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Effect of atorvastatin on plasma apoE metabolism in patients with combined hyperlipidemia

2002; Elsevier BV; Volume: 43; Issue: 9 Linguagem: Inglês

10.1194/jlr.m200016-jlr200

1539-7262

Jeffrey S. Cohn, Michel J. Tremblay, Rami Batal, Hélène Jacques, Lyne Veilleux, Claudia Rodríguez, P. Hugh R. Barrett, Denise Dubreuil, Madeleine Roy, Lise Bernier, Orval Mamer, Jean Davignon,

Peroxisome Proliferator-Activated Receptors

Atorvastatin, a synthetic HMG-CoA reductase inhibitor used for the treatment of hyperlipidemia and the prevention of coronary artery disease, significantly lowers plasma cholesterol and low-density lipoprotein cholesterol (LDL-C) levels. It also reduces total plasma triglyceride and apoE concentrations. In view of the direct involvement of apoE in the pathogenesis of atherosclerosis, we have investigated the effect of atorvastatin treatment (40 mg/day) on in vivo rates of plasma apoE production and catabolism in six patients with combined hyperlipidemia using a primed constant infusion of deuterated leucine. Atorvastatin treatment resulted in a significant decrease (i.e., 30–37%) in levels of total triglyceride, cholesterol, LDL-C, and apoB in all six patients. Total plasma apoE concentration was reduced from 7.4 ± 0.9 to 4.3 ± 0.2 mg/dl (−38 ± 8%, P < 0.05), predominantly due to a decrease in VLDL apoE (3.4 ± 0.8 vs. 1.7 ± 0.2 mg/dl; −42 ± 11%) and IDL/LDL apoE (1.9 ± 0.3 vs. 0.8 ± 0.1 mg/dl; −57 ± 6%). Total plasma lipoprotein apoE transport (i.e., production) was significantly reduced from 4.67 ± 0.39 to 3.04 ± 0.51 mg/kg/day (−34 ± 10%, P < 0.05) and VLDL apoE transport was reduced from 3.82 ± 0.67 to 2.26 ± 0.42 mg/kg/day (−36 ± 10%, P = 0.057). Plasma and VLDL apoE residence times and HDL apoE kinetic parameters were not significantly affected by drug treatment. Percentage decreases in VLDL apoE concentration and VLDL apoE production were significantly correlated with drug-induced reductions in VLDL triglyceride concentration (r = 0.99, P < 0.001; r = 0.88, P < 0.05, respectively, n = 6).Our results demonstrate that atorvastatin causes a pronounced decrease in total plasma and VLDL apoE concentrations and a significant decrease in plasma and VLDL apoE rates of production in patients with combined hyperlipidemia. Atorvastatin, a synthetic HMG-CoA reductase inhibitor used for the treatment of hyperlipidemia and the prevention of coronary artery disease, significantly lowers plasma cholesterol and low-density lipoprotein cholesterol (LDL-C) levels. It also reduces total plasma triglyceride and apoE concentrations. In view of the direct involvement of apoE in the pathogenesis of atherosclerosis, we have investigated the effect of atorvastatin treatment (40 mg/day) on in vivo rates of plasma apoE production and catabolism in six patients with combined hyperlipidemia using a primed constant infusion of deuterated leucine. Atorvastatin treatment resulted in a significant decrease (i.e., 30–37%) in levels of total triglyceride, cholesterol, LDL-C, and apoB in all six patients. Total plasma apoE concentration was reduced from 7.4 ± 0.9 to 4.3 ± 0.2 mg/dl (−38 ± 8%, P < 0.05), predominantly due to a decrease in VLDL apoE (3.4 ± 0.8 vs. 1.7 ± 0.2 mg/dl; −42 ± 11%) and IDL/LDL apoE (1.9 ± 0.3 vs. 0.8 ± 0.1 mg/dl; −57 ± 6%). Total plasma lipoprotein apoE transport (i.e., production) was significantly reduced from 4.67 ± 0.39 to 3.04 ± 0.51 mg/kg/day (−34 ± 10%, P < 0.05) and VLDL apoE transport was reduced from 3.82 ± 0.67 to 2.26 ± 0.42 mg/kg/day (−36 ± 10%, P = 0.057). Plasma and VLDL apoE residence times and HDL apoE kinetic parameters were not significantly affected by drug treatment. Percentage decreases in VLDL apoE concentration and VLDL apoE production were significantly correlated with drug-induced reductions in VLDL triglyceride concentration (r = 0.99, P < 0.001; r = 0.88, P < 0.05, respectively, n = 6). Our results demonstrate that atorvastatin causes a pronounced decrease in total plasma and VLDL apoE concentrations and a significant decrease in plasma and VLDL apoE rates of production in patients with combined hyperlipidemia. Apolipoprotein E (apoE) is a 34 kDa glycoprotein that is synthesized and secreted by most human tissues, including the liver, large intestine, brain, kidney, spleen, adrenal gland, and lung (1Driscoll D.M. Getz G.S. Extrahepatic synthesis of apolipoprotein E.J. Lipid Res. 1984; 25: 1368-1379Google Scholar, 2Elshourbagy N.A. Liao W.S. Mahley R.W. Taylor J.M. Apolipoprotein E mRNA is abundant in the brain and adrenals, as well as in the liver, and is present in other peripheral tissues of rats and marmosets.Proc. Natl. Acad. Sci. USA. 1985; 82: 203-207Google Scholar). It is associated in plasma with several classes of lipoproteins, including chylomicrons, VLDL, IDL, and a subclass of HDL (3Mahley R.W. Rall Jr., S.C. Type III hyperlipoproteinemia (dysbetalipoproteinemia): the role of apolipoprotein E in normal and abnormal lipoprotein metabolism.in: Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic Basis of Inherited Disease. McGraw-Hill Publishing Co., New York, NY.1989: 1195-1213Google Scholar). A pivotal role of apoE in plasma is to mediate hepatic and extrahepatic uptake of plasma lipoproteins by binding with high affinity to all members of the LDL-receptor family, including the LDL-receptor, the LDL-receptor-related protein (LRP), the VLDL receptor, GP330/megalin, and ApoER-2 (4Nimpf J. Schneider W.J. From cholesterol transport to signal transduction: low density lipoprotein receptor, very low density lipoprotein receptor, and apolipoprotein E receptor-2.Biochim. Biophys. Acta. 2000; 1529: 287-298Google Scholar). ApoE thus plays a central role in determining plasma cholesterol and triglyceride levels, a function that is best exemplified by the pronounced accumulation of cholesterol-rich lipoproteins and triglyceride-rich lipoprotein remnants in the plasma of both humans and mice lacking functional apoE (3Mahley R.W. Rall Jr., S.C. Type III hyperlipoproteinemia (dysbetalipoproteinemia): the role of apolipoprotein E in normal and abnormal lipoprotein metabolism.in: Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic Basis of Inherited Disease. McGraw-Hill Publishing Co., New York, NY.1989: 1195-1213Google Scholar, 5Schaefer E.J. Gregg R.E. Ghiselli G. Forte T.M. Ordovas J.M. Zech L.A. Brewer Jr., H.B. Familial apolipoprotein E deficiency.J. Clin. Invest. 1986; 78: 1-14Google Scholar, 6Plump A.S. Smith J.D. Hayek T. Aalto-Setälä K. Walsh A. Verstuyft J.G. Rubin E.M. Breslow J.L. Severe hypercholesterolemia and atherosclerosis in apolipoprotein E-deficient mice created by homologous recombination in ES cells.Cell. 1992; 71: 343-353Google Scholar). ApoE also modulates cellular cholesterol metabolism by facilitating cholesterol efflux (7Mazzone T. Apolipoprotein E secretion by macrophages: its potential physiological functions.Curr. Opin. Lipidol. 1996; 7: 303-307Google Scholar). Endogenous apoE facilitates the transport of cholesterol from intracellular compartments to the cell membrane, or alternatively, secreted apoE can act as an extracellular acceptor of cellular cholesterol (8Lin C.Y. Duan H. Mazzone T. Apolipoprotein E-dependent cholesterol efflux from macrophages: kinetic study and divergent mechanisms for endogenous versus exogenous apolipoprotein E.J. Lipid Res. 1999; 40: 1618-1627Google Scholar). ApoE-mediated cholesterol efflux from macrophages may be important in protecting these cells from the deleterious effects of cholesterol overload and transformation into atherosclerotic foam cells.As a consequence of the aforementioned functions, apoE plays a critical role in the onset and development of atherosclerosis (9Mahley R.W. Huang Y. Apolipoprotein E: from atherosclerosis to Alzheimer's disease and beyond.Curr. Opin. Lipidol. 1999; 10: 207-217Google Scholar, 10Davignon J. Cohn J.S. Mabile L. Bernier L. Apolipoprotein E and atherosclerosis: insight from animal and human studies.Clin. Chim. Acta. 1999; 286: 115-143Google Scholar, 11Curtiss L.K. Boisvert W.A. ApoE and atherosclerosis.Curr. Opin. Lipidol. 2000; 11: 243-251Google Scholar). This is a powerful protective role best exemplified by the severe hypercholesterolemia and pronounced atherosclerosis that occurs in apoE knockout (apoE−/−) mice (6Plump A.S. Smith J.D. Hayek T. Aalto-Setälä K. Walsh A. Verstuyft J.G. Rubin E.M. Breslow J.L. Severe hypercholesterolemia and atherosclerosis in apolipoprotein E-deficient mice created by homologous recombination in ES cells.Cell. 1992; 71: 343-353Google Scholar). Elevated levels of proatherogenic plasma lipoproteins clearly contribute to the development of arterial disease in these animals. This does not, however, entirely explain the protective role of apoE. Secretion of apoE by macrophages is also important, since selective expression of apoE in macrophages of apoE−/− mice (through bone marrow transplantation or transgenic expression) significantly reduces the extent of atherosclerosis (12Linton M.F. Atkinson J.B. Fazio S. Prevention of atherosclerosis in apolipoprotein E-deficient mice by bone marrow transplantation.Science. 1995; 267: 1034-1037Google Scholar, 13Kashyap V.S. Santamarina-Fojo S. Brown D.R. Parrott C.L. Applebaum-Bowden D. Meyn S. Talley G. Paigen B. Maeda N. Brewer Jr., H.B. Apolipoprotein E deficiency in mice: gene replacement and prevention of atherosclerosis using adenovirus vectors.J. Clin. Invest. 1995; 96: 1612-1620Google Scholar). Even low levels of apoE made in insufficient quantities to correct the hyperlipidemia of apoE−/− mice (and not made in the liver or in macrophages) can protect against arterial atherosclerosis (14Thorngate F.E. Rudel L.L. Walzem R.L. Williams D.L. Low levels of extrahepatic nonmacrophage apoE inhibit atherosclerosis withoiut correcting hypercholesterolemia in apoE-deficient mice.Arterioscler. Thromb. Vasc. Biol. 2000; 20: 1939-1945Google Scholar). ApoE may therefore have additional anti-atherogenic qualities, including inhibition of platelet aggregation (15Riddell D.R. Owen J.S. Inhibition of ADP-induced platelet aggregation by apoE is not mediated by membrane cholesterol depletion.Thromb. Res. 1996; 81: 597-606Google Scholar), stimulation of NO production (16Riddell D.R. Graham A. Owen J.S. Apolipoprotein E inhibits platelet aggregation through the L-arginine:nitric oxide pathway.J. Biol. Chem. 1997; 272: 89-95Google Scholar), reduction of smooth muscle cell migration and proliferation (17Swertfeger D.K. Hui D.Y. Apolipoprotein e receptor binding versus heparan sulfate proteoglycan binding in its regulation of smooth muscle cell migration and proliferation.J. Biol. Chem. 2001; 276: 25043-25048Google Scholar), and inhibition of lymphocyte proliferation (18Kelly M.E. Clay M.A. Mistry M.J. Hsieh-Li H.M. Harmony J.A. Apolipoprotein E inhibition of proliferation of mitogen-activated T lymphocytes: production of interleukin 2 with reduced biological activity.Cell. Immunol. 1994; 159: 124-139Google Scholar). These latter functions are thought to be dependent on the ability of apoE to act as a cell signaling molecule, and to be independent of its role as a lipid transport protein (19Swertfeger D.K. Hui D.Y. Apolipoprotein E: a cholesterol transport protein with lipid transport-independent cell signaling properties.Front. Biosci. 2001; 6: D526-D535Google Scholar).Treatment of hyperlipidemic patients with 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase inhibitors (or statins) has clearly been shown to reduce primary and secondary risk of cardiovascular morbidity and mortality (20LaRosa J.C. He J. Vupputuri S. Effect of statins on risk of coronary disease: a meta-analysis of randomized controlled trials.JAMA. 1999; 282: 2340-2346Google Scholar, 21Gotto Jr., A.M. Statin therapy: where are we? Where do we go next?.Am. J. Cardiol. 2001; 87: 13B-18BGoogle Scholar). Atorvastatin, a widely used synthetic HMG-CoA reductase inhibitor, significantly lowers plasma concentration of total cholesterol, LDL cholesterol (LDL-C), and apoB, and can also reduce plasma triglyceride levels (22Lea A.P. McTavish D. Atorvastatin. A review of its pharmacology and therapeutic potential in the management of hyperlipidaemias.Drugs. 1997; 53: 828-847Google Scholar, 23Davignon J. Advances in drug treatment of dyslipidemia: focus on atorvastatin.Can. J. Cardiol. 1998; 14: 28B-38BGoogle Scholar). Atorvastatin has also been shown to reduce plasma concentrations of apoE in patients with hypercholesterolemia (24Dallongeville J. Fruchart J.C. Maigret P. Bertolini S. Bon G.B. Campbell M.M. Farnier M. Langan J. Mahla G. Pauciullo P. Sirtori C. Double-blind comparison of apolipoprotein and lipoprotein particle lowering effects of atorvastatin and pravastatin monotherapy in patients with primary hypercholesterolemia.J. Cardiovasc. Pharmacol. Ther. 1998; 3: 103-110Google Scholar) or hypertriglyceridemia (25Le N.A. Innis-Whitehouse W. Li X. Bakker-Arkema R. Black D. Brown W.V. Lipid and apolipoprotein levels and distribution in patients with hypertriglyceridemia: effect of triglyceride reductions with atorvastatin.Metabolism. 2000; 49: 167-177Google Scholar). This may be due to reduced production or increased catabolism of plasma apoE. Although a number of previous studies have investigated the plasma kinetics of apoE in normolipidemic and hyperlipidemic subjects (26Gregg R.E. Zech L.A. Schaefer E.J. Brewer Jr., H.B. Type III hyperlipoproteinemia: defective metabolism of an abnormal apolipoproteinE.Science. 1981; 211: 584-586Google Scholar, 27Gregg R.E. Zech L.A. Schaefer E.J. Brewer Jr., H.B. Apolipoprotein E metabolism in normolipidemic human subjects.J. Lipid Res. 1984; 25: 1167-1176Google Scholar, 28Gregg R.E. Zech L.A. Schaefer E.J. Stark D. Wilson D. Brewer Jr., H.B. Abnormal in vivo metabolism of apolipoprotein E4 in humans.J. Clin. Invest. 1986; 78: 815-821Google Scholar, 29Ghiselli G. Beigel Y. Soma M. Gotto Jr., A.M. Plasma catabolism of human apolipoprotein E isoproteins: lack of conversion of the doubly sialilated form to the asialo form in plasma.Metabolism. 1986; 35: 399-403Google Scholar, 30Millar J.S. Lichtenstein A.H. Dolnikowski G.G. Ordovas J.M. Schaefer E.J. Proposal of a multicompartmental model for use in the study of apolipoprotein E metabolism.Metabolism. 1998; 47: 922-928Google Scholar, 31Batal R. Tremblay M. Barrett P.H.R. Jacques H. Fredenrich A. Mamer O. Davignon J. Cohn J.S. Plasma kinetics of apoC-III and apoE in normolipidemic and hyperlipidemic subjects.J. Lipid Res. 2000; 41: 706-718Google Scholar, 32Millar J.S. Lichtenstein A.H. Ordovas J.M. Dolnikowski G.G. Schaefer E.J. Human triglyceride-rich lipoprotein apo E kinetics and its relationship to LDL apo B-100 metabolism.Atherosclerosis. 2001; 155: 477-485Google Scholar), no previous work has investigated the effect of drug treatment on plasma or lipoprotein apoE kinetics. We have therefore carried out the present study using a primed constant infusion of deuterated leucine, in which plasma apoE metabolism, i.e., rates of plasma apoE transport (production) and fractional catabolism (i.e., residence time) were determined in combined hyperlipidemic patients treated with atorvastatin.MethodsPatients and treatmentSix male patients with combined hyperlipidemia were selected from our lipid clinic. Each patient gave their informed consent to participate in the study, which was approved by the Ethics Committee of the Clinical Research Institute of Montreal. At screening, they had a fasting plasma triglyceride concentration greater than 2.3 mmol/l (200 mg/dl), but less than 9.10 mmol/l (800 mg/dl), and an LDL-C concentration greater than 4.1 mmol/l (160 mg/dl). At screening, their plasma triglyceride concentration was 3.48 ± 0.23 mmol/l and their LDL-C was 5.21 ± 0.23 mmol/l. Their age (mean ± SE) was 47 ± 6 years and BMI was 26.8 ± 0.9. Two patients had an apoE-4/3 phenotype (numbers 2 and 3 in Tables) and four had an apoE-3/3 phenotype. They had no evidence nor history of diabetes mellitus, or liver or thyroid disease, and at screening were not taking medications known to affect plasma lipid metabolism. Patients were studied on two occasions: once while being treated with atorvastatin (40 mg/day) and then on a second occasion while not taking medication. For five patients, the atorvastatin treatment period (a minimum of six months) preceeded the non-treatment period (a minimum of 5 weeks). For one patient (number 3 in Tables), the first infusion was carried out while untreated, and the second infusion was carried out after 6 weeks of atorvastatin therapy.Protocol for apoE kinetic studyAfter a 12-h overnight fast, patients were given a primed constant intravenous infusion of deuterium-labeled leucine (l-[D3]leucine 98%, Cambridge Isotope Laboratories, MA), as described previously (33Cohn J.S. Wagner D.A. Cohn S.D. Millar J.S. Schaefer E.J. Measurement of very low density and low density lipoprotein apolipoprotein (apo) B-100 and high density lipoprotein apoA-I production in human subjects using deuterated leucine: effect of fasting and feeding.J. Clin. Invest. 1990; 85: 804-811Google Scholar, 34Lichtenstein A.H. Cohn J.S. Hachey D.L. Millar J.S. Ordovas J.M. Schaefer E.J. Comparison of deuterated leucine, valine, and lysine in the measurement of human apolipoprotein A-I and B-100 kinetics.J. Lipid Res. 1990; 31: 1693-1701Google Scholar). They were injected via a needle inserted into a left forearm vein with 10 μmol per kg body weight of l-[D3]leucine dissolved in physiological saline, followed by a 12-h constant infusion (given by peristaltic pump) of 10 μmol l-[D3]leucine per kg per h. Subjects remained fasted during the infusion but had free access to drinking water. Blood samples (20 ml) were collected from an antecubital vein of the right arm at regular intervals (0, 15, 30, and 45 min, and 1, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 h) in tubes containing EDTA to a final concentration of 0.1%. Plasma was immediately separated by centrifugation at 3,500 rpm for 15 min at 4°C. An antimicrobial agent (sodium azide) and a protease inhibitor (aprotinin) were added to plasma samples to give a final concentration of 0.02% and 1.67 μg/ml, respectively.Isolation of lipoproteins and apolipoproteinsVLDL, IDL together with LDL, and HDL, were isolated from 5 ml plasma by sequential ultracentrifugation in an XL-90 ultracentrifuge using a 50.4 Ti rotor (Beckman) at 50,000 rpm for 10 h, at densities (d) of 1.006, 1.063, and 1.21 g/ml, respectively. Total lipoproteins were isolated from plasma by ultracentrifugation (50,000 rpm, 10 h) of 2 ml of plasma, adjusted to d = 1.25 g/ml with KBr. Lipoproteins were recovered in the supernate by tube-slicing. ApoE was isolated from VLDL, HDL, and total plasma lipoproteins (d < 1.25 g/ml fractions) by preparative isoelectric focusing (IEF) on 7.5% polyacrylamide-urea (8M) gels (pH gradient 4–7), as described previously (35Batal R. Tremblay M. Krimbou L. Mamer O. Davignon J. Genest Jr., J. Cohn J.S. Familial HDL deficiency characterized by hypercatabolism of mature apoA-I but not proapoA-I.Arterioscler. Thromb. Vasc. Biol. 1998; 18: 655-664Google Scholar). Before IEF separation of HDL and the d < 1.25 g/ml fractions, they were dialyzed against 10 mM ammonium bicarbonate, preincubated with cysteamine (β-mercaptoethylamine, Sigma-Aldrich) in a ratio of 6 mg for every milligram of protein for 4 h at 37°C, and then delipidated. The aim of cysteamine treatment was to separate apoE from isoforms of apoA-I, which normally co-migrate to the same position on IEF gels. Cysteamine treatment causes an amino group to bind to the single cysteine residue of apoE3. ApoA-I and apoE-4 are not affected since they do not contain cysteine. Cysteamine-modified apoE-3 consequently migrates to a higher position in IEF gels due to its increased positive charge (35Batal R. Tremblay M. Krimbou L. Mamer O. Davignon J. Genest Jr., J. Cohn J.S. Familial HDL deficiency characterized by hypercatabolism of mature apoA-I but not proapoA-I.Arterioscler. Thromb. Vasc. Biol. 1998; 18: 655-664Google Scholar). VLDL samples were delipidated, but were not treated with cysteamine prior to electrophoresis. Coumassie blue staining was used to identify the position of apolipoproteins in gels after electrophoresis. The VLDL apoE-3 band was analyzed for enrichment in all six patients. In apoE-3/3 patients, approximately 80% of VLDL apoE migrated in the E3 position and was analyzed. In apoE4/3 patients, approximately 40% of VLDL apoE migrated in the E3 position and was analyzed. This meant that samples from apoE-4/3 and apoE-3/3 patients were analyzed slightly differently. Importantly, however, the type and amount of VLDL apoE analyzed for a given patient was the same, whether he was on or off statin therapy.Plasma lipids and apolipoproteinsApoE phenotypes were determined by isoelectric focusing of delipidated VLDL (35Batal R. Tremblay M. Krimbou L. Mamer O. Davignon J. Genest Jr., J. Cohn J.S. Familial HDL deficiency characterized by hypercatabolism of mature apoA-I but not proapoA-I.Arterioscler. Thromb. Vasc. Biol. 1998; 18: 655-664Google Scholar). Plasma and lipoprotein fractions were assayed for total (free and esterified) cholesterol and triglyceride with a COBAS MIRA-S automated analyzer (Hoffman-LaRoche) using enzymatic reagents. HDL-C was determined by assaying cholesterol in the supernatant after precipitation of apoB-containing lipoproteins in the d > 1.006 g/ml fraction with heparin/manganese. LDL-C was calculated as d > 1.006 g/ml cholesterol minus cholesterol in HDL. Plasma apoA-I and apoB were measured by nephelometry on a Behring Nephelometer 100 (Behring) using Behring protocol and reagents. Plasma and lipoprotein apoE concentrations were measured with an ELISA developed in our laboratory (36Cohn J.S. Tremblay M. Amiot M. Bouthillier D. Roy M. Genest Jr. J. Davignon J. Plasma concentration of apolipoprotein E in intermediate-sized remnant-like lipoproteins in normolipidemic and hyperlipidemic subjects.Arterioscler. Thromb. Vasc. Biol. 1996; 16: 149-159Google Scholar) using immunopurified polyclonal antibody (Biodesign, Kennebunk, ME). Recovery of apoE after sequential ultracentrifugation was determined as a percentage: (VLDL apoE + IDL/LDL apoE + HDL apoE + d > 1.21 g/ml apoE) × 100/total plasma apoE. ApoE recovery was 74.5 ± 2.0% for plasma isolated from untreated patients and 71.3 ± 1.5% for plasma isolated from treated patients. Final lipoprotein apoE concentrations were corrected proportionately to give 100% recovery.Determination of isotopic enrichmentApolipoprotein bands, as well as blank (non-protein containing) gel slices, were excised from polyacrylamide gels as described previously (35Batal R. Tremblay M. Krimbou L. Mamer O. Davignon J. Genest Jr., J. Cohn J.S. Familial HDL deficiency characterized by hypercatabolism of mature apoA-I but not proapoA-I.Arterioscler. Thromb. Vasc. Biol. 1998; 18: 655-664Google Scholar). The band corresponding to non-sialylated apoE3 was analyzed in VLDL. For HDL and d < 1.25 g/ml fractions, apoE isoforms migrated together as one band due to cystamine treatment. Each slice was added to a borosilicate sample vial containing 600 μl of 6 N HCL, and an internal standard (250 ng norleucine, Sigma-Aldrich) dissolved in 50 μl double distilled water. Gel slices were hydrolyzed at 110°C for 24 h, cooled to −20°C for 20 min, and centrifuged at 3,500 rpm for 5 min. Free amino acids in the hydrolysate were separated from precipitated polyacrylamide, purified by cation exchange chromatography using AG 50 W-X8 resin (BioRad), and derivatized by treatment with 200 μl of acetyl chloride-acidified 1-propanol (1:5, v/v) for 1 h at 100°C, and 50 μl of heptaflurobutyric anhydride (Supelco) for 20 min at 60°C (33Cohn J.S. Wagner D.A. Cohn S.D. Millar J.S. Schaefer E.J. Measurement of very low density and low density lipoprotein apolipoprotein (apo) B-100 and high density lipoprotein apoA-I production in human subjects using deuterated leucine: effect of fasting and feeding.J. Clin. Invest. 1990; 85: 804-811Google Scholar). Plasma amino acids were also separated by cation exchange chromatography and derivatized to allow for the determination of plasma leucine isotopic enrichment. Enrichment of samples with deuterium-labeled leucine was measured by gas chromatography/mass spectrometry (Hewlett-Packard, 5988 GC-MS) using negative chemical ionization and methane as the moderator gas. Selective ion monitoring at m/z =352 and 349 (ionic species corresponding to derivatized deuterium-labeled and derivatized non-deuterium-labeled leucine, respectively) was performed, and tracer to tracee ratios were derived from isotopic ratios for each sample. Tracer to tracee ratios were corrected for background leucine in gel slices (i.e., trace amounts of leucine introduced during the amino acid purification and derivitization procedures) by estimating the amount of leucine in processed blank gel slices relative to the norleucine internal standard. Background leucine varied from 0.5% to 10% of total leucine recovered from each gel slice containing apoE.Kinetic analysisStable isotope enrichment curves for apoE in total plasma lipoproteins (d < 1.25 g/ml), VLDL, and HDL were fitted to a three compartment model using SAAM II computer software (SAAM II institute, WA). The first compartment represented the plasma amino acid precursor pool. The second compartment was a delay compartment, which accounted for the synthesis, assembly, and secretion of apolipoprotein. The third compartment was the plasma protein compartment. Plasma leucine enrichment (measured eight times during the course of the infusion experiment) was used as a measure of precursor pool enrichment. Mean (± SE) plasma leucine tracer to tracee ratio was 11.4 ± 0.5% in untreated patients and was 10.9 ± 0.5% in treated patients. Modeling of tracer to tracee ratio data allowed for the determination of fractional transport rates (FTR) (i.e., the fraction of protein pools being renewed per day). Residence time (RT) was calculated as the reciprocal of FTR (1/FTR), and transport rate (TR) was calculated (in mg/kg.day) as:TR = FTR (pools/day) × apolipoprotein pool size (mg) ÷body weight (kg)where: pool size = plasma concentration (mg/dl) × plasma volume (0.045 l/kg).For VLDL and HDL, apoE transport rates were a reflection of the amount of apoE becoming associated with either plasma VLDL or HDL per unit time. The majority of this apoE in the fasted state was newly synthesized and was acquired directly from tissue. This parameter, however, also measured apoE that was transported into VLDL or HDL indirectly from other lipoproteins due to lipoprotein conversion or apolipoprotein exchange. In the case of total plasma lipoprotein apoE, transport rates were a reflection of the amount of apoE entering the circulation per unit time and were thus a measure of overall tissue synthesis and secretion (i.e., production) of plasma apoE.Statistical analysisStatistical significance of differences between mean values was assessed by paired t-tests using SigmaStat software (Jandel Scientific, CA). Pearson correlation coefficients (r) were calculated to describe the correlation between different kinetic and mass parameters.ResultsAtorvastatin treatment was associated with a decrease in the plasma concentration of total triglyceride, cholesterol, VLDL-C, LDL-C, and apoB in all six patients. These parameters were on average reduced by ∼35%, except for VLDL-C, which was reduced by 60% (Table 1). VLDL triglyceride concentration was 2.20 ± 0.39 mg/dl in untreated patients and 1.36 ± 0.16 mg/dl in treated patients (−33 ± 9%, P = 0.064). VLDL apoB concentration was 15.5 ± 2.3 mg/dl in untreated patients and 9.3 ± 1.6 mg/dl in treated patients (−38 ± 11%, P < 0.05). There was no significant difference in HDL-C or plasma apoA-I levels. Total plasma apoE concentrations were also significantly decreased (−38 ± 8%, P < 0.05). This reduction was predominantly due to less apoE in the VLDL and IDL/LDL fractions (Table 2). The one patient (number 3) who was studied in the order "untreated–treated," and who was therefore on atorvastatin treatment for a shorter period of time (6 weeks vs. 6 months), had a similar decrease in plasma lipids and apolipoproteins as the other patients. The two patients (numbers 2 and 3) with an apoE-4/3 phenotype responded to therapy, even though in men the apoE-4 allele is generally associated with a poorer response to statin therapy and the apoE-2 allele is associated with a better response (37Ordovas J.M. Mooser V. The APOE locus and the pharmacogenetics of lipid response.Curr. Opin. Lipidol. 2002; 13: 113-117Google Scholar).TABLE 1Plasma lipid and apolipoprotein concentrations in patients untreated or treated with atorvastatinTotal TriglycerideTotal CholesterolVLDL CholesterolLDL CholesterolHDL CholesterolApoBApoA-Immol/lmg/dlUntreated12.368.340.736.281.3320710722.166.860.934.731.2015113433.196.811.284.331.2020111641.428.020.486.770.7822911952.067.530.766.060.711928964.177.181.983.911.28177112Mean ± SE2.56 ± 0.407.46 ± 0.26 1.03 ± 0.225.35 ± 0.481.08 ± 0.11 193 ± 11113 ± 6Treated11.615.490.324.001.1711911122.094.800.672.861.2713311331.793.890.442.101.3510311940.945.160.074.100.9912713051.915.260.434.120.711499061.644.460.393.110.96109108Mean ± SE 1.66 ± 0.164.84 ± 0.24 0.39 ± 0.083.38 ± 0.341.08 ± 0.10 123 ± 7112 ± 5Percent changeaPercent change due to treatment was calculated for each patient and then averaged.−30 ± 9%−35 ± 2%−60 ± 9%−37 ± 4% 1 ± 7%−35 ± 6% 0 ± 3%SignificancebStatistical significance between untreated and treated values was determined by paired Student's t -test and is given as a P value; ns = not significant.0.065< 0.001< 0.05< 0.001ns< 0.01nsValues are for individual patients (numbered 1–6). Each value is the mean of five measurements (at 3 h intervals) during the infusion experiment.a Percent change due to treatment was calculated for each patient and then averaged.b Statistical significance between untreated and treated values was determined by paired Student's t -test and is given as a P value; ns = not significant. Open table in a new tab TABLE 2Total plasma and lipoprotein apoE concentrations in patients untreated or treated with atorvastatinVLDL A