Published online before print October 6, 2005, doi: 10.1161/01.ATV.0000189158.28455.94
© 2005 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
A Novel Inhibitor of Oxidosqualene:Lanosterol Cyclase Inhibits Very Low–Density Lipoprotein Apolipoprotein B100 (ApoB100) Production and Enhances Low-Density Lipoprotein ApoB100 Catabolism Through Marked Reduction in Hepatic Cholesterol Content
From the Robarts Research Institute, Vascular Biology Group (D.E.T., S.M.L., B.G.S., J.Y.E., M.W.H.); Departments of Medicine (D.E.T., J.Y.E., M.W.H.) and Biochemistry (S.M.L., M.W.H.), The University of Western Ontario, London, Canada; the School of Medicine and Pharmacology (P.H.R.B.), University of Western Australia, Perth, West Australia, Australia; and the Pharmaceuticals Division (J.D.A., H.D., O.H.M.), F. Hoffmann-La Roche Ltd, Basel, Switzerland. O.H.M.’s current address: Actelion Pharmaceuticals Ltd, Gewerbestrasse 16, Allschwil, CH, 4123, Switzerland.
Correspondence to Dr Murray W. Huff, Robarts Research Institute, Room 416, 100 Perth Dr, London, Ontario, Canada, N6A 5K8. E-mail mhuff{at}uwo.ca
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Abstract |
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Objective— Inhibition of 2,3-oxidosqualene:lanosterol cyclase (OSC), an enzyme in the cholesterol synthesis pathway, has the unique ability to inhibit cholesterol synthesis while simultaneously enhancing oxysterol synthesis. Our objectives were to determine, in vivo, if a novel OSC inhibitor reduced low-density lipoprotein (LDL) cholesterol and to define the mechanism(s) involved.
Methods and Results— Miniature pigs received the OSC inhibitor RO0717625 or placebo and a diet containing fat (34% of energy) and 400 mg per day of cholesterol. Treatment decreased plasma total cholesterol (–20%) and LDL cholesterol (–29%). Apolipoprotein B (apoB) kinetic parameters were determined. Very low–density lipoprotein (VLDL) apoB pool size decreased 22% because of inhibition of VLDL production (–43%). LDL apoB pool size decreased 22% because of a 1.5-fold increase in fractional catabolic rate (FCR). The increased FCR was associated with a 2-fold increase in hepatic LDL receptor mRNA. Hepatic total and microsomal cholesterol were reduced by 16% and 27%, respectively. Plasma lathosterol concentrations decreased 57%, reflecting inhibition of hepatic cholesterol synthesis. Treatment reduced plasma plant sterols and decreased postprandial cholesterol transport in chylomicrons.
Conclusions— A novel OSC inhibitor, RO0717625, decreased VLDL and LDL apoB100 through decreased VLDL production and enhanced LDL clearance. Thus, OSC represents a potential therapeutic target for dyslipidemia.
In miniature pigs, a novel OSC inhibitor, RO0717625, decreased VLDL and LDL apoB100 through decreased VLDL production and increased LDL receptor–mediated LDL apoB clearance. Cholesterol synthesis and postprandial cholesterol transport in chylomicrons decreased, resulting in a marked reduction in hepatic cholesterol concentration. Thus, OSC is a potential therapeutic target for dyslipidemia.
Key Words: oxidosqualene:lanosterol cyclase • apoB kinetics • LDL receptor • ABCG5 • ABCG8
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Introduction |
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Landmark trials using 3-hydroxy-3-methylglutaryl–coenzyme A (HMG-CoA) reductase inhibitors (statins) revealed reductions in cardiovascular mortality and morbidity associated with low-density lipoprotein (LDL) cholesterol lowering.1 Statins are well tolerated, although reductions in nonsterol intermediates in the cholesterol-synthesis pathway, such as isoprenoids and coenzyme Q, may theoretically be associated with adverse clinical events.2,3 This has stimulated the development of compounds that inhibit cholesterol biosynthesis yet act distal to the synthesis of these nonsterol intermediates. 2,3-oxidosqualene:lanosterol cyclase (OSC; enzyme collection 5.4.99.7, also known as lanosterol synthase), a microsomal enzyme, represents a unique target for cholesterol-lowering drugs.4,5 OSC is downstream of isoprenoid synthesis. Furthermore, compounds that decrease plasma concentrations of atherogenic lipoproteins by more than 1 mechanism are likely to be more efficacious.
OSC catalyzes the highly selective cyclization of 2,3-monoepoxysqualene (MOS) to lanosterol, the first sterol to be formed.4 OSC also catalyzes cyclization of 2,3;22,23-diepoxysqualene (DOS), which itself is derived from MOS, to 24(S),25-epoxylanosterol, the immediate precursor of 24(S),25-epoxycholesterol. Synthesis of 24(S),25-epoxycholesterol is favored over cholesterol synthesis under conditions of partial OSC inhibition, whereas complete OSC inhibition results in decreased synthesis of cholesterol and 24(S),25-epoxycholesterol. Because 24(S),25-epoxycholesterol not only represses HMG-CoA reductase activity, but also enhances its degradation, this theoretically results in the generation of a synergistic, self-limited, negative regulatory loop.4,6–10 Thus, an OSC inhibitor (OSCi) can uniquely stimulate endogenous synthesis of oxysterols while simultaneously inhibiting endogenous cholesterol synthesis. For a diagrammatical representation of this pathway, see Figure I (available online at http://atvb.ahajournals.org).
Increased synthesis of 24(S),25-epoxycholesterol has the potential to block the activation of sterol response element-binding proteins (SREBPs), as has been shown in vitro for SREBP-111 and SREBP-2.12 Decreased SREBP-2 activation of HMG-CoA reductase would increase the cholesterol-lowering potential of OSC inhibition.9 However, decreased SREBP-2 activation of the LDL receptor would have the opposite effect. 24(S),25-epoxycholesterol is a potent activator of the liver X receptor (LXR).13 LXR activation is known to increase transcription of a number of genes important in the regulation of hepatic lipid metabolism, including SREBP-1c, fatty acid synthase (FAS), ATP-binding cassette A1 (ABCA1), ABCG5, ABCG8, and CYP7A1 (but only in rodents). Therefore, an OSCi-induced increase in 24(S),25-epoxycholesterol could, through SREBP-1c and FAS activation, increase the synthesis and accumulation of hepatic triglycerides,14 detracting from the plasma lipid-lowering potential. Conversely, LXR activation of ABCG5 and ABCG8 has been shown in mice to inhibit net cholesterol absorption and increase the transport of plant sterols and cholesterol from the liver into bile, yielding diminished stores of hepatic cholesterol.15 This could potentially enhance reductions in LDL cholesterol through decreased very low–density lipoprotein (VLDL) production or upregulation of hepatic LDL receptors. How these complex regulatory mechanisms interact, in vivo, in response to OSC inhibition is unknown.
Administration of a prototypic OSCi, RO0488071, to hamsters, squirrel monkeys, and Göttingen minipigs decreased LDL cholesterol by 30% to 35%;8 however, the mechanisms have not been completely defined. In hamsters, RO0488071 did not trigger increased activities of HMG-CoA reductase, squalene synthase, or OSC itself, an effect assumed to be secondary to an increase in oxysterol synthesis.8 RO0488071 inhibited triglyceride secretion into plasma by 44%, suggesting inhibition of hepatic VLDL apolipoprotein B (apoB), whereas the fractional catabolic rate (FCR) of iodinated LDL was unaffected, suggesting no change in hepatic LDL receptor expression.8 Although these compounds appear to be effective for lowering serum and LDL cholesterol in small animal models fed diets high in fat and cholesterol, the mechanism whereby these inhibitors modulate the kinetics of apoB metabolism has not been investigated in a large animal model fed a physiologically relevant diet. Furthermore, the impact of OSC inhibition on the expression of genes in the liver and small intestine known to regulate plasma lipoprotein metabolism remains unknown.
In the present study, regulation of apoB metabolism was examined in miniature pigs administered RO0717625, a recently developed OSCi. OSC inhibition decreased LDL cholesterol and apoB through a dual mechanism of action. OSCi treatment inhibited cholesterol synthesis, reduced plasma plant sterols, and decreased postprandial transport of cholesterol in chylomicrons, which were associated with a marked reduction in hepatic cholesterol. Plasma apoB was significantly decreased through inhibition of VLDL apoB production and enhanced LDL receptor–mediated LDL apoB clearance, both of which are attributed to the reduction in hepatic cholesterol.
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Methods |
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Please see the online data supplement (available at http://atvb.ahajournals.org) for materials and methods concerning animals and diets, dose of OSCi, lipoprotein turnover studies, apoB kinetic analysis, tissue lipids and enzyme activities, tissue mRNA abundance, plasma and lipoprotein lipid determinations, and statistical analysis.
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Results |
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The effect of RO0717625 at 3 mg/kg per day on plasma and lipoprotein lipid concentrations after 21 days of treatment is shown in Figure 1. Treatment significantly reduced plasma total cholesterol by 20% (P<0.007) and LDL cholesterol by 29% (P<0.005). Other lipids were unchanged. RO0717625 decreased VLDL apoB by 22% (P<0.03) and LDL apoB by 22% (P<0.002). Plasma lathosterol concentrations were decreased by 57% (P<0.001), reflective of inhibition of hepatic cholesterol synthesis (Figure 2). Plasma plant sterol concentrations were decreased by 37% (P<0.05), which implies a reduction in cholesterol absorption.
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Trideuterated leucine was injected as a bolus after a 22-hour fast into each pig. Kinetic parameters of apoB were determined from the simultaneous analysis of all isotope enrichment data using the model described in Figure IIA (available online at http://atvb.ahajournals.org).16 A fit of the model to the VLDL, intermediate-density lipoprotein (IDL), and LDL apoB enrichment curves is shown for a representative pair of animals in supplemental Figure IIB and IIC. Kinetic parameters for VLDL and LDL apoB are summarized in the Table. RO0717625 treatment significantly decreased the VLDL apoB pool size by 22% (P<0.03), which was largely attributable to a 43% decrease in the VLDL apoB production rate (P<0.02). The VLDL apoB FCR decreased by 27%, but this was not significant. Neither the amount of VLDL apoB converted to LDL apoB nor the amount cleared directly from the circulation was affected. IDL apoB kinetic parameters were unchanged (data not shown).
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RO0717625 significantly decreased the LDL apoB pool size by 22% (P<0.002; Table). This was primarily attributable to a 47% (P<0.027) increase in the LDL apoB FCR because total LDL apoB production was unaffected. LDL apoB direct synthesis (LDL apoB entering the plasma compartment without initial metabolism through the VLDL or IDL pools) was <10% of total apoB production and was unaffected by treatment. Although VLDL apoB production was decreased, total production of apoB into plasma (VLDL plus LDL direct synthesis) did not change with RO0717625 treatment.
VLDL and LDL were analyzed for lipid and protein content (see Table I, available online at http://atvb.ahajournals.org).RO0717625 treatment did not change VLDL composition. Treatment decreased the LDL percentage of cholesteryl ester by 9% (P<0.02) and increased the percentage of protein by 11% (P<0.0003). The ratio of triglyceride/cholesteryl ester (wt/wt) in LDL and the ratio of surface to core lipids were unchanged, suggesting that treatment did not alter LDL particle size.
Approximately 24 hours after the last dose of the drug was administered, pigs were euthanized and sections of liver and jejunum were removed and stored at –80°C before analysis. RO0717625 decreased hepatic concentrations of total cholesterol and cholesteryl ester by 16% (P<0.045) and 39% (P<0.025), respectively (Figure 3), whereas liver triglycerides and free cholesterol were unchanged. Significant reductions in hepatic microsomal total cholesterol (–28%; P<0.00001), free cholesterol (–27%; P<0.00001), and cholesteryl ester (–38%; P<0.042) were observed (Figure 3). Oleate incorporation into cholesteryl ester decreased by 47% (P<0.021) in liver, whereas oleate incorporation into triglyceride was unaffected. Intestinal lipid concentrations and the incorporation of oleate into cholesteryl ester or triglyceride were unchanged (Figure III, available online at http://atvb.ahajournals.org).
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The activity of HMG-CoA reductase increased 1.75-fold (P<0.006) in microsomes prepared from livers of pigs treated with RO0717625 (Figure 3). This was associated with a 4.8-fold (P<0.003) increase in liver HMG-CoA reductase mRNA (Figure 4). However, net cholesterol synthesis was decreased as estimated from the significant decrease in plasma lathosterol concentrations (Figure 2). Liver mRNA abundance of the LDL receptor increased 2-fold (P<0.040; Figure 4).
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OSCi treatment increased the mRNA abundance of ABCG5 and ABCG8 in the small intestine by 1.4- and 1.6-fold, respectively (P<0.04 for both; Figure 4). Hepatic ABCG5 and ABCG8 expression increased 1.3-fold (P<0.03 for both). Because the expression of these 2 transporters is regulated through a common promoter, their expressions were correlated. The correlation coefficient for ABCG5 compared with ABCG8 mRNA abundance was R=0.76 (P<0.0001) and R=0.84 (P<0.0001) for the liver and intestine, respectively. LXR
was increased 1.2-fold (P<0.05) but was unaffected in the intestine. Liver ABCA1 and apoB mRNA were unchanged (data not shown).
In mice treated with the synthetic LXR agonist TO901317, the expression of ABCG5 and ABCG8 in liver and intestine was increased, leading to decreased fractional cholesterol absorption and diminished hepatic cholesterol stores.15 To determined whether the increase in ABCG5 and ABCG8 in pigs treated with RO0717625 was consistent with a decrease in cholesterol absorption, we examined the increase in plasma chylomicron cholesterol in response to a fat- and cholesterol-containing meal in a separate set of animals (n=3 per group). As shown in Figure IV (available online at http://atvb.ahajournals.org), treatment of pigs with RO0717625 (3 mg/kg per day) resulted in a 34% reduction in the area under the plasma chylomicron cholesterol curve (1.49±0.26 versus 2.51±0.42) compared with control animals. Together with the reduction in plasma plant sterols, this response is consistent with a decrease in net cholesterol absorption.
Figure 5A summarizes the major apoB concentrations and kinetic parameters observed in this study after RO0717625 treatment. VLDL apoB concentrations decreased primarily because of a decrease in the appearance of newly synthesized VLDL apoB into plasma. LDL apoB concentrations decreased significantly, primarily because of a significant increase in FCR. For comparison purposes, Figure 5B summarizes the percent change from control (no treatment) in apoB kinetic parameters obtained in the present study compared with a previous study using atorvastatin monotherapy (3 mg/kg per day),17 which produced similar reductions in LDL cholesterol to those observed in the present study. RO0717625 and atorvastatin significantly decreased VLDL apoB concentrations primarily because of reduced VLDL apoB production rates. The LDL apoB pool size was decreased by both treatments; however, the primary mechanisms differed. Atorvastatin decreased total LDL apoB production but had no significant effect on LDL apoB FCR. The OSCi had no significant effect on LDL apoB production but induced a marked increase in LDL apoB FCR, which was attributed to a greater decrease in hepatic cholesterol concentrations (–28%; Figure 3) compared with the statin (no change).17
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Discussion |
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Inhibition of OSC within the liver has the potential to decrease cholesterol synthesis and increase 24(S),25-epoxycholesterol synthesis, thus providing an attractive target for the treatment of hypercholesterolemia through a dual mechanism of action. This study demonstrates in a large animal model of human lipoprotein metabolism that inhibition of OSC leads to significant reductions in the plasma concentrations of VLDL apoB and LDL apoB. The decrease in VLDL apoB was attributable entirely to inhibition of VLDL apoB secretion, and the marked decrease in LDL apoB concentrations was attributable to increased LDL apoB clearance mediated by increased hepatic expression of LDL receptors. OSC inhibition markedly decreased plasma lathosterol levels, reflecting a significant reduction in hepatic cholesterol synthesis. Liver total and esterified cholesterol declined markedly, which was attributed to the decrease in cholesterol synthesis and reduced delivery of cholesterol to the liver from the intestine.
A reduction in VLDL apoB secretion has been observed previously using a similar protocol in pigs treated with 10 mg/kg per day of atorvastatin (–50%),18 3 mg/kg per day of atorvastatin (–34%),17 or 10 mg/kg per day of simvastatin (–33%).18 The reduction in VLDL apoB secretion has been linked to inhibition of hepatic cholesterol synthesis, thereby limiting cholesterol availability for hepatic lipoprotein assembly.18 The decrease in liver cholesterol and cholesteryl ester in OSCi-treated pigs is consistent with this hypothesis. Although VLDL secretion can be influenced by hepatic triglyceride synthesis, the latter was unaffected by OSCi treatment. Our results are in accord with studies in fat-fed hamsters treated with the OSCi RO0488071, in which the Triton WR-1339 technique revealed a decreased triglyceride secretion rate, suggesting reduced hepatic production of VLDL apoB.8
Inhibition of OSC significantly decreased LDL apoB concentrations attributable entirely to an enhanced clearance of LDL apoB from plasma, consistent with the significant increase in hepatic LDL receptor mRNA. Enhanced LDL apoB clearance has been observed previously using a similar protocol in pigs treated with high-dose (10 mg/kg per day) atorvastatin (15%)18 but was unaffected in pigs treated with a lower dose of atorvastatin (3 mg/kg per day)17 or simvastatin (10 mg/kg per day).18 However, in these studies, LDL apoB decreased primarily because of inhibition of LDL apoB production.17,18 In contrast, an inhibitor of the apical sodium-dependent bile acid transporter (ASBT) decreased LDL apoB concentrations primarily because of a significant increase in LDL apoB clearance: 18% as monotherapy and 45% in combination with atorvastatin.19,20
OSCi treatment significantly increased the hepatic expression of the LDL receptor and HMG-CoA reductase. Although consistent with the significant decrease in hepatic cholesterol content, these findings were unanticipated. In hamsters treated with the prototype OSCi RO0488071, HMG-CoA reductase activity and plasma clearance of radiolabeled LDL were unaffected. Although HMG-CoA reductase and LDL receptor expression were not measured, RO0488071 had no effect on liver cholesterol, providing a potential explanation for the results obtained. Whether these differences between OSCi-treated pigs and hamsters reflect differences in species response, the chemical class of OSCi used or the higher fat content and saturated fat composition of the diet, is unclear. We initially hypothesized that synthesis of 24(S),25-epoxycholesterol would repress HMG-CoA reductase expression, possibly through inhibition of SREBP-2 processing, which has been observed in cultured cells.9 Similarly, LDL receptor activity is decreased in OSCi-treated cells, consistent with an inhibition of SREBP-2 processing.11,12 One potential explanation is that the dose of RO0717625 used in the present study was above the level required for optimal 24(S),25-epoxycholesterol synthesis. However, we observed that MOS levels were elevated, whereas DOS levels were only marginally increased in the livers of OSCi-treated pigs, a favorable ratio for 24(S),25-epoxycholesterol synthesis.
A more plausible explanation is that 24(S),25-epoxycholesterol activated LXR, which we have demonstrated previously in OSCi-treated HepG2 cells.11 LXR activation could enhance the expression of genes involved in hepatic and intestinal cholesterol homeostasis. In mice, activation of LXR with the synthetic agonist TO901317 increased ABCG5 and ABCG8 expression in liver and intestine, leading to increased transport of cholesterol from the liver into bile and decreased cholesterol absorption, yielding diminished stores of hepatic cholesterol.15 Similar findings were reported for ABCG5/ABCG8 transgenic mice.21 Our observation that ABCG5 and ABCG8 expression increased in liver and intestine of treated pigs suggests activation of LXR by 24(S),25-epoxycholesterol. Cholesterol absorption was not directly measured in this study. However, the reduction in plasma plant sterol concentrations, together with the significant reduction of postprandial transport of cholesterol within chylomicrons, implies a net decrease in intestinal cholesterol absorption. Furthermore, the increased ABCG5 and ABCG8 mRNA were associated with decreased VLDL apoB production and increased LDL apoB FCR, a result supported by a recent human study. Chan et al demonstrated decreased VLDL apoB production and increased LDL apoB FCR in overweight subjects carrying the 400K allelic variant of ABCG8,22 which has been predicted to enhance its function.23
A net decrease in cholesterol transport to the liver from the intestine would enhance the reduction in hepatic cholesterol achieved through inhibition of cholesterol synthesis by the OSCi. Indeed, the reduction in hepatic cholesterol concentrations in OSCi-treated pigs (–28%) was greater than that observed with atorvastatin or simvastatin (no change)18 but similar to reductions observed with atorvastatin combined with an ASBT inhibitor (–14%).20 Therefore, it is possible that the OSCi-induced diminished hepatic cholesterol concentrations did not allow for the inactivation of SREBP-2 by 24(S),25-epoxycholesterol. Oxysterols are hypothesized to regulate SREBP-2 by causing the influx of plasma membrane cholesterol into the endoplasmic reticulum (ER) membrane. In turn, this facilitates the interaction of SREBP cleavage-activating protein (SCAP) with Insig proteins,24 retaining the SCAP/SREBP complex within the ER and preventing activation of SREBPs to their active processed forms. Thus, hepatocytes depleted in cholesterol may be unable to respond to the increase in 24(S),25-epoxycholesterol, resulting in insufficient cholesterol influx into the ER membrane to block SREBP-2 processing.
If OSCi treatment increased hepatic LXR activation, it might be anticipated that other LXR-regulated genes would also be upregulated. Increased expression of FAS or SREBP-1c could potentially lead to hepatic triglyceride accumulation, as observed in mice treated with the synthetic LXR activator TO901317.14 However, we did not detect any change in the synthesis or concentration of liver triglyceride. Although not measured, this implies that FAS and SREBP-1c were not activated. The reason for this finding is not entirely clear; however, it is possible that there is differential sensitivity of various genes to LXR activation. Recently, 2 groups demonstrated induction of intestinal ABCA1 with relatively little induction of liver FAS or SREBP-1c by selective synthetic LXR-activators GW396525 and N,N-dimethyl-3ß-hydroxycholenamide.26 The former study reported that GW3965-liganded LXR recruited selected coactivators less efficiently than the strong FAS and SREBP-1c inducer TO901317.25 Therefore, it is possible that in liver and intestine, ABCG5 and ABCG8 expression are more sensitive to LXR activation by the endogenous oxysterol 24(S),25-epoxycholesterol compared with FAS or SREBP-1c.
In conclusion, partial inhibition of OSC by RO0717625 in a large animal model of human lipoprotein metabolism significantly reduces LDL cholesterol and apoB. RO0717265 leads to inhibition of cholesterol synthesis. Our results are consistent with a mechanism whereby 24(S),25-epoxycholesterol increased the expression of ABCG5 and ABCG8 in liver and intestine, resulting in decreased postprandial transport of cholesterol in chylomicrons, thereby potentiating the reduction in hepatic cholesterol achieved through inhibition of cholesterol synthesis. The primary mechanism for reductions in plasma apoB is a substantial reduction in VLDL apoB production and enhanced LDL receptor–mediated LDL apoB clearance, both of which are linked to the reduction in hepatic cholesterol. Thus, OSC inhibition represents a potential target for the treatment of hypercholesterolemia via a dual mechanism of action.
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Acknowledgments |
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This work was supported by grants from the Heart and Stroke Foundation of Ontario (Operating T-5603 and Program PRG-4854) to M.W.H., the National Institutes of Health (NCRR grant RR-12609) to P.H.R.B. and F. Hoffmann-La Roche Ltd, Basel, Switzerland. P.H.R.B. is a career development fellow of the National Heart Foundation of Australia. The authors thank Kevin Dwyer for GC-MS analyses, Kim Thomaes for performing surgeries, and Shannon Bull for expert technical assistance.
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Footnotes |
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Murray W. Huff received a grant from F. Hoffmann-La Roche Pharmaceutical Company, which supported, in part, the work described in this manuscript. Johannes D. Aebi and Henrietta Dehmlow are employees of F. Hoffmann-La Roche Pharmaceutical Company. At the time this work was carried out, Olivier H. Morand was an employee of F. Hoffmann-La Roche Pharmaceutical Company.
Received April 12, 2005; accepted September 26, 2005.
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M. M. Beyea, C. L. Heslop, C. G. Sawyez, J. Y. Edwards, J. G. Markle, R. A. Hegele, and M. W. Huff Selective Up-regulation of LXR-regulated Genes ABCA1, ABCG1, and APOE in Macrophages through Increased Endogenous Synthesis of 24(S),25-Epoxycholesterol J. Biol. Chem., February 23, 2007; 282(8): 5207 - 5216. [Abstract] [Full Text] [PDF]
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F. Basso, L. A. Freeman, C. Ko, C. Joyce, M. J. Amar, R. D. Shamburek, T. Tansey, F. Thomas, J. Wu, B. Paigen, et al. Hepatic ABCG5/G8 overexpression reduces apoB-lipoproteins and atherosclerosis when cholesterol absorption is inhibited J. Lipid Res., January 1, 2007; 48(1): 114 - 126. [Abstract] [Full Text] [PDF]
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