D-4F-mediated reduction in metabolites of arachidonic and linoleic acids in the small intestine is associated with decreased inflammation in low-density lipoprotein receptor-null mice
2011; Elsevier BV; Volume: 53; Issue: 3 Linguagem: Inglês
10.1194/jlr.m023523
ISSN1539-7262
AutoresMohamad Navab, Srinivasa T. Reddy, G.M. Anantharamaiah, Greg Hough, Georgette M. Buga, Jan Danciger, Alan M. Fogelman,
Tópico(s)Peroxisome Proliferator-Activated Receptors
ResumoTo test the hypothesis that intestine is a major site of action for D-4F, LDLR−/− mice were fed a Western diet (WD) and administered the peptide subcutaneously (SQ) or orally. Plasma and liver D-4F levels were 298-fold and 96-fold higher, respectively, after SQ administration, whereas peptide levels in small intestine only varied by 1.66 ± 0.33-fold. Levels of metabolites of arachidonic and linoleic acids known to bind with high affinity to D-4F were significantly reduced in intestine, liver and hepatic bile to a similar degree whether administered SQ or orally. However, levels of 20-HETE, which is known to bind the peptide with low affinity, were unchanged. D-4F treatment reduced plasma serum amyloid A (SAA) and triglyceride levels (P < 0.03) and increased HDL-cholesterol levels (P < 0.04) similarly after SQ or oral administration. Plasma levels of metabolites of arachidonic and linoleic acids significantly correlated with SAA levels (P < 0.0001). Feeding 15-HETE in chow (without WD) significantly increased plasma SAA and triglyceride levels and decreased HDL-cholesterol and paraoxonase activity (P < 0.05), all of which were significantly ameliorated by SQ D-4F (P < 0.05). We conclude that D-4F administration reduces levels of free metabolites of arachidonic and linoleic acids in the small intestine and this is associated with decreased inflammation in LDL receptor deficient mice. To test the hypothesis that intestine is a major site of action for D-4F, LDLR−/− mice were fed a Western diet (WD) and administered the peptide subcutaneously (SQ) or orally. Plasma and liver D-4F levels were 298-fold and 96-fold higher, respectively, after SQ administration, whereas peptide levels in small intestine only varied by 1.66 ± 0.33-fold. Levels of metabolites of arachidonic and linoleic acids known to bind with high affinity to D-4F were significantly reduced in intestine, liver and hepatic bile to a similar degree whether administered SQ or orally. However, levels of 20-HETE, which is known to bind the peptide with low affinity, were unchanged. D-4F treatment reduced plasma serum amyloid A (SAA) and triglyceride levels (P < 0.03) and increased HDL-cholesterol levels (P < 0.04) similarly after SQ or oral administration. Plasma levels of metabolites of arachidonic and linoleic acids significantly correlated with SAA levels (P < 0.0001). Feeding 15-HETE in chow (without WD) significantly increased plasma SAA and triglyceride levels and decreased HDL-cholesterol and paraoxonase activity (P < 0.05), all of which were significantly ameliorated by SQ D-4F (P < 0.05). We conclude that D-4F administration reduces levels of free metabolites of arachidonic and linoleic acids in the small intestine and this is associated with decreased inflammation in LDL receptor deficient mice. Apolipoprotein A-I (apoA-I) mimetic peptides have been studied in animals and humans for their ability to improve biomarkers of inflammation and in animal models of atherosclerosis for their ability to decrease lesions (1Navab M. Reddy S.T. Anantharamaiah G.M. Imaizumi S. Hough G. Hama S. Fogelman A.M. Intestine may be a major site of action for the apoA-I mimetic peptide 4F whether administered subcutaneously or orally.J. Lipid Res. 2011; 52: 1200-1210Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The mechanism of action of these peptides has been thought to be due to their remarkable ability to bind oxidized lipids compared with apoA-I (2Van Lenten B.J. Wagner A.C. Jung C.L. Ruchala P. Waring A.J. Lehrer R.I. Watson A.D. Hama S. Navab M. Anantharamaiah G.M. et al.Anti-inflammatory apoA-I mimetic peptides bind oxidized lipids with much higher affinity than human apoA-I.J. Lipid Res. 2008; 49: 2302-2311Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). The classes of oxidized lipids bound with high affinity by these mimetic peptides include oxidized phospholipids, oxidized metabolites of arachidonic and linoleic acids, and oxidized sterols (2Van Lenten B.J. Wagner A.C. Jung C.L. Ruchala P. Waring A.J. Lehrer R.I. Watson A.D. Hama S. Navab M. Anantharamaiah G.M. et al.Anti-inflammatory apoA-I mimetic peptides bind oxidized lipids with much higher affinity than human apoA-I.J. Lipid Res. 2008; 49: 2302-2311Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). Oxidized metabolites of arachidonic and linoleic acids can be generated by enzymatic systems such as lipoxygenases or cyclooxygenases, or they can be generated by nonspecific oxidation. Except for 20-hydroxyeicosatetraenoic acid (HETE), all of the oxidized metabolites of arachidonic and linoleic acids that we studied bound to the peptide with much higher affinity than was the case for the binding to apoA-I (2Van Lenten B.J. Wagner A.C. Jung C.L. Ruchala P. Waring A.J. Lehrer R.I. Watson A.D. Hama S. Navab M. Anantharamaiah G.M. et al.Anti-inflammatory apoA-I mimetic peptides bind oxidized lipids with much higher affinity than human apoA-I.J. Lipid Res. 2008; 49: 2302-2311Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 3Imaizumi S. Grijalva V. Navab M. Van Lenten B.J. Wagner A.C. Anantharamaiah G.M. Fogelman A.M. Reddy S.T. L-4F differentially alters plasma levels of oxidized fatty acids resulting in more anti-inflammatory HDL in mice.Drug Metab. Lett. 2010; 4: 139-148Crossref PubMed Scopus (49) Google Scholar). We recently reported that the dosage of the apoA-I mimetic peptide 4F administered to apoE−/− mice determined efficacy, but plasma and hepatic levels of peptide did not (1Navab M. Reddy S.T. Anantharamaiah G.M. Imaizumi S. Hough G. Hama S. Fogelman A.M. Intestine may be a major site of action for the apoA-I mimetic peptide 4F whether administered subcutaneously or orally.J. Lipid Res. 2011; 52: 1200-1210Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Because efficacy was similar at the same dosages but plasma and hepatic levels were dramatically higher when the peptide was administered by subcutaneous injection (SQ) compared with oral administration, we suspected that there might be a compartment outside of the liver or plasma where peptide concentration would be similar. We found that the concentration of D-4F in the feces was the same regardless of whether the peptide was administered SQ or orally, suggesting that the intestine maybe a major site of action for the peptide regardless of the route of administration (1Navab M. Reddy S.T. Anantharamaiah G.M. Imaizumi S. Hough G. Hama S. Fogelman A.M. Intestine may be a major site of action for the apoA-I mimetic peptide 4F whether administered subcutaneously or orally.J. Lipid Res. 2011; 52: 1200-1210Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The concentration of free 15-HETE and 13-hydroxyoctadecadienoic acid (HODE) in the plasma of apoE−/− mice was significantly higher than that of wild-type mice (3Imaizumi S. Grijalva V. Navab M. Van Lenten B.J. Wagner A.C. Anantharamaiah G.M. Fogelman A.M. Reddy S.T. L-4F differentially alters plasma levels of oxidized fatty acids resulting in more anti-inflammatory HDL in mice.Drug Metab. Lett. 2010; 4: 139-148Crossref PubMed Scopus (49) Google Scholar). After administration of the 4F peptide, plasma levels of free oxidized fatty acids that bound with higher affinity to the mimetic peptide compared with apoA-I (e.g., 5-HETE, 15-HETE, 9-HODE, and 13-HODE) significantly decreased, but the levels of 20-HETE, which bound with equal low affinity to apoA-I and 4F, did not decrease (3Imaizumi S. Grijalva V. Navab M. Van Lenten B.J. Wagner A.C. Anantharamaiah G.M. Fogelman A.M. Reddy S.T. L-4F differentially alters plasma levels of oxidized fatty acids resulting in more anti-inflammatory HDL in mice.Drug Metab. Lett. 2010; 4: 139-148Crossref PubMed Scopus (49) Google Scholar). These studies focused on the plasma levels of free oxidized fatty acids, which are only a small fraction (<10%) of the total plasma oxidized fatty acids. Interestingly, only the free oxidized fatty acid plasma levels decreased after the administration of the apoA-I mimetic peptide; esterified oxidized fatty acid levels were unchanged (3Imaizumi S. Grijalva V. Navab M. Van Lenten B.J. Wagner A.C. Anantharamaiah G.M. Fogelman A.M. Reddy S.T. L-4F differentially alters plasma levels of oxidized fatty acids resulting in more anti-inflammatory HDL in mice.Drug Metab. Lett. 2010; 4: 139-148Crossref PubMed Scopus (49) Google Scholar). In other studies, apoE−/− mice were made diabetic, resulting in a significant increase in the hepatic content of free arachidonic acid and free 12-HETE, 15-HETE, 13-HODE, PGD2, and PGE2. This was associated with a significant increase in aortic atherosclerosis. Oral administration of D-4F significantly decreased the hepatic content of free arachidonic acid and free oxidized fatty acids derived from arachidonic and linoleic acids and significantly decreased aortic atherosclerosis without affecting other plasma lipid or lipoprotein levels (4Morgantini C. Imaizumi S. Grijalva V. Navab M. Fogelman A.M. Reddy S.T. ApoAI mimetic peptides prevent atherosclerosis development and reduce plaque inflammation in a mouse model of diabetes.Diabetes. 2010; 59: 3223-3228Crossref PubMed Scopus (57) Google Scholar). Subsequently, we reported that HDL from type 2 diabetics contained significantly more free 5-HETE, 12-HETE, 15-HETE, 9-HODE, and 13-HODE than HDL from healthy volunteers. The type 2 diabetic HDL was also proinflammatory in a cell-based assay and was abnormal in a cell-free assay. The HDL content of free 5-HETE, 12-HETE, 15-HETE, 9-HODE, and 13-HODE significantly correlated with the values obtained in the cell-free assay (5Morgantini C. Natali A. Boldrini B. Imaizumi S. Navab M. Fogelman A.M. Ferrannini E. Reddy S.T. Anti-inflammatory and antioxidant properties of HDLs are impaired in type 2 diabetes.Diabetes. 2011; 60: 2617-2623Crossref PubMed Scopus (137) Google Scholar). The experiments reported here were designed to extend our previous studies on the interaction of the D-4F peptide with the intestine (1Navab M. Reddy S.T. Anantharamaiah G.M. Imaizumi S. Hough G. Hama S. Fogelman A.M. Intestine may be a major site of action for the apoA-I mimetic peptide 4F whether administered subcutaneously or orally.J. Lipid Res. 2011; 52: 1200-1210Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). These studies confirm our previous studies (1Navab M. Reddy S.T. Anantharamaiah G.M. Imaizumi S. Hough G. Hama S. Fogelman A.M. Intestine may be a major site of action for the apoA-I mimetic peptide 4F whether administered subcutaneously or orally.J. Lipid Res. 2011; 52: 1200-1210Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 2Van Lenten B.J. Wagner A.C. Jung C.L. Ruchala P. Waring A.J. Lehrer R.I. Watson A.D. Hama S. Navab M. Anantharamaiah G.M. et al.Anti-inflammatory apoA-I mimetic peptides bind oxidized lipids with much higher affinity than human apoA-I.J. Lipid Res. 2008; 49: 2302-2311Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 3Imaizumi S. Grijalva V. Navab M. Van Lenten B.J. Wagner A.C. Anantharamaiah G.M. Fogelman A.M. Reddy S.T. L-4F differentially alters plasma levels of oxidized fatty acids resulting in more anti-inflammatory HDL in mice.Drug Metab. Lett. 2010; 4: 139-148Crossref PubMed Scopus (49) Google Scholar, 4Morgantini C. Imaizumi S. Grijalva V. Navab M. Fogelman A.M. Reddy S.T. ApoAI mimetic peptides prevent atherosclerosis development and reduce plaque inflammation in a mouse model of diabetes.Diabetes. 2010; 59: 3223-3228Crossref PubMed Scopus (57) Google Scholar–5Morgantini C. Natali A. Boldrini B. Imaizumi S. Navab M. Fogelman A.M. Ferrannini E. Reddy S.T. Anti-inflammatory and antioxidant properties of HDLs are impaired in type 2 diabetes.Diabetes. 2011; 60: 2617-2623Crossref PubMed Scopus (137) Google Scholar) and demonstrate for the first time that D-4F-mediated reductions in free oxidized metabolites of arachidonic and linoleic acids in the small intestine are associated with reduced inflammation in LDLR−/− mice. The peptide 4F (Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2) was synthesized from all D-amino acids (D-4F) by solid-phase synthesis as described (6Navab M. Anantharamaiah G.M. Reddy S.T. Hama S. Hough G. Grijalva V.R. Wagner A.C. Frank J.S. Datta G. Garber D. et al.Oral D-4F causes formation of pre-beta high-density lipoprotein and improves high-density lipoprotein-mediated cholesterol efflux and reverse cholesterol transport from macrophages in apolipoprotein E-null mice.Circulation. 2004; 109: 3215-3220Crossref PubMed Scopus (312) Google Scholar). Protease inhibitors were purchased from Roche Diagnostics Gmbh (Complete, Mini, EDTA-free tablet) (Catalogue No. 11 836 179 001). In the experiments in which a HETE or HODE were added to mouse chow without the Western diet (WD), 12(S)-HETE (Catalog #34570), 15(S)-HETE (Catalog #34720), and 13(S)-HODE (Catalog #38610) were purchased from Cayman Chemical Co. (Ann Arbor, MI). All other materials were purchased from sources previously described (2Van Lenten B.J. Wagner A.C. Jung C.L. Ruchala P. Waring A.J. Lehrer R.I. Watson A.D. Hama S. Navab M. Anantharamaiah G.M. et al.Anti-inflammatory apoA-I mimetic peptides bind oxidized lipids with much higher affinity than human apoA-I.J. Lipid Res. 2008; 49: 2302-2311Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 3Imaizumi S. Grijalva V. Navab M. Van Lenten B.J. Wagner A.C. Anantharamaiah G.M. Fogelman A.M. Reddy S.T. L-4F differentially alters plasma levels of oxidized fatty acids resulting in more anti-inflammatory HDL in mice.Drug Metab. Lett. 2010; 4: 139-148Crossref PubMed Scopus (49) Google Scholar, 7Navab M. Ruchala P. Waring A.J. Lehrer R.I. Hama S. Hough G. Palgunachari M.N. Anantharamaiah G.M. Fogelman A.M. A novel method for oral delivery of apolipoprotein mimetic peptides synthesized from all L-amino acids.J. Lipid Res. 2009; 50: 1538-1547Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Female LDLR−/− mice originally purchased from Jackson laboratories on a C57BL/6J background were obtained from the breeding colony of the Department of Laboratory and Animal Medicine at the David Geffen School of Medicine at UCLA. The mice used in these studies were of different ages, which are stated in each Table legend. The mice were maintained on a chow diet (Ralston Purina) before being switched to a WD (Teklad, Harlan, catalog # TD88137). The WD was kept frozen until it was administered to the mice each night. Peptide was administered to the mice by daily SQ) on the back or was administered orally by providing the peptide in the drinking water. For oral administration, the peptide was freshly prepared by dissolving in water, and the drinking water was changed three times per week. For SQ administration, the peptide was uniformly dissolved in normal saline (pH 7.4) using a glass-glass homogenizer, and each mouse received a daily injection of 1 ml containing the peptide in normal saline (pH 7.4). In all experiments, the daily dose of peptide administered by either route of administration was 900 μg/mouse/day (45 mg/kg/day). The following protocol was used to conveniently and accurately provide free oxidized fatty acids in the chow diet. Three hundred milliliters of water were added to 400 g of chow pellets. The pellets were allowed to soften for 30 min, and a highly uniform mixture was generated using a small industrial-type mixer. The desired quantity of the free oxidized fatty acid was first mixed in saline and was then carefully and gradually added and mixed into the chow using the mixer. Mixing was continued at high speed for 1 min, at which time the mixer was stopped and the material was mixed in the opposite direction using a spatula to ensure a highly uniform distribution of the added free oxidized fatty acid. This process was continued for a total of 5–10 cycles. The resulting mixture containing the oxidized fatty acid was spread and flattened uniformly on a sheet of aluminum foil in a tray to achieve a height of ∼2 mm. The mixture was then cut into blocks of ∼5 g each using a rotary knife. These blocks were stored at −20°C. Each evening, four blocks of the frozen diet containing the oxidized fatty acids were provided to each cage containing four mice. The mice ate all of the diet by morning. The frozen diet contained 1 μg free oxidized fatty acid per gram frozen diet. Thus, on average each mouse received 5 μg of the free oxidized fatty acid each day. All experiments were performed using protocols approved by the Animal Research Committee at UCLA. The levels of free arachidonic acid and free metabolites of arachidonic and linoleic acids were determined by LC-ESI-MS/MS as previously described (3Imaizumi S. Grijalva V. Navab M. Van Lenten B.J. Wagner A.C. Anantharamaiah G.M. Fogelman A.M. Reddy S.T. L-4F differentially alters plasma levels of oxidized fatty acids resulting in more anti-inflammatory HDL in mice.Drug Metab. Lett. 2010; 4: 139-148Crossref PubMed Scopus (49) Google Scholar). In each instance, a deuterium-labeled internal standard was included to correct for extraction efficiency and to facilitate quantification (3Imaizumi S. Grijalva V. Navab M. Van Lenten B.J. Wagner A.C. Anantharamaiah G.M. Fogelman A.M. Reddy S.T. L-4F differentially alters plasma levels of oxidized fatty acids resulting in more anti-inflammatory HDL in mice.Drug Metab. Lett. 2010; 4: 139-148Crossref PubMed Scopus (49) Google Scholar). After an overnight fast, mouse plasma was immediately prepared from heparinized blood obtained from the retroorbital sinus under mild isoflurane anesthesia. For mice receiving subcutaneous injections, the blood was removed 1.5–3 h after the last injection. Plasma was immediately frozen and stored at −80°C until analysis. Plasma serum amyloid A (SAA) levels were determined by ELISA (, catalog # KMA0011; Invitrogen) according to the manufacturer's instructions. Lipid and lipoprotein and protein determinations were determined by methods described previously (1Navab M. Reddy S.T. Anantharamaiah G.M. Imaizumi S. Hough G. Hama S. Fogelman A.M. Intestine may be a major site of action for the apoA-I mimetic peptide 4F whether administered subcutaneously or orally.J. Lipid Res. 2011; 52: 1200-1210Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 6Navab M. Anantharamaiah G.M. Reddy S.T. Hama S. Hough G. Grijalva V.R. Wagner A.C. Frank J.S. Datta G. Garber D. et al.Oral D-4F causes formation of pre-beta high-density lipoprotein and improves high-density lipoprotein-mediated cholesterol efflux and reverse cholesterol transport from macrophages in apolipoprotein E-null mice.Circulation. 2004; 109: 3215-3220Crossref PubMed Scopus (312) Google Scholar). Under anesthesia, a nick was made in the inferior vena cava, and ice-cold physiological saline with 2 mM EDTA and 20 μM butylhydroxytoluene (BHT) was infused for 10 min at approximately 0.5 ml/min through a needle placed in the left ventricle. The organs appeared pale within a few minutes. After removal of blood as described above, the small intestine (i.e., the portion of intestine between the stomach and the cecum) was removed and kept on ice. Enterocytes were isolated using a minor modification of the method of Merchant and Heller (8Merchant J.L. Heller R.A. 3-Hydroxy-3-methylglutaryl coenzyme A reductase in isolated villous and crypt cells of the rat ileum.J. Lipid Res. 1977; 18: 722-733Abstract Full Text PDF PubMed Google Scholar). The lumen of the small intestine was thoroughly washed with ice-cold PBS containing 0.1 mM PMSF, 1 mM benzamidine, 2 mM EDTA, and 20 μM BHT. The small intestine was carefully cannulated with a steel rod, the distal end was tied, and the rod was carefully removed, inverting the small intestine. The ends of the inverted small intestine were tied. The tied inverted small intestine was incubated in a 15-ml centrifuge tube in 10 ml of buffer A (1.5 mM KC1, 96 mM NaCl, 8 mM KH2PO4, 27 mM sodium citrate, 5.6 mM Na2HPO4 [pH 7.3], 0.1 mM PMSF, and 1 mM benzamidine) for 15 min at 37°C on an eppendorf MixMate™ set at 300 rpm. Buffer A was replaced with buffer B (2.7 mM KC1, 137 mM NaCl, 1.5 mM EDTA, 1.5 mM KH2PO4, 8.1 mM Na2HPO4 (pH 7.4), 0.5 mM dithiothreitol, 0.1 mM PMSF, and 1 mM benzamidine) and incubated for 30 min at 37°C on the MixMate™ at 300 rpm. The small intestine was removed from the centrifuge tube, the tube was centrifuged at 1500 rpm for 15 min at 4°C, the supernatant above the enterocytes was removed, and the pelleted enterocytes were snap frozen. After all of the mice were processed, the enterocyte pellets were disrupted on ice in the presence of 2 mM EDTA, 20 μM BHT, and protease inhibitors using a tissue grinder and centrifuged at 4°C. The supernatant was collected and extracted using solid-phase extraction (Waters Oasis HLB cartridge and methanol). The cartridge eluent was evaporated under argon, resuspended in methanol, and centrifuged, and a portion of the supernatant was analyzed. The remaining supernatant was saved for determination of cell protein. The small intestine was collected as described above. The contents within the lumen of the small intestine (133 ± 62 mg per mouse) were collected on ice. Pyrogen-free water containing 20 μM BHT and protease inhibitors were added, and the contents were ground on ice and centrifuged at 4°C. The supernatant was extracted and analyzed. The livers of the mice were removed, snap frozen, and kept at −80°C. The frozen livers were removed from the freezer and kept on ice while samples were collected from each lobe, minced, homogenized, extracted, and analyzed. Mice were fasted overnight with free access to water and were anesthetized with a mixture of ketamine (100–150 mg/kg) and xylazine (5–10 mg/kg) using a total maximum volume of 10 ml/kg administered by intraperitoneal injection. Laparotomy was performed through an upper midline incision. After the liver and gallbladder were exposed, the lower end of the cystic duct was ligated, and the common bile duct was cannulated below the entrance of the cystic duct using PE-01 tubing (internal diameter, 0.12 mm; outer diameter, 0.25 mm). After successful cannulation using a dissecting microscope, hepatic bile was collected by gravity for 30–60 min into a vessel maintained in ice. Animal body temperature was maintained at 37°C by placing the mouse 18 inches below a heat lamp. The temperature observed at this distance was 37°C. The bile was rapidly frozen and stored at −80°C until analysis. Plasma was collected as described above. The tissues were rendered free of blood by perfusion with ice-cold saline for 10 min as described above. The liver was collected and processed as described above. The small intestine was removed, the contents within the lumen of the small intestine were collected, the lumen was washed with ice-cold saline, adventitial fat was rapidly removed, and the length of the small intestine was determined. The duodenum (the proximal 34% of the length of the small intestine between the stomach and the ligament of Treitz), the jejunum (the middle 36% of the length of the small intestine), and the ileum (the most distal 30% of the length of the small intestine) were separated, snap frozen, and kept at −80°C. The plasma, intestinal contents, and tissues were extracted and analyzed for D-4F content by LC-ESI/MS/MS using an internal standard of 15N13C-labeled 4F peptide as previously described (1Navab M. Reddy S.T. Anantharamaiah G.M. Imaizumi S. Hough G. Hama S. Fogelman A.M. Intestine may be a major site of action for the apoA-I mimetic peptide 4F whether administered subcutaneously or orally.J. Lipid Res. 2011; 52: 1200-1210Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 9Buga G.M. Navab M. Imaizumi S. Reddy S.T. Yekta B. Hough G. Chanslor S. Anantharamaiah G.M. Fogelman A.M. L-4F alters hyperlipidemic (but not healthy) mouse plasma to reduce platelet aggregation.Arterioscler. Thromb. Vasc. Biol. 2010; 30: 283-289Crossref PubMed Scopus (26) Google Scholar, 10Bloedon L.T. Dunbar R. Duffy D. Pinell-Salles P. Norris R. DeGroot B.J. Movva R. Navab M. Fogelman A.M. Rader D.J. Safety, pharmacokinetics, and pharmacodynamics of oral apoA-I mimetic peptide D-4F in high-risk cardiovascular patients.J. Lipid Res. 2008; 49: 1344-1352Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar–11Watson C.E. Weissbach N. Kjems L. Ayalasomayajula S. Zhang Y. Chang I. Navab M. Hama S. Hough G. Reddy S.T. et al.Treatment of patients with cardiovascular disease with L-4F, an apoA-1 mimetic, did not improve select biomarkers of HDL function.J. Lipid Res. 2011; 52: 361-373Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Statistical analyses were performed by ANOVA or unpaired two-tail t test or by linear regression using GraphPad Prism version 5.03 (GraphPad Software, San Diego, CA). SQ administration of D-4F resulted in ∼300-fold and ∼100-fold higher levels of peptide in the plasma and liver, respectively, compared with the levels found after oral administration (Table 1). In contrast, peptide levels in the contents of the small intestine or in the tissue of the small intestine were similar whether the peptide was administered orally or SQ. The average fold difference in peptide levels in the small intestine and in the contents of the small intestine was only 1.66 ± 0.33-fold (Table 1).TABLE 1D-4F levels in plasma and liver.D-4F levels in plasma and liver were orders of magnitude higher after SQ administration compared with oral administration, while D-4F levels in the small intestine were similar after SQ and oral administration. Female LDLR−/− mice (11–12 months of age) were placed on a WD and treated with D-4F. Mice (n = 5 per group) received drinking water with D-4F sufficient to give a daily dose of 900 μg/mouse/day or daily SQ injections of the same dosage on the back. After 2 weeks, the mice were fasted overnight and allowed access to water that did not contain D-4F (SQ group) or did contain D-4F (oral group). In the morning, 90 min after the SQ group received their injection, the mice were anesthetized, plasma was obtained, tissues were rendered free of blood by perfusion, and liver and small intestine were collected, processed, and analyzed by LC-ESI-MS/MS for D-4F as described in Materials and Methods. The data shown are mean ± SD. NS = not significant. D-4F levels in plasma and liver were orders of magnitude higher after SQ administration compared with oral administration, while D-4F levels in the small intestine were similar after SQ and oral administration. Female LDLR−/− mice (11–12 months of age) were placed on a WD and treated with D-4F. Mice (n = 5 per group) received drinking water with D-4F sufficient to give a daily dose of 900 μg/mouse/day or daily SQ injections of the same dosage on the back. After 2 weeks, the mice were fasted overnight and allowed access to water that did not contain D-4F (SQ group) or did contain D-4F (oral group). In the morning, 90 min after the SQ group received their injection, the mice were anesthetized, plasma was obtained, tissues were rendered free of blood by perfusion, and liver and small intestine were collected, processed, and analyzed by LC-ESI-MS/MS for D-4F as described in Materials and Methods. The data shown are mean ± SD. NS = not significant. Oral administration of D-4F reduced free 5-HETE, 15-HETE, 9-HODE, and 13-HODE in the contents of the small intestine of LDLR−/− mice fed a WD for 1 week (Table 2). The oral and SQ experiments were performed sequentially due to the number of mice processed and the amount of work required for flushing the blood from the mice before removing the small intestine and the time required for collecting the contents of the small intestine from each mouse. Administration of D-4F SQ also reduced the content of free 5-HETE, 15-HETE, 9-HODE, and 13-HODE in the contents of the small intestine of LDLR−/− mice fed a WD (Table 3). Although the results of the experiment described in TABLE 2, TABLE 3 were qualitatively similar, the values obtained in the second experiment (SQ) were much higher than in the first experiment (oral), suggesting variability between different mice studied at different times or variability in the processing of the contents of the small intestine. Supplementary Table I indicates that the free HETEs and HODEs measured in the contents of the small intestine could not be accounted for by undigested WD.TABLE 2Oral D-4F reduced free 5-HETE, 15-HETE, 9-HODE, and 13-HODE in the contents of the small intestineFemale LDLR−/− mice 6–7 months of age were placed on a WD and treated or not treated with D-4F. Mice (n = 8 per group) received drinking water without or with D-4F sufficient to give a daily dose of 900 μg/mouse/day. After 1 week the mice were anesthetized, and the contents of the small intestine were collected and analyzed by LC-ESI-MS/MS for free 5-HETE, 15-HETE, 9-HODE, and 13-HODE as described in Materials and Methods. Data are mean ± SD.TABLE 3SQ D-4F reduced free 5-HETE, 15-HETE, 9-HODE, and 13-HODE in the contents of the small intestineTABLE 3SQ D-4F reduced free 5-HETE, 15-HETE, 9-HODE, and 13-HODE in the contents of the small intestineFemale LDLR−/− mice 6–7 months of age were placed on a WD and treated or not treated with D-4F. Mice (n = 20 per group) received drinking water without D-4F and received SQ saline without or with D-4F at a daily dose of 900 μg/mouse/day. After 1 week, 2–3 h after the last SQ injection, the mice were anesthetized, and the contents of the small intestine were collected and analyzed by LC-ESI-MS/MS for free 5-HETE, 15-HETE, 9-HODE, and 13-HODE as described in Materials and Methods. The data shown are mean ± SD. Female LDLR−/− mice 6–7 months of age were placed on a WD and treated or not treated with D-4F. Mice (n = 8 per group) receiv
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