HDL-CE turnover (0.062 F 0.010 h 1) and CE selective uptake (0.038 F 0.014 h 1). Our results show that CE in dogs is mainly eliminated by selective uptake of HDL-CE (60% of HDL-CE turnover), unlike in other species studied by similar methods in our laboratory. This study shows that among species used to analyze cholesterol metabolism, the dog appears to be the animal in whom HDL-CE selective uptake represents the largest part of HDL-CE turnover. D 2004 Elsevier Inc. All rights reserved. Keywords: Cholesterol; Reverse cholesterol transport; Lecithin-cholesterol acyltransferase; Kinetic analysis; Dog
1. Introduction Cholesterol transport by plasma high density lipoproteins (HDL) plays a key role in reverse cholesterol transport (RCT). Cholesterol HDL metabolism is complex, involving multiple metabolic pathways (Fielding and Fielding, 1995). Apo A-I is mainly secreted by the liver and forms preß-HDL with phospholipids (Barter et al., 2003; Lawn et al., 1999). Abbreviations: HDL, high density lipoproteins; VLDL, very low density lipoproteins; LDL, low density lipoproteins; apoAI, apolipoprotein AI; RCT, reverse cholesterol transport; LCAT, lecithin cholesterol acyl transferase; CETP, cholesteryl ester transfer protein; SR-BI, scavenger receptor class B type I; CE, cholesteryl ester. * Corresponding author. Tel.: +33-240-087535; fax: +33-240-087544. E-mail address: [email protected] (K. Ouguerram). 1096-4959/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2004.04.011
This form of nascent HDL removes cholesterol from peripheral tissues through a recently identified ABCA1 mediated process (Oram and Vaughan, 2000). ATP Binding Cassette transporter A1 (ABCA1) is an essential transporter in the RCT because its deficiency results in very low or no plasma HDL (Attie et al., 2001). Deficiency of ABCA1 gives rise to Tangier disease, where cholesteryl esters accumulate in macrophage-derived foam-cells. This reduction in the rate of RCT increases the risk for developing atherosclerosis (Hobbs and Rader, 1999). The ABCA1 process produces spherical HDL particles through the activity of plasma lecithin cholesterol acyl transferase (LCAT). LCAT converts cholesterol to cholesteryl esters on the surface of HDL particles (Glomset et al., 1966). Thus, this enzyme promotes cholesterol efflux from cell to HDL by an accumulation of the cholesteryl esters in the core of the
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lipoprotein (Santamarina-Fojo et al., 2000). Neo-formed HDL-cholesteryl ester (HDL-CE) can have different metabolic fates including (i) apolipoprotein AI (apoAI)-dependent tissue uptake, (ii) cholesteryl ester transfer protein (CETP)-mediated transfers to lower density lipoproteins, and (iii) direct delivery to liver by the selective HDL-CE uptake pathway mediated by SR-BI receptor. Kinetic studies have shown in the rat, a species with a low CETP activity, that the fates of CE and apoAI were uncoupled (Glass et al., 1983). In this species a receptor has been found (scavenger receptor class B type I, SR-BI) allowing specific uptake of HDL-CE by the liver (Rigotti et al., 1997), and this pathway has been the purpose of numerous studies (Silver and Tall, 2001). SR-BI has potential anti-atherogenic activities because this HDL receptor promotes hepatic uptake of HDL cholesterol. This cholesterol is then directly secreted into bile (Ji et al., 1999). In species with CETP activity, such as in humans, the process involving SR-BI could coexist with the transfer of CE in the plasma from HDL to very low density lipoproteins (VLDL) and low density lipoproteins (LDL). Therefore, in these species with CETP activity such as rabbits and humans, in vivo quantitative analysis of both processes is difficult as it requires a complex multicompartmental approach (Goldberg et al., 1991; Ouguerram et al., 2002). However, the physiological difference between these two pathways is important. Indeed, it is reasonable to assume that the CETPmediated transfer of CE from HDL to lower density lipoproteins is an atherogenic pathway (Okamoto et al., 2000) because it provides cholesterol to LDL. On the contrary, CE selective uptake from HDL does not enrich the atherogenic lipoproteins in CE and should be anti-atherogenic. Thus, it would be of crucial interest to possess an animal model exhibiting CE selective uptake as the main process involved in HDL-CE turnover. The aim of this study was to test the dog model in order to develop a protocol allowing a quantification of selective uptake of HDL-CE. The dog model was selected because this animal is lacking CE transfer activity (Ha and Barter, 1982; Guyard-Dangremont et al., 1998; Tsutsumi et al., 2001) and because large animals, such as dogs, make it possible to obtain high amounts of plasma in order to determine as much information as humans.
2. Materials and methods 2.1. Animals Five adult dogs (beagles, 11– 23 kg) were supplied by the National Veterinary School of Nantes after approval of the protocol by the animal ethic committee. Dogs were fed under standard laboratory conditions: they consumed in a single meal a commercial dry dog food (25%, 17% and 58% of calories, respectively, as protein, fat and carbohydrates, 3730 kcal metabolizable energy/kg, on a dry mass basis)
and were fed according to the NRC recommendation (132 kcal metabolizable energy/kg BW0.75). Two catheters (Vasocan Braunu¨le, 20G 11/4U, Melsungen, Germany) were inserted into the cephalic vein of both forelimbs of each dog: one for double infusion of tracers ([1-13C]acetate and [D3-leucine] and the other for collection of venous blood. 2.2. Experimental design Stable isotope tracers were weighed on a high precision scale and dissolved in known volumes of sterile 0.9% NaCl. Solutions were prepared no earlier than 24 h before use, sterilized by passing through a 0.22 Am filter, stored in sterile sealed containers, and kept at 4 jC until required. The study was then conducted after an overnight fast. Each animal received intravenously a prime of 10 Amol kg 1 of [5,5,5-2H3]leucine (as endogenous marker of apoAI, 99.8 at.%; Mass Trace, Woburn, MA, USA), and 2 Amol kg 1 for [1,213C]acetate as endogenous marker of cholesterol, 99% 13C enrichment, Tracer Technologies, Somerville, MA, USA) immediately followed by a constant tracer infusion (10 Amol kg 1 h 1 for [5,5,5-2H3]leucine and 2 Amol kg 1 min 1 for [1,213C]acetate) for 8 h as previously published (Frenais et al., 1997; Ouguerram et al., 2002). Blood samples were collected at 10, 30, 60 min and hourly until 10 h during the perfusion and 25 h after the beginning of the perfusion. The samples were collected in tubes containing EDTA (0.2 mmol/l final concentration) and dithiobisnitrobenzoic acid as inhibitor of LCAT reaction (0.2 mmol/l final concentration). Plasma was immediately separated by centrifugation at 4 jC and stored frozen at 20 jC for later determination of cholesterol concentration and isotopic enrichments of cholesterol and apoAI. HDL were isolated by ultracentrifugation between 1.063 and 1.21 g/ml as previously described (Frenais et al., 1997). For measurement of unesterified enrichment in lipoproteins of density lower than 1.063, two fractions were isolated (d < 1.006 g/ ml and 1.006 < d < 1.063 g/ml). 2.3. Analytical methods Measurement of apoAI enrichment was performed as previously described (Frenais et al., 1997). Briefly, HDL were delipidated with diethyl ether and apolipoproteins concentrated with trichloroacetic acid (4.9 mM) and desoxycholic acid (3.6 mM). ApoAI was isolated from other apolipoproteins by PAGE-SDS using a 4.5– 10% discontinuous gradient (Mindham and Mayes, 1992). Apolipoproteins were identified by comparing migration distances with known molecular weight standards (cross-linked phosphorylase b markers, Sigma, St. Louis, MO, and electrophoresis calibration kit, Pharmacia LKB, Biotechnology, Piscataway, NJ, USA). Apolipoprotein bands were excised from polyacrylamide gels and dried in vacuum for 1 to 2 h (RC 10-10 Jouan, Saint Herblain, France). The desiccated gel slices were hydrolyzed with 1 ml 4 mol/l HCl (Sigma, St. Quentin
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Fallavier, France) at 110 jC for 24 h. Hydrolysates were then evaporated to dryness and the amino acids were purified by cation exchange chromatography using a Temex 50W-X8 resin (Bio-Rad, Richmond, CA, USA). Amino acids and plasma leucine were esterified with propanol/ acetyl chloride, and further derivatized using heptafluorobutyric anhydride (Fluka Chemie, Buchs, Switzerland) prior to analysis. Samples were analyzed for isotopic enrichment of leucine using gas chromatography coupled-mass spectrometry (GC-MS) as previously described (Frenais et al., 1997). Briefly, chromatographic separations were carried out on a 30 m 0.25 mm i.d. DB-5 capillary column (J&W Scientific, Rancho Cordova, CA, USA). The column temperature was as follows: initial temperature was held at 80 jC, then increased at 10 jC/min to a final temperature of 180 jC. Electron-impact gas chromatography-mass spectrometry was performed on a 5890 gas chromatograph connected with a 5971A quadrupole mass spectrometer (Hewlett-Packard, Palo Alto, CA, USA). The isotopic ratio was determined by selected ion-monitoring at m/z 282 and 285. Results were expressed as tracer/tracee ratio for modeling analysis (Cobelli et al., 1992). Analysis of cholesterol was performed as previously described (Ouguerram et al., 2002). Unesterified cholesterol and CE were isolated from lipoproteins as previously reported (Ouguerram et al., 1996). Briefly, lipid extraction was performed with chloroform – methanol (2:1, v/v) according to Folch et al. (1957). Unesterified cholesterol and CE were separated by chromatography on silicic acid (Hirsh and Ahrens, 1958), using microcolumns (Sep Pack cartridges, Waters, Milford, MA, USA). The cholesterol samples were derivatized with a mixture of acetic anhydride (500 Al) –pyridine (100 Al) (Aldrich, France). The samples were heated to 90 jC for 10 min. After cooling to room temperature, the derivatizing reagents were evaporated under nitrogen, and the residue was dissolved in hexane. Due to low 13C-enrichments in cholesterol and to allow us a higher sensitivity we have used gas chromatography – combustion-isotope ratio mass spectrometry (GC-C-IRMS) with a Finnigan Mat Delta S isotope ratio mass spectrometer coupled to a HP 5890 series II gas chromatograph (HewlettPackard) with a DB-1 capillary column (30 m 0.32 mm i.d., 0.25 Am film thickness; J&W Scientific). The temperature of the injection port was 280 jC and the oven temperature was initially set at 70 jC, then increased at 50 jC/min up to 280 jC and was held for 15 min at 280 jC. The samples were injected in splitless mode. The cholesterol peak eluted from the GC column was introduced on line into a combustion furnace which operated at 940 jC. The furnace is a non-porous alumina tube packed with a wire of copper, nickel and platinum. Combustion water was removed by passing the effluent through a water-permeable Nafion tube. The CO2 was driven in the electron impact ion source. The same samples were analyzed in triplicate by GC/C/IRMS and measurements were highly reproducible ( < 0.5%).
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Isotopic enrichment was expressed relative to pulse peaks of CO2 reference, calibrated against the international standard (Pee Dee Belemnite, PDB). Isotopic calculations: The isotopic abundance in 13C in cholesterol is given by the GC-C-IRMS as dx versus the international standard PDB as: d13 C ðxÞ ¼ ððRsample Rstd Þ=Rstd Þ 1000; where R is the 13C/12C ratio in the sample or the international standard PDB (Rstd = 0.0112372). Therefore, Rsample = Rstd*[1+(0.001*d13C)]. The observed fractional abundance F (atom%) was calculated as: F ¼ 100Rstd *½ð0:001*d13 Csample Þ þ 1 The isotopic enrichment E (atom% excess) was calculated as: E ¼ Fsamplej Fbasal value where Fsamplej and Fbasal value are the observed fractional abundance in the sample at time j and at baseline, respectively. E ¼ 100Rstd *ð0:001*d13 Csample þ 1Þ APE ¼ atom% sample atom% basal value ðAtom Percent ExcessÞ 2.4. Concentration measurement Unesterified and esterified cholesterol concentrations in samples were measured using commercially available enzymatic kits (Cholesterol RTU, BioMe´rieux, Lyon, France and ‘‘choleste´rol libre enzymatique’’ color, Biotrol Diagnostic, Lognes, France). 2.5. Modeling methods Data were treated by compartmental analysis using computer software (Barrett et al., 1998) for simulation, analysis and modeling (SAAMII v 1.01, Resource Facility for Kinetic Analysis, Department of Bioengineering, SAAM Institute, Seattle, WA, USA). HDL-apoAI model was a one-compartment model (Fig. 1A). Labeling input was done after a delay from plasma free leucine tracer-to-tracee ratio (as forcing function). In this model, k01 represented the parameter of HDL particle uptake by tissues including liver. HDL-CE model was also a one-compartment model (Fig. 1B). Labeling input was done from unesterified cholesterol enrichments (as forcing function) through cholesterol esterification kLCAT. In this model k01 and k01V represented the two output parameters, uptake of HDL particle and CE selective uptake by tissues including liver, respectively. In these two models, k01 described the simultaneous uptake of both apoAI and CE
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Fig. 1. Study model of apoAI-HDL (A) and cholesteryl ester-HDL (B) turnover.
through HDL particle uptake. Parameters were simultaneously identified from data of HDL-apoAI and CE. The rate constant of cholesterol esterification (kLCAT) was calculated as the ratio between the absolute rate of cholesterol ester input in HDL cholesteryl ester identified by modeling and plasma unesterified cholesterol concentration. 2.6. Statistical analysis Results are reported as mean F S.D. The Wilcoxon unpaired test (two tail p-value) was used to compare turnover rate of HDL-apoAI and HDL-CE. Analysis was performed using the Instat statistical Software package (GraphPad, San Diego, CA, USA). A two-tail p-value < 0.05 was considered as statistically significant. The goodness of fit was assessed using the run test (Sokal and Rohlf, 1995). Different models were compared using the AIC test (Akaike, 1974) and the F-test (Boxenbaum et al., 1974).
Fig. 2A shows time courses of enrichment in HDL-apoAI and plasma leucine. During perfusion, the tracer/tracee ratio curve was approximately constant for plasma leucine and then fell after the end of the perfusion. ApoAI curve regularly increased during perfusion and then remained constant after the end of perfusion. The time course of 13 C enrichment in plasma unesterified cholesterol was virtually identical in lipoproteins of density lower than 1.006, of density 1.006 – 1.063 and in HDL during the experiment (results not shown). Therefore, the results were only expressed as plasma unesterified cholesterol (Fig. 2B) and used as forcing function to take into account the time course of labeling input into HDL-CE. Labeling appeared first in unesterified cholesterol, increased during the perfu-
3. Results Amounts of unesterified cholesterol and CE in plasma and HDL fractions are provided in Table 1. In plasma, cholesterol was mainly present in esterified form (77%). The major part of cholesterol (76%) was recovered in HDL. Table 1 Concentration (mmol/l) of cholesterol in plasma and in HDL (HDL-CE) in the dog Dogs Plasma
kLCAT
k01
k01V
0.130 F 0.004 0.110 F 0.007 0.100 F 0.01 0.150 F 0.012 0.180 F 0.002 0.130 0.032
0.015 F 0.003 0.021 F 0.004 0.027 F 0.004 0.044 F 0.007 0.014 F 0.004 0.024 0.012
0.055 F 0.002 0.039 F 0.002 0.020 F 0.001 0.029 F 0.005 0.045 F 0.002 0.038 0.014
TC UC CE HDLCE 1 2 3 4 5 Mean S.D.
4.4 4.0 4.8 3.6 3.2 4.0 1.0
1.1 1.0 1.1 0.8 0.6 0.9 0.2
3.3 3.1 3.7 2.8 2.6 3.1 0.4
2.1 1.8 2.3 1.6 1.7 1.9 0.3
Kinetic parameters (h 1, F S.D. obtained during fitting) identified using models of Fig. 1: kLCAT, cholesterol esterification by LCAT, k01, HDL particle uptake, k01V, HDL cholesteryl ester selective uptake. Total cholesterol (TC), unesterified cholesterol (UC), cholesteryl ester (CE).
Fig. 2. Semilogarithmic representation of time course of enrichments during perfusion of tracers for Dog 2: in apolipoprotein AI (AI-HDL) (curve A), in cholesteryl ester (CE-HDL) (curve B). Points represent experimental data and curves represent the best fit from the model in Fig. 1. Dashed curves represent plasma leucine (A) and unesterified cholesterol (UC) enrichment (B) used as forcing function.
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sion and decreased after interruption of the perfusion. The HDL-CE curve was regularly increasing during perfusion. This increase was maintained at the end of perfusion, but at a lower rate. Kinetic data for CE and apoAI in HDL are reported in Table 1. Methods provided identified values F S.D. as obtained by iterative least squares fitting for individual kinetic parameters. For each dog, fitting provided the parameter values with a S.D. smaller than 25% for all of them. The turnover rate was 3 times higher ( p < 0.05) for CE (k01 + k01V equals to 0.062 F 0.010 h 1) compared to apoAI (k01 equals to 0.024 F 0.012 h 1). The rate of selective CE uptake was 0.038 F 0.014 h 1, i.e. about 60% of total turnover. The measured esterification rate (kLCAT) was 0.13 F 0.032 h 1.
4. Discussion In our study, dogs were normocholesterolemic (Watson, 1996). As previously described (Maldonado et al., 2001), HDL are the main carrier of cholesterol in plasma (76%). This situation had already been reported in rats (Ouguerram et al., 1992), in contrast to that observed in humans, where plasma cholesterol is mainly carried by LDL (Ouguerram et al., 2002). In dogs, it was shown, in vitro, that the cholesteryl ester transfer protein activity is very small (Ha and Barter, 1982; Guyard-Dangremont et al., 1998; Tsutsumi et al., 2001). After apoB100 and cholesteryl ester labeling by stable isotopes, enrichment of both these molecules in VLDL and LDL allows simple precursor product kinetics showing that their fate is linked and then that CETP activity in vivo in dogs is negligible (data not shown). These data argue that the dog constitutes a model to study selective uptake with a simple protocol including labeling of both HDL apolipoprotein AI and cholesteryl ester. The present study is the first to measure in vivo processes involved in reverse cholesterol transport in dogs by studying the HDL cholesterol ester uptake. During tracer administration, rapid equilibration between lipoprotein fractions was obtained for unesterified cholesterol enrichments, as already reported in other species and by using various ways of labeling (Magot et al., 1985; Ouguerram et al., 2002). This observation demonstrates again the rapidity of unesterified cholesterol exchanges between lipoproteins and argues for the use of plasma unesterified cholesterol as a single compartment. The model used in this study is consistent as attested by the run-test and the low coefficient of variation (S.D. < 25%) for all the individual parameters obtained during the fitting. Moreover, we tested the hypothesis of more complex models including either heterogeneity into HDL or exchange of plasma HDL with a non-plasma pool (Blum et al., 1977) and no significant improvement was found. The esterification rate was 0.130 F 0.032 h 1 (158 + 37 Amol/l/h) and was higher compared to in vitro reported
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LCAT activity measurement (0.072 – 0.076 h 1) in the same species (Lacko et al., 1974; Stokke, 1974; Blomhof et al., 1978; Julien et al., 1988). This difference has already been observed (Magot et al., 1987) in rats for LCAT activity between in vitro (Dobiasova, 1983) and in vivo methods (Magot et al., 1987). Our results can be compared with in vivo data obtained in similar experimental conditions in other species. In humans, the esterification rate in vivo was estimated at 0.033 – 0.049 h 1(30 – 83 Amol/l/h; Monroe et al., 1983; Ouguerram et al., 2002). In vivo, experiments in rats gave higher values, 0.44 h 1, i.e. 300 Amol/l/h (Magot et al., 1987). Our data suggest that in vivo LCAT activity is 2.5 – 4 times higher in dogs compared to humans but 3 times lower compared to rats. The role of this enzyme in reverse cholesterol transport has been previously highlighted (Dobiasova and Frohlich, 1999; Santamarina-Fojo et al., 2000), underlining the major role of LCAT in RCT. The LCAT activity measured in this study suggests a higher level of reverse cholesterol transport in dogs than in humans. Kinetic analysis allows in vivo measurement of the rate of total HDL-CE turnover (0.062 F 0.010 h 1), HDL apoAI-mediated CE turnover (0.024 F 0.012 h 1) and CE selective uptake (0.038 F 0.014 h 1). Our results show that in dogs, the HDL-CE are mainly removed from the plasma through selective uptake (60% of the total). Although our dogs presented a wide range in body mass, no relation was detectable between mass and any measured parameter in our study (results not shown) including for apoAI uptake that showed a great variability in our dogs. Kinetic studies using non-hydrolyzable tracers in the rat have shown differential tissue fate for the two moieties: towards liver mainly through HDL-CE uptake and towards extrahepatic tissues mainly through apoAI uptake (Glass et al., 1983; Ouguerram et al., 1996). Using non-transferable markers, this last process has been shown to be performed by HDL particle uptake (Ponsin et al., 1993; Khoo et al., 1995), except for the kidneys whose apoAI represents only a low part of total apoAI uptake (Glass et al., 1983; Ouguerram et al., 1996). Moreover, it has been shown in hamsters, species with low CETP activity, that HDL-CE selective Table 2 Comparison of HDL-CE turnover data (in h 1, and % of total turnover rate) from dog, rat, and human obtained in vivo in our laboratory with similar kinetic methods Species
HDL-AI mediated CE turnover
HDL-CE selective uptake
CETP mediated transfer
Doga Ratb Humanc Miced Rabbite
0.024 0.066 0.008 0.06 0.038
0.038 0.022 0.013 0.023 0.089
– – 0.093 (82%) – 0.25 (66%)
a
(39%) (75%) (7%) (72%) (10%)
Present study. Ouguerram et al. (1996). c Ouguerram et al. (2002). d Khoo et al. (1995). e Goldberg et al. (1991). b
(61%) (25%) (11%) (28%) (24%)
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uptake by the liver was equal to cholesterol acquisition by all extrahepatic cells (through synthesis, LDL and HDL uptake) suggesting that HDL cholesteryl ester uptake by the liver accurately reflects the rate of reverse cholesterol transport (Woolet and Spady, 1997). In the same way, it has been shown that selective uptake of cholesteryl esters from HDL by rat liver is efficiently coupled to bile acid synthesis (Pieters et al., 1991). Table 2 shows a comparison between the data we have obtained in dogs in the current study and previously published data in rats and humans from our laboratory obtained by similar in vivo methods. The only difference concerns the kind of labeling, i.e. radioisotopes for studies in rats (Ouguerram et al., 1996), and stable isotopes in humans (Ouguerram et al., 2002) and in the present study. Our data may also be compared with literature data obtained by similar in vivo methods in rats (Rinninger and Pittman, 1987), mice (Khoo et al., 1995) and rabbits (Goldberg et al., 1991). Comparison of apoAI turnover between species shows that the intensity of this process in dogs is close to the rabbit (0.038 h 1; Goldberg et al., 1991) and intermediate between humans (0.0083 h 1; Ouguerram et al., 2002) and rats (0.066 –0.154 h 1; Ouguerram et al., 1996; Rinninger and Pittman, 1987) or mice (0.06 h 1; Khoo et al., 1995). Comparison between species of selective CE uptake shows that this pathway is high in dogs compared to the other species (1.5 times higher than rats Ouguerram et al., 1996; Rinninger and Pittman, 1987) and mice (Khoo et al., 1995), 3 times higher than humans (Ouguerram et al., 2002), but 2 times lower than rabbits (Goldberg et al., 1991). Then in dogs, species devoid in CETP activity, the main process involved in HDL-CE removal from plasma is HDLCE selective uptake (60% of HDL-CE turnover). In mice and rats, a species also devoid in CETP activity, the main process is HDL particle uptake (75 – 85% of HDL-CE turnover for rats and 70% for mice). In humans and rabbits, the main process is CETP mediated CE transfers toward lipoproteins of lower density (80% of the total for humans and 65% for rabbit). In agreement with low atherosclerosis risk observed in dogs compared to humans (Khoo et al., 1995), our results suggest that the dog is a species presenting an active RCT performed by a high LCAT activity followed by an efficient selective HDL-CE uptake independent of apoAI catabolism. In dogs, HDL-CE are mainly eliminated from the plasma by selective uptake (near 2/3 of the total), in sharp contrast to the other species lacking CETP activity such as rats. This uptake could be performed by a process close to SR-BI receptor activity reported in the rat. Indeed, through this study, among species used to analyze cholesterol metabolism, the dog appears to be the animal in whom HDL-CE selective uptake represents the largest part of HDL-CE turnover. Further studies are needed to explore mechanisms involved in HDL cholesterol ester selective uptake in dogs, particularly SR-BI receptor pathway. Then the model and protocol presented here could be useful to study modulation
of HDL selective uptake by various nutritional and pharmacological factors.
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K. Ouguerram et al. / Comparative Biochemistry and Physiology, Part B 138 (2004) 339–345 Ji, Y., Wang, N., Ramakrishnan, R., Sehayek, E., Huszar, D., Breslow, J.L., Tall, A.R., 1999. Hepatic scavenger receptor BI promotes rapid clearance of high density lipoprotein free cholesterol and its transport into bile. J. Biol. Chem. 274, 33398 – 33402. Julien, P., Fong, B., Angel, A., 1988. Composition, morphology and distribution of high density lipoproteins in plasma and peripheral lymph: effect of feeding cholesterol and saturated fat. Biochim. Biophys. Acta 960, 275 – 285. Khoo, J.C., Pittman, R.C., Rubin, E.M., 1995. Selective uptake of HDL cholesteryl esters is active in transgenic mice expressing human apolipoprotein A-I. J. Lipid Res. 36, 593 – 600. Lacko, A.G., Rutenberg, H.L., Soloff, L.A., 1974. Serum cholesterol esterification in species resistant and susceptible to atherosclerosis. Atherosclerosis 19, 297 – 305. Lawn, R.M., Wade, D.P., Garvin, M.R., Wang, X., Schwartz, K., Porter, J.G., Seilhamer, J.J., Vaughan, A.M., Oram, J.F., 1999. The Tangier disease gene product ABC1 controls the cellular apolipoprotein-mediated lipid removal pathway. J. Clin. Invest. 104, 25 – 31. Magot, T., Champarnaud, G., Anfreville, R., Lutton, C., Chevallier, F., 1985. Origin and fate of cholesterol in rat plasma lipoproteins in vivo: I. Qualitative analysis. Ann. Nutr. Metab. 29, 160 – 174. Magot, T., Frein, Y., Champarnaud, G., Chervy, A., Lutton, C., 1987. Origin and fate of rat plasma cholesterol in vivo. Modelling of cholesterol movements between plasma and organs. Biochim. Biophys. Acta 921, 587 – 594. Maldonado, E.N., Romero, J.R., Ochoa, B., Aveldano, M.I., 2001. Lipid and fatty acid composition of canine lipoproteins. Comp. Biochem. Physiol., B 128, 719 – 729. Mindham, M.A., Mayes, P.A., 1992. A simple and rapid method for preparation of apolipoproteins for electrophoresis. J. Lipid Res. 33, 1084 – 1088. Monroe, P., Vlahcevic, Z.R., Swell, L., 1983. In vivo evaluation of lipoprotein cholesterol ester metabolism in patient with liver disease. Gastroenterology 85, 820 – 829. Okamoto, H., Yonemori, F., Wakitani, K., Minowa, T., Maeda, K., Shinkai, H., 2000. A cholesteryl ester transfer protein inhibitor attenuates atherosclerosis in rabbits. Nature 406, 203 – 207. Oram, J.F., Vaughan, A.M., 2000. ABCA-1 mediated transport of cellular cholesterol and phospholipids to HDL-apolipoproteins. Curr. Opin. Lipidol. 11, 253 – 260.
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Ouguerram, K., Magot, T., Lutton, C., 1996. Metabolism of plasma lipoproteins in the genetically hypercholesterolemic rat (Rico). Metabolism 45, 4 – 11. Ouguerram, K., Krempf, M., Maugeais, C., Maugere, P., Darmaun, D., Magot, T., 2002. A new labeling approach using stable isotopes to study in vivo plasma cholesterol metabolism in humans. Metabolism 51, 5 – 11. Pieters, M.N., Schouten, D., Bakkeren, H.F., Esbach, B., Brouwer, A., Knook, D.L., van Berkel, T.J., 1991. Selective uptake of cholesteryl esters from apolipoprotein-E-free high-density lipoproteins by rat parenchymal cells in vivo is efficiently coupled to bile acid synthesis. Biochem. J. 280, 359 – 365. Ponsin, G., Pulcini, T., Sparrow, J.T., Gotto Jr., A.M., Pownall, H.J., 1993. High density lipoprotein interconversions in rat and man as assessed with a novel nontransferable apolipopeptide. J. Biol. Chem. 268, 3114 – 3119. Rigotti, A., Trigatti, B., Babitt, J., Penman, M., Xu, S., Krieger, M., 1997. Scavenger receptor BI—a cell surface receptor for high density lipoprotein. Curr. Opin. Lipidol. 8, 181 – 188. Rinninger, F., Pittman, R.C., 1987. Regulation of the selective uptake of high density lipoprotein associated cholesteryl esters. J. Lipid Res. 28, 1313 – 1325. Santamarina-Fojo, S., Lambert, G., Hoeg, J.M., Brewer Jr., H.B., 2000. Lecithin-cholesterol acyltransferase: role in lipoprotein metabolism, reverse cholesterol transport and atherosclerosis. Curr. Opin. Lipidol. 11, 267 – 275. Silver, D.L., Tall, A.R., 2001. The cellular biology of scavenger receptor class B type I. Curr. Opin. Lipidol. 12, 497 – 504. Sokal, R.R., Rohlf, F.J., 1995. Biometry, 3rd ed. Freeman, New York, pp. 797 – 803. Stokke, K.J., 1974. Cholesteryl ester metabolism in liver and blood plasma of various animal species. Atherosclerosis 19, 393 – 406. Tsutsumi, K., Hagi, A., Inoue, Y., 2001. The relationship between plasma high density lipoprotein cholesterol levels and cholesterol ester transfer protein activity in six species of healthy experimental animals. Biol. Pharm. Bull. 24, 579 – 581. Watson, T.D.G., 1996. Lipoprotein metabolism in dogs and cats. Comp. Haematol. Int. 6, 17 – 23. Woolet, L.A., Spady, D.K., 1997. Kinetic parameters for high density lipoprotein apoprotein AI and cholesteryl ester transport in the hamster. J. Clin. Invest. 99, 1704 – 1713.
Selective uptake of high density lipoproteins cholesteryl ester in the dog, a species lacking in cholesteryl ester transfer protein activity An in vivo approach using stable isotopes Khadija Ouguerram a,*, Patrick Nguyen b, Michel Krempf a, Etienne Pouteau b, Francßois Briand a,b, Edwige Bailhache a,b, Thierry Magot a a
Centre de Recherche en Nutrition Humaine, INSERM U539, CHU Nantes, 1 place Alexis Ricordeau, 44093 Nantes Cedex 01, France b USC INRA de Nutrition et Endocrinologie, Ecole nationale ve´te´rinaire de Nantes, France Received 16 December 2003; received in revised form 9 April 2004; accepted 13 April 2004
Abstract Amongst the processes involved in the reverse cholesterol transport (RCT) from organs to liver, including high density lipoproteinsapolipoprotein AI (HDL-apoAI) dependent tissue uptake and cholesteryl ester transfer protein (CETP)-mediated transfers, the selective uptake of cholesteryl ester (CE) is of increasing interest through its antiatherogenic role. The purpose of this report is to develop a simple protocol allowing study of this process in an animal model with easier quantification of CE selective uptake. The dog was chosen essentially because this animal has a low CETP activity and an appropriate size to conduce a kinetic study. Tracer kinetics were performed to estimate in vivo the contributions of the pathways involved in HDL-CE turnover in dogs. Stable isotopes, 13C-acetate and D3-leucine as labeled precursors of CE and apoAI, were infused to fasting dogs. Isotopic enrichments were monitored in plasma unesterified cholesterol and in HDL-CE and apoAI by mass spectrometry. Kinetics were analyzed using compartmental modeling. Results concerned the measurement of the activity of cholesterol esterification (0.13 F 0.032 h 1), rate of HDL-apoAI catabolism (0.024 F 0.012 h 1), HDL-CE turnover (0.062 F 0.010 h 1) and CE selective uptake (0.038 F 0.014 h 1). Our results show that CE in dogs is mainly eliminated by selective uptake of HDL-CE (60% of HDL-CE turnover), unlike in other species studied by similar methods in our laboratory. This study shows that among species used to analyze cholesterol metabolism, the dog appears to be the animal in whom HDL-CE selective uptake represents the largest part of HDL-CE turnover. D 2004 Elsevier Inc. All rights reserved. Keywords: Cholesterol; Reverse cholesterol transport; Lecithin-cholesterol acyltransferase; Kinetic analysis; Dog
1. Introduction Cholesterol transport by plasma high density lipoproteins (HDL) plays a key role in reverse cholesterol transport (RCT). Cholesterol HDL metabolism is complex, involving multiple metabolic pathways (Fielding and Fielding, 1995). Apo A-I is mainly secreted by the liver and forms preß-HDL with phospholipids (Barter et al., 2003; Lawn et al., 1999). Abbreviations: HDL, high density lipoproteins; VLDL, very low density lipoproteins; LDL, low density lipoproteins; apoAI, apolipoprotein AI; RCT, reverse cholesterol transport; LCAT, lecithin cholesterol acyl transferase; CETP, cholesteryl ester transfer protein; SR-BI, scavenger receptor class B type I; CE, cholesteryl ester. * Corresponding author. Tel.: +33-240-087535; fax: +33-240-087544. E-mail address: [email protected] (K. Ouguerram). 1096-4959/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2004.04.011
This form of nascent HDL removes cholesterol from peripheral tissues through a recently identified ABCA1 mediated process (Oram and Vaughan, 2000). ATP Binding Cassette transporter A1 (ABCA1) is an essential transporter in the RCT because its deficiency results in very low or no plasma HDL (Attie et al., 2001). Deficiency of ABCA1 gives rise to Tangier disease, where cholesteryl esters accumulate in macrophage-derived foam-cells. This reduction in the rate of RCT increases the risk for developing atherosclerosis (Hobbs and Rader, 1999). The ABCA1 process produces spherical HDL particles through the activity of plasma lecithin cholesterol acyl transferase (LCAT). LCAT converts cholesterol to cholesteryl esters on the surface of HDL particles (Glomset et al., 1966). Thus, this enzyme promotes cholesterol efflux from cell to HDL by an accumulation of the cholesteryl esters in the core of the
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lipoprotein (Santamarina-Fojo et al., 2000). Neo-formed HDL-cholesteryl ester (HDL-CE) can have different metabolic fates including (i) apolipoprotein AI (apoAI)-dependent tissue uptake, (ii) cholesteryl ester transfer protein (CETP)-mediated transfers to lower density lipoproteins, and (iii) direct delivery to liver by the selective HDL-CE uptake pathway mediated by SR-BI receptor. Kinetic studies have shown in the rat, a species with a low CETP activity, that the fates of CE and apoAI were uncoupled (Glass et al., 1983). In this species a receptor has been found (scavenger receptor class B type I, SR-BI) allowing specific uptake of HDL-CE by the liver (Rigotti et al., 1997), and this pathway has been the purpose of numerous studies (Silver and Tall, 2001). SR-BI has potential anti-atherogenic activities because this HDL receptor promotes hepatic uptake of HDL cholesterol. This cholesterol is then directly secreted into bile (Ji et al., 1999). In species with CETP activity, such as in humans, the process involving SR-BI could coexist with the transfer of CE in the plasma from HDL to very low density lipoproteins (VLDL) and low density lipoproteins (LDL). Therefore, in these species with CETP activity such as rabbits and humans, in vivo quantitative analysis of both processes is difficult as it requires a complex multicompartmental approach (Goldberg et al., 1991; Ouguerram et al., 2002). However, the physiological difference between these two pathways is important. Indeed, it is reasonable to assume that the CETPmediated transfer of CE from HDL to lower density lipoproteins is an atherogenic pathway (Okamoto et al., 2000) because it provides cholesterol to LDL. On the contrary, CE selective uptake from HDL does not enrich the atherogenic lipoproteins in CE and should be anti-atherogenic. Thus, it would be of crucial interest to possess an animal model exhibiting CE selective uptake as the main process involved in HDL-CE turnover. The aim of this study was to test the dog model in order to develop a protocol allowing a quantification of selective uptake of HDL-CE. The dog model was selected because this animal is lacking CE transfer activity (Ha and Barter, 1982; Guyard-Dangremont et al., 1998; Tsutsumi et al., 2001) and because large animals, such as dogs, make it possible to obtain high amounts of plasma in order to determine as much information as humans.
2. Materials and methods 2.1. Animals Five adult dogs (beagles, 11– 23 kg) were supplied by the National Veterinary School of Nantes after approval of the protocol by the animal ethic committee. Dogs were fed under standard laboratory conditions: they consumed in a single meal a commercial dry dog food (25%, 17% and 58% of calories, respectively, as protein, fat and carbohydrates, 3730 kcal metabolizable energy/kg, on a dry mass basis)
and were fed according to the NRC recommendation (132 kcal metabolizable energy/kg BW0.75). Two catheters (Vasocan Braunu¨le, 20G 11/4U, Melsungen, Germany) were inserted into the cephalic vein of both forelimbs of each dog: one for double infusion of tracers ([1-13C]acetate and [D3-leucine] and the other for collection of venous blood. 2.2. Experimental design Stable isotope tracers were weighed on a high precision scale and dissolved in known volumes of sterile 0.9% NaCl. Solutions were prepared no earlier than 24 h before use, sterilized by passing through a 0.22 Am filter, stored in sterile sealed containers, and kept at 4 jC until required. The study was then conducted after an overnight fast. Each animal received intravenously a prime of 10 Amol kg 1 of [5,5,5-2H3]leucine (as endogenous marker of apoAI, 99.8 at.%; Mass Trace, Woburn, MA, USA), and 2 Amol kg 1 for [1,213C]acetate as endogenous marker of cholesterol, 99% 13C enrichment, Tracer Technologies, Somerville, MA, USA) immediately followed by a constant tracer infusion (10 Amol kg 1 h 1 for [5,5,5-2H3]leucine and 2 Amol kg 1 min 1 for [1,213C]acetate) for 8 h as previously published (Frenais et al., 1997; Ouguerram et al., 2002). Blood samples were collected at 10, 30, 60 min and hourly until 10 h during the perfusion and 25 h after the beginning of the perfusion. The samples were collected in tubes containing EDTA (0.2 mmol/l final concentration) and dithiobisnitrobenzoic acid as inhibitor of LCAT reaction (0.2 mmol/l final concentration). Plasma was immediately separated by centrifugation at 4 jC and stored frozen at 20 jC for later determination of cholesterol concentration and isotopic enrichments of cholesterol and apoAI. HDL were isolated by ultracentrifugation between 1.063 and 1.21 g/ml as previously described (Frenais et al., 1997). For measurement of unesterified enrichment in lipoproteins of density lower than 1.063, two fractions were isolated (d < 1.006 g/ ml and 1.006 < d < 1.063 g/ml). 2.3. Analytical methods Measurement of apoAI enrichment was performed as previously described (Frenais et al., 1997). Briefly, HDL were delipidated with diethyl ether and apolipoproteins concentrated with trichloroacetic acid (4.9 mM) and desoxycholic acid (3.6 mM). ApoAI was isolated from other apolipoproteins by PAGE-SDS using a 4.5– 10% discontinuous gradient (Mindham and Mayes, 1992). Apolipoproteins were identified by comparing migration distances with known molecular weight standards (cross-linked phosphorylase b markers, Sigma, St. Louis, MO, and electrophoresis calibration kit, Pharmacia LKB, Biotechnology, Piscataway, NJ, USA). Apolipoprotein bands were excised from polyacrylamide gels and dried in vacuum for 1 to 2 h (RC 10-10 Jouan, Saint Herblain, France). The desiccated gel slices were hydrolyzed with 1 ml 4 mol/l HCl (Sigma, St. Quentin
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Fallavier, France) at 110 jC for 24 h. Hydrolysates were then evaporated to dryness and the amino acids were purified by cation exchange chromatography using a Temex 50W-X8 resin (Bio-Rad, Richmond, CA, USA). Amino acids and plasma leucine were esterified with propanol/ acetyl chloride, and further derivatized using heptafluorobutyric anhydride (Fluka Chemie, Buchs, Switzerland) prior to analysis. Samples were analyzed for isotopic enrichment of leucine using gas chromatography coupled-mass spectrometry (GC-MS) as previously described (Frenais et al., 1997). Briefly, chromatographic separations were carried out on a 30 m 0.25 mm i.d. DB-5 capillary column (J&W Scientific, Rancho Cordova, CA, USA). The column temperature was as follows: initial temperature was held at 80 jC, then increased at 10 jC/min to a final temperature of 180 jC. Electron-impact gas chromatography-mass spectrometry was performed on a 5890 gas chromatograph connected with a 5971A quadrupole mass spectrometer (Hewlett-Packard, Palo Alto, CA, USA). The isotopic ratio was determined by selected ion-monitoring at m/z 282 and 285. Results were expressed as tracer/tracee ratio for modeling analysis (Cobelli et al., 1992). Analysis of cholesterol was performed as previously described (Ouguerram et al., 2002). Unesterified cholesterol and CE were isolated from lipoproteins as previously reported (Ouguerram et al., 1996). Briefly, lipid extraction was performed with chloroform – methanol (2:1, v/v) according to Folch et al. (1957). Unesterified cholesterol and CE were separated by chromatography on silicic acid (Hirsh and Ahrens, 1958), using microcolumns (Sep Pack cartridges, Waters, Milford, MA, USA). The cholesterol samples were derivatized with a mixture of acetic anhydride (500 Al) –pyridine (100 Al) (Aldrich, France). The samples were heated to 90 jC for 10 min. After cooling to room temperature, the derivatizing reagents were evaporated under nitrogen, and the residue was dissolved in hexane. Due to low 13C-enrichments in cholesterol and to allow us a higher sensitivity we have used gas chromatography – combustion-isotope ratio mass spectrometry (GC-C-IRMS) with a Finnigan Mat Delta S isotope ratio mass spectrometer coupled to a HP 5890 series II gas chromatograph (HewlettPackard) with a DB-1 capillary column (30 m 0.32 mm i.d., 0.25 Am film thickness; J&W Scientific). The temperature of the injection port was 280 jC and the oven temperature was initially set at 70 jC, then increased at 50 jC/min up to 280 jC and was held for 15 min at 280 jC. The samples were injected in splitless mode. The cholesterol peak eluted from the GC column was introduced on line into a combustion furnace which operated at 940 jC. The furnace is a non-porous alumina tube packed with a wire of copper, nickel and platinum. Combustion water was removed by passing the effluent through a water-permeable Nafion tube. The CO2 was driven in the electron impact ion source. The same samples were analyzed in triplicate by GC/C/IRMS and measurements were highly reproducible ( < 0.5%).
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Isotopic enrichment was expressed relative to pulse peaks of CO2 reference, calibrated against the international standard (Pee Dee Belemnite, PDB). Isotopic calculations: The isotopic abundance in 13C in cholesterol is given by the GC-C-IRMS as dx versus the international standard PDB as: d13 C ðxÞ ¼ ððRsample Rstd Þ=Rstd Þ 1000; where R is the 13C/12C ratio in the sample or the international standard PDB (Rstd = 0.0112372). Therefore, Rsample = Rstd*[1+(0.001*d13C)]. The observed fractional abundance F (atom%) was calculated as: F ¼ 100Rstd *½ð0:001*d13 Csample Þ þ 1 The isotopic enrichment E (atom% excess) was calculated as: E ¼ Fsamplej Fbasal value where Fsamplej and Fbasal value are the observed fractional abundance in the sample at time j and at baseline, respectively. E ¼ 100Rstd *ð0:001*d13 Csample þ 1Þ APE ¼ atom% sample atom% basal value ðAtom Percent ExcessÞ 2.4. Concentration measurement Unesterified and esterified cholesterol concentrations in samples were measured using commercially available enzymatic kits (Cholesterol RTU, BioMe´rieux, Lyon, France and ‘‘choleste´rol libre enzymatique’’ color, Biotrol Diagnostic, Lognes, France). 2.5. Modeling methods Data were treated by compartmental analysis using computer software (Barrett et al., 1998) for simulation, analysis and modeling (SAAMII v 1.01, Resource Facility for Kinetic Analysis, Department of Bioengineering, SAAM Institute, Seattle, WA, USA). HDL-apoAI model was a one-compartment model (Fig. 1A). Labeling input was done after a delay from plasma free leucine tracer-to-tracee ratio (as forcing function). In this model, k01 represented the parameter of HDL particle uptake by tissues including liver. HDL-CE model was also a one-compartment model (Fig. 1B). Labeling input was done from unesterified cholesterol enrichments (as forcing function) through cholesterol esterification kLCAT. In this model k01 and k01V represented the two output parameters, uptake of HDL particle and CE selective uptake by tissues including liver, respectively. In these two models, k01 described the simultaneous uptake of both apoAI and CE
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Fig. 1. Study model of apoAI-HDL (A) and cholesteryl ester-HDL (B) turnover.
through HDL particle uptake. Parameters were simultaneously identified from data of HDL-apoAI and CE. The rate constant of cholesterol esterification (kLCAT) was calculated as the ratio between the absolute rate of cholesterol ester input in HDL cholesteryl ester identified by modeling and plasma unesterified cholesterol concentration. 2.6. Statistical analysis Results are reported as mean F S.D. The Wilcoxon unpaired test (two tail p-value) was used to compare turnover rate of HDL-apoAI and HDL-CE. Analysis was performed using the Instat statistical Software package (GraphPad, San Diego, CA, USA). A two-tail p-value < 0.05 was considered as statistically significant. The goodness of fit was assessed using the run test (Sokal and Rohlf, 1995). Different models were compared using the AIC test (Akaike, 1974) and the F-test (Boxenbaum et al., 1974).
Fig. 2A shows time courses of enrichment in HDL-apoAI and plasma leucine. During perfusion, the tracer/tracee ratio curve was approximately constant for plasma leucine and then fell after the end of the perfusion. ApoAI curve regularly increased during perfusion and then remained constant after the end of perfusion. The time course of 13 C enrichment in plasma unesterified cholesterol was virtually identical in lipoproteins of density lower than 1.006, of density 1.006 – 1.063 and in HDL during the experiment (results not shown). Therefore, the results were only expressed as plasma unesterified cholesterol (Fig. 2B) and used as forcing function to take into account the time course of labeling input into HDL-CE. Labeling appeared first in unesterified cholesterol, increased during the perfu-
3. Results Amounts of unesterified cholesterol and CE in plasma and HDL fractions are provided in Table 1. In plasma, cholesterol was mainly present in esterified form (77%). The major part of cholesterol (76%) was recovered in HDL. Table 1 Concentration (mmol/l) of cholesterol in plasma and in HDL (HDL-CE) in the dog Dogs Plasma
kLCAT
k01
k01V
0.130 F 0.004 0.110 F 0.007 0.100 F 0.01 0.150 F 0.012 0.180 F 0.002 0.130 0.032
0.015 F 0.003 0.021 F 0.004 0.027 F 0.004 0.044 F 0.007 0.014 F 0.004 0.024 0.012
0.055 F 0.002 0.039 F 0.002 0.020 F 0.001 0.029 F 0.005 0.045 F 0.002 0.038 0.014
TC UC CE HDLCE 1 2 3 4 5 Mean S.D.
4.4 4.0 4.8 3.6 3.2 4.0 1.0
1.1 1.0 1.1 0.8 0.6 0.9 0.2
3.3 3.1 3.7 2.8 2.6 3.1 0.4
2.1 1.8 2.3 1.6 1.7 1.9 0.3
Kinetic parameters (h 1, F S.D. obtained during fitting) identified using models of Fig. 1: kLCAT, cholesterol esterification by LCAT, k01, HDL particle uptake, k01V, HDL cholesteryl ester selective uptake. Total cholesterol (TC), unesterified cholesterol (UC), cholesteryl ester (CE).
Fig. 2. Semilogarithmic representation of time course of enrichments during perfusion of tracers for Dog 2: in apolipoprotein AI (AI-HDL) (curve A), in cholesteryl ester (CE-HDL) (curve B). Points represent experimental data and curves represent the best fit from the model in Fig. 1. Dashed curves represent plasma leucine (A) and unesterified cholesterol (UC) enrichment (B) used as forcing function.
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sion and decreased after interruption of the perfusion. The HDL-CE curve was regularly increasing during perfusion. This increase was maintained at the end of perfusion, but at a lower rate. Kinetic data for CE and apoAI in HDL are reported in Table 1. Methods provided identified values F S.D. as obtained by iterative least squares fitting for individual kinetic parameters. For each dog, fitting provided the parameter values with a S.D. smaller than 25% for all of them. The turnover rate was 3 times higher ( p < 0.05) for CE (k01 + k01V equals to 0.062 F 0.010 h 1) compared to apoAI (k01 equals to 0.024 F 0.012 h 1). The rate of selective CE uptake was 0.038 F 0.014 h 1, i.e. about 60% of total turnover. The measured esterification rate (kLCAT) was 0.13 F 0.032 h 1.
4. Discussion In our study, dogs were normocholesterolemic (Watson, 1996). As previously described (Maldonado et al., 2001), HDL are the main carrier of cholesterol in plasma (76%). This situation had already been reported in rats (Ouguerram et al., 1992), in contrast to that observed in humans, where plasma cholesterol is mainly carried by LDL (Ouguerram et al., 2002). In dogs, it was shown, in vitro, that the cholesteryl ester transfer protein activity is very small (Ha and Barter, 1982; Guyard-Dangremont et al., 1998; Tsutsumi et al., 2001). After apoB100 and cholesteryl ester labeling by stable isotopes, enrichment of both these molecules in VLDL and LDL allows simple precursor product kinetics showing that their fate is linked and then that CETP activity in vivo in dogs is negligible (data not shown). These data argue that the dog constitutes a model to study selective uptake with a simple protocol including labeling of both HDL apolipoprotein AI and cholesteryl ester. The present study is the first to measure in vivo processes involved in reverse cholesterol transport in dogs by studying the HDL cholesterol ester uptake. During tracer administration, rapid equilibration between lipoprotein fractions was obtained for unesterified cholesterol enrichments, as already reported in other species and by using various ways of labeling (Magot et al., 1985; Ouguerram et al., 2002). This observation demonstrates again the rapidity of unesterified cholesterol exchanges between lipoproteins and argues for the use of plasma unesterified cholesterol as a single compartment. The model used in this study is consistent as attested by the run-test and the low coefficient of variation (S.D. < 25%) for all the individual parameters obtained during the fitting. Moreover, we tested the hypothesis of more complex models including either heterogeneity into HDL or exchange of plasma HDL with a non-plasma pool (Blum et al., 1977) and no significant improvement was found. The esterification rate was 0.130 F 0.032 h 1 (158 + 37 Amol/l/h) and was higher compared to in vitro reported
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LCAT activity measurement (0.072 – 0.076 h 1) in the same species (Lacko et al., 1974; Stokke, 1974; Blomhof et al., 1978; Julien et al., 1988). This difference has already been observed (Magot et al., 1987) in rats for LCAT activity between in vitro (Dobiasova, 1983) and in vivo methods (Magot et al., 1987). Our results can be compared with in vivo data obtained in similar experimental conditions in other species. In humans, the esterification rate in vivo was estimated at 0.033 – 0.049 h 1(30 – 83 Amol/l/h; Monroe et al., 1983; Ouguerram et al., 2002). In vivo, experiments in rats gave higher values, 0.44 h 1, i.e. 300 Amol/l/h (Magot et al., 1987). Our data suggest that in vivo LCAT activity is 2.5 – 4 times higher in dogs compared to humans but 3 times lower compared to rats. The role of this enzyme in reverse cholesterol transport has been previously highlighted (Dobiasova and Frohlich, 1999; Santamarina-Fojo et al., 2000), underlining the major role of LCAT in RCT. The LCAT activity measured in this study suggests a higher level of reverse cholesterol transport in dogs than in humans. Kinetic analysis allows in vivo measurement of the rate of total HDL-CE turnover (0.062 F 0.010 h 1), HDL apoAI-mediated CE turnover (0.024 F 0.012 h 1) and CE selective uptake (0.038 F 0.014 h 1). Our results show that in dogs, the HDL-CE are mainly removed from the plasma through selective uptake (60% of the total). Although our dogs presented a wide range in body mass, no relation was detectable between mass and any measured parameter in our study (results not shown) including for apoAI uptake that showed a great variability in our dogs. Kinetic studies using non-hydrolyzable tracers in the rat have shown differential tissue fate for the two moieties: towards liver mainly through HDL-CE uptake and towards extrahepatic tissues mainly through apoAI uptake (Glass et al., 1983; Ouguerram et al., 1996). Using non-transferable markers, this last process has been shown to be performed by HDL particle uptake (Ponsin et al., 1993; Khoo et al., 1995), except for the kidneys whose apoAI represents only a low part of total apoAI uptake (Glass et al., 1983; Ouguerram et al., 1996). Moreover, it has been shown in hamsters, species with low CETP activity, that HDL-CE selective Table 2 Comparison of HDL-CE turnover data (in h 1, and % of total turnover rate) from dog, rat, and human obtained in vivo in our laboratory with similar kinetic methods Species
HDL-AI mediated CE turnover
HDL-CE selective uptake
CETP mediated transfer
Doga Ratb Humanc Miced Rabbite
0.024 0.066 0.008 0.06 0.038
0.038 0.022 0.013 0.023 0.089
– – 0.093 (82%) – 0.25 (66%)
a
(39%) (75%) (7%) (72%) (10%)
Present study. Ouguerram et al. (1996). c Ouguerram et al. (2002). d Khoo et al. (1995). e Goldberg et al. (1991). b
(61%) (25%) (11%) (28%) (24%)
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uptake by the liver was equal to cholesterol acquisition by all extrahepatic cells (through synthesis, LDL and HDL uptake) suggesting that HDL cholesteryl ester uptake by the liver accurately reflects the rate of reverse cholesterol transport (Woolet and Spady, 1997). In the same way, it has been shown that selective uptake of cholesteryl esters from HDL by rat liver is efficiently coupled to bile acid synthesis (Pieters et al., 1991). Table 2 shows a comparison between the data we have obtained in dogs in the current study and previously published data in rats and humans from our laboratory obtained by similar in vivo methods. The only difference concerns the kind of labeling, i.e. radioisotopes for studies in rats (Ouguerram et al., 1996), and stable isotopes in humans (Ouguerram et al., 2002) and in the present study. Our data may also be compared with literature data obtained by similar in vivo methods in rats (Rinninger and Pittman, 1987), mice (Khoo et al., 1995) and rabbits (Goldberg et al., 1991). Comparison of apoAI turnover between species shows that the intensity of this process in dogs is close to the rabbit (0.038 h 1; Goldberg et al., 1991) and intermediate between humans (0.0083 h 1; Ouguerram et al., 2002) and rats (0.066 –0.154 h 1; Ouguerram et al., 1996; Rinninger and Pittman, 1987) or mice (0.06 h 1; Khoo et al., 1995). Comparison between species of selective CE uptake shows that this pathway is high in dogs compared to the other species (1.5 times higher than rats Ouguerram et al., 1996; Rinninger and Pittman, 1987) and mice (Khoo et al., 1995), 3 times higher than humans (Ouguerram et al., 2002), but 2 times lower than rabbits (Goldberg et al., 1991). Then in dogs, species devoid in CETP activity, the main process involved in HDL-CE removal from plasma is HDLCE selective uptake (60% of HDL-CE turnover). In mice and rats, a species also devoid in CETP activity, the main process is HDL particle uptake (75 – 85% of HDL-CE turnover for rats and 70% for mice). In humans and rabbits, the main process is CETP mediated CE transfers toward lipoproteins of lower density (80% of the total for humans and 65% for rabbit). In agreement with low atherosclerosis risk observed in dogs compared to humans (Khoo et al., 1995), our results suggest that the dog is a species presenting an active RCT performed by a high LCAT activity followed by an efficient selective HDL-CE uptake independent of apoAI catabolism. In dogs, HDL-CE are mainly eliminated from the plasma by selective uptake (near 2/3 of the total), in sharp contrast to the other species lacking CETP activity such as rats. This uptake could be performed by a process close to SR-BI receptor activity reported in the rat. Indeed, through this study, among species used to analyze cholesterol metabolism, the dog appears to be the animal in whom HDL-CE selective uptake represents the largest part of HDL-CE turnover. Further studies are needed to explore mechanisms involved in HDL cholesterol ester selective uptake in dogs, particularly SR-BI receptor pathway. Then the model and protocol presented here could be useful to study modulation
of HDL selective uptake by various nutritional and pharmacological factors.
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