Evidence for a complex relationship between apoA-V and apoC-III in patients with severe hypertriglyceridemia
2006; Elsevier BV; Volume: 47; Issue: 10 Linguagem: Inglês
10.1194/jlr.m500533-jlr200
ISSN1539-7262
AutoresFrank G. Schaap, Melchior C. Nierman, Jimmy F.P. Berbée, Hiroaki Hattori, Philippa J. Talmud, Stefan F.C. Vaessen, Patrick C.N. Rensen, Robert A.F.M. Chamuleau, Jan Albert Kuivenhoven, Albert K. Groen,
Tópico(s)Diabetes, Cardiovascular Risks, and Lipoproteins
ResumoThe relevance of apolipoprotein A-V (apoA-V) for human lipid homeostasis is underscored by genetic association studies and the identification of truncation-causing mutations in the APOA5 gene as a cause of type V hyperlipidemia, compatible with an LPL-activating role of apoA-V. An inverse correlation between plasma apoA-V and triglyceride (TG) levels has been surmised from animal data. Recent studies in human subjects using (semi)quantitative immunoassays, however, do not provide unambiguous support for such a relationship. Here, we used a novel, validated ELISA to measure plasma apoA-V levels in patients (n = 28) with hypertriglyceridemia (HTG; 1.8–78.7 mmol TG/l) and normolipidemic controls (n = 42). Unexpectedly, plasma apoA-V levels were markedly increased in the HTG subjects compared with controls (1,987 vs. 258 ng/ml; P < 0.001). In the HTG group, apoA-V and TG were positively correlated (r = +0.44, P = 0.02). In addition, we noted an increased level of the LPL-inhibitory protein apoC-III in the HTG group (45.8 vs. 10.6 mg/dl in controls; P < 0.001). The correlation between apoA-V and TG levels in the HTG group disappeared (partial r = +0.09, P = 0.65) when controlling for apoC-III levels. In contrast, apoC-III and TG remained positively correlated in this group when controlling for apoA-V (partial r = +0.43, P = 0.025). Our findings suggest that in HTG patients, increased TG levels are accompanied by high plasma levels of apoA-V and apoC-III, apolipoproteins with opposite modes of action. This study provides evidence for a complex interaction between apoA-V and apoC-III in patients with severe HTG. The relevance of apolipoprotein A-V (apoA-V) for human lipid homeostasis is underscored by genetic association studies and the identification of truncation-causing mutations in the APOA5 gene as a cause of type V hyperlipidemia, compatible with an LPL-activating role of apoA-V. An inverse correlation between plasma apoA-V and triglyceride (TG) levels has been surmised from animal data. Recent studies in human subjects using (semi)quantitative immunoassays, however, do not provide unambiguous support for such a relationship. Here, we used a novel, validated ELISA to measure plasma apoA-V levels in patients (n = 28) with hypertriglyceridemia (HTG; 1.8–78.7 mmol TG/l) and normolipidemic controls (n = 42). Unexpectedly, plasma apoA-V levels were markedly increased in the HTG subjects compared with controls (1,987 vs. 258 ng/ml; P < 0.001). In the HTG group, apoA-V and TG were positively correlated (r = +0.44, P = 0.02). In addition, we noted an increased level of the LPL-inhibitory protein apoC-III in the HTG group (45.8 vs. 10.6 mg/dl in controls; P < 0.001). The correlation between apoA-V and TG levels in the HTG group disappeared (partial r = +0.09, P = 0.65) when controlling for apoC-III levels. In contrast, apoC-III and TG remained positively correlated in this group when controlling for apoA-V (partial r = +0.43, P = 0.025). Our findings suggest that in HTG patients, increased TG levels are accompanied by high plasma levels of apoA-V and apoC-III, apolipoproteins with opposite modes of action. This study provides evidence for a complex interaction between apoA-V and apoC-III in patients with severe HTG. Abbreviations apoA-Vapolipoprotein A-VHTG, hypertriglyceridemiahypertriglyceridemicMAbmonoclonal antibodyTCtotal cholesterolTGtriglyceride apolipoprotein A-V hypertriglyceridemic monoclonal antibody total cholesterol triglyceride The recognition of hypertriglyceridemia (HTG) as an independent risk factor for cardiovascular pathologies (1Cullen P. Evidence that triglycerides are an independent coronary heart disease risk factor..Am. J. 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Vrins C. van der Vliet H.N. Chamuleau R.A. Havekes L.M. Groen A.K. van Dijk K.W. ApoAV reduces plasma triglycerides by inhibiting very low density lipoprotein-triglyceride (VLDL-TG) production and stimulating lipoprotein lipase-mediated VLDL-TG hydrolysis..J. Biol. Chem. 2004; 279: 27941-27947Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 10Fruchart-Najib J. Bauge E. Niculescu L.S. Pham T. Thomas B. Rommens C. Majd Z. Brewer B. Pennacchio L.A. Fruchart J.C. Mechanism of triglyceride lowering in mice expressing human apolipoprotein A5..Biochem. Biophys. Res. Commun. 2004; 319: 397-404Crossref PubMed Scopus (177) Google Scholar). ApoA-V has readily become recognized as an important determinant of plasma TG levels in humans and mice since its discovery 5 years ago (11Pennacchio L.A. Olivier M. Hubacek J.A. Cohen J.C. Cox D.R. Fruchart J.C. Krauss R.M. Rubin E.M. 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Chamuleau R.A. Adenoviral overexpression of apolipoprotein A-V reduces serum levels of triglycerides and cholesterol in mice..Biochem. Biophys. Res. Commun. 2002; 295: 1156-1159Crossref PubMed Scopus (153) Google Scholar) [e.g., adenoviral expression of apoa5 in mice resulted in a dose-dependent reduction of plasma TG levels (9Schaap F.G. Rensen P.C. Voshol P.J. Vrins C. van der Vliet H.N. Chamuleau R.A. Havekes L.M. Groen A.K. van Dijk K.W. ApoAV reduces plasma triglycerides by inhibiting very low density lipoprotein-triglyceride (VLDL-TG) production and stimulating lipoprotein lipase-mediated VLDL-TG hydrolysis..J. Biol. Chem. 2004; 279: 27941-27947Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar)]. In humans, genetic variation at the APOA5 locus has been associated with HTG (11Pennacchio L.A. Olivier M. Hubacek J.A. Cohen J.C. Cox D.R. Fruchart J.C. Krauss R.M. Rubin E.M. An apolipoprotein influencing triglycerides in humans and mice revealed by comparative sequencing..Science. 2001; 294: 169-173Crossref PubMed Scopus (801) Google Scholar, 14Pennacchio L.A. Olivier M. Hubacek J.A. Krauss R.M. Rubin E.M. Cohen J.C. Two independent apolipoprotein A5 haplotypes influence human plasma triglyceride levels..Hum. Mol. Genet. 2002; 11: 3031-3038Crossref PubMed Google Scholar, 15Kao J.T. Wen H.C. Chien K.L. Hsu H.C. Lin S.W. A novel genetic variant in the apolipoprotein A5 gene is associated with hypertriglyceridemia..Hum. Mol. Genet. 2003; 12: 2533-2539Crossref PubMed Scopus (155) Google Scholar, 16Olivier M. Wang X. Cole R. Gau B. Kim J. Rubin E.M. Pennacchio L.A. Haplotype analysis of the apolipoprotein gene cluster on human chromosome 11..Genomics. 2004; 83: 912-923Crossref PubMed Scopus (94) Google Scholar, 17Talmud P.J. Hawe E. Martin S. Olivier M. Miller G.J. Rubin E.M. Pennacchio L.A. Humphries S.E. Relative contribution of variation within the APOC3/A4/A5 gene cluster in determining plasma triglycerides..Hum. Mol. Genet. 2002; 11: 3039-3046Crossref PubMed Scopus (341) Google Scholar). Moreover, homozygosity for truncation-causing mutations (Q148X and Q139X) in the APOA5 gene was recently shown to be associated with severe HTG in humans (18Oliva C.P. Pisciotta L. Li V.G. Sambataro M.P. Cantafora A. Bellocchio A. Catapano A. Tarugi P. Bertolini S. Calandra S. Inherited apolipoprotein A-V deficiency in severe hypertriglyceridemia..Arterioscler. Thromb. Vasc. Biol. 2005; 25: 411-417Crossref PubMed Scopus (168) Google Scholar, 19Marcais C. Verges B. Charriere S. Pruneta V. Merlin M. Billon S. Perrot L. Drai J. Sassolas A. Pennacchio L.A. et al.Apoa5 Q139X truncation predisposes to late-onset hyperchylomicronemia due to lipoprotein lipase impairment..J. Clin. Invest. 2005; 115: 2862-2869Crossref PubMed Scopus (140) Google Scholar). Hence, besides the deficiency of either LPL or apoC-II, mutations in the APOA5 gene were identified as a novel cause of type I hyperlipidemia (OMIM 238600), although it may be noted that patients with apoA-V mutations primarily present with type V hyperlipidemia (OMIM 144650). Although the underlying mechanism is still obscure, several lines of evidence indicate that apoA-V stimulates LPL activity (8Merkel M. Loeffler B. Kluger M. Fabig N. Geppert G. Pennacchio L.A. Laatsch A. Heeren J. Apolipoprotein AV accelerates plasma hydrolysis of triglyceride-rich lipoproteins by interaction with proteoglycan-bound lipoprotein lipase..J. Biol. Chem. 2005; 280: 21553-21560Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar, 9Schaap F.G. Rensen P.C. Voshol P.J. Vrins C. van der Vliet H.N. Chamuleau R.A. Havekes L.M. Groen A.K. van Dijk K.W. ApoAV reduces plasma triglycerides by inhibiting very low density lipoprotein-triglyceride (VLDL-TG) production and stimulating lipoprotein lipase-mediated VLDL-TG hydrolysis..J. Biol. Chem. 2004; 279: 27941-27947Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 10Fruchart-Najib J. Bauge E. Niculescu L.S. Pham T. Thomas B. Rommens C. Majd Z. Brewer B. Pennacchio L.A. Fruchart J.C. Mechanism of triglyceride lowering in mice expressing human apolipoprotein A5..Biochem. Biophys. Res. Commun. 2004; 319: 397-404Crossref PubMed Scopus (177) Google Scholar, 20Grosskopf I. Baroukh N. Lee S.J. Kamari Y. Harats D. Rubin E.M. Pennacchio L.A. Cooper A.D. Apolipoprotein A-V deficiency results in marked hypertriglyceridemia attributable to decreased lipolysis of triglyceride-rich lipoproteins and removal of their remnants..Arterioscler. Thromb. Vasc. Biol. 2005; 25: 2573-2579Crossref PubMed Scopus (118) Google Scholar, 21Lookene A. Beckstead J.A. Nilsson S. Olivecrona G. Ryan R.O. Apolipoprotein A-V-heparin interactions: implications for plasma lipoprotein metabolism..J. Biol. Chem. 2005; 280: 25383-25387Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 22Merkel M. Heeren J. Give me A5 for lipoprotein hydrolysis!.J. Clin. Invest. 2005; 115: 2694-2696Crossref PubMed Scopus (87) Google Scholar). Whether this effect is direct, as suggested by dose-dependent stimulation of LPL activity in vitro (9Schaap F.G. Rensen P.C. Voshol P.J. Vrins C. van der Vliet H.N. Chamuleau R.A. Havekes L.M. Groen A.K. van Dijk K.W. ApoAV reduces plasma triglycerides by inhibiting very low density lipoprotein-triglyceride (VLDL-TG) production and stimulating lipoprotein lipase-mediated VLDL-TG hydrolysis..J. Biol. Chem. 2004; 279: 27941-27947Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar), or indirect, through improved capturing of TG-rich lipoproteins by heparan sulfate proteoglycan-bound LPL (8Merkel M. Loeffler B. Kluger M. Fabig N. Geppert G. Pennacchio L.A. Laatsch A. Heeren J. Apolipoprotein AV accelerates plasma hydrolysis of triglyceride-rich lipoproteins by interaction with proteoglycan-bound lipoprotein lipase..J. Biol. Chem. 2005; 280: 21553-21560Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar, 22Merkel M. Heeren J. Give me A5 for lipoprotein hydrolysis!.J. Clin. Invest. 2005; 115: 2694-2696Crossref PubMed Scopus (87) Google Scholar) or through stabilization of the active dimeric form of LPL, remains to be determined. It is of interest that apoA-V and apoC-III appear to have opposite modes of action (7van Dijk K.W. Rensen P.C. Voshol P.J. Havekes L.M. The role and mode of action of apolipoproteins CIII and AV: synergistic actors in triglyceride metabolism?.Curr. Opin. Lipidol. 2004; 15: 239-246Crossref PubMed Scopus (126) Google Scholar). Accordingly, although gene disruption or overexpression of either apoa5 or apoc3 has a large impact on plasma TG levels, simultaneous deletion of both endogenous genes or simultaneous transgenic overexpression of human APOA5 and APOC3 has no effect on plasma TG levels in mice (23Baroukh N. Bauge E. Akiyama J. Chang J. Afzal V. Fruchart J.C. Rubin E.M. Fruchart-Najib J. Pennacchio L.A. Analysis of apolipoprotein A5, c3, and plasma triglyceride concentrations in genetically engineered mice..Arterioscler. Thromb. Vasc. Biol. 2004; 24: 1297-1302Crossref PubMed Scopus (73) Google Scholar). In agreement with this, recombinant apoA-V was able to fully overcome the LPL-inhibitory effect of apoC-III in vitro (9Schaap F.G. Rensen P.C. Voshol P.J. Vrins C. van der Vliet H.N. Chamuleau R.A. Havekes L.M. Groen A.K. van Dijk K.W. ApoAV reduces plasma triglycerides by inhibiting very low density lipoprotein-triglyceride (VLDL-TG) production and stimulating lipoprotein lipase-mediated VLDL-TG hydrolysis..J. Biol. Chem. 2004; 279: 27941-27947Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). The relative amounts of apoA-V and apoC-III in plasma, and most likely their distribution over lipoproteins, may influence LPL activity and ultimately TG levels. With the recent development of immunoassays for the quantification of apoA-V, it became apparent that plasma levels of apoA-V in normolipidemic subjects are rather low (157–198 ng/ml) compared with other apolipoproteins (24Ishihara M. Kujiraoka T. Iwasaki T. Nagano M. Takano M. Ishii J. Tsuji M. Ide H. Miller I.P. Miller N.E. et al.A sandwich enzyme-linked immunosorbent assay for human plasma apolipoprotein A-V concentration..J. Lipid Res. 2005; 46: 2015-2022Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 25O'Brien P.J. Alborn W.E. Sloan J.H. Ulmer M. Boodhoo A. Knierman M.D. Schultze A.E. Konrad R.J. The novel apolipoprotein A5 is present in human serum, is associated with VLDL, HDL, and chylomicrons, and circulates at very low concentrations compared with other apolipoproteins..Clin. Chem. 2005; 51: 351-359Crossref PubMed Scopus (182) Google Scholar). In this study, we aimed to determine apoA-V levels and study the correlation with lipid parameters in human subjects with varying degrees of HTG. Plasma apoA-V levels were determined using a newly developed ELISA. Patients referred to our lipid clinic (Academic Medical Center, Amsterdam, The Netherlands) were recruited for this study cohort when fasting plasma TG levels were >10 mmol/l on the initial consultation. Subjects with mutations in the LPL gene, as determined by LPL gene sequencing, were excluded from the cohort. The subjects studied here were of European ethnicity and presented with type V hyperlipidemia. At the time of blood sampling for the current lipid and apolipoprotein measurements, the majority of the subjects (18 of 26) were using various lipid-lowering regimens (pharmaceutical or dietary intervention) to manage their hyperlipidemia. Fasted plasma obtained from normolipidemic volunteers (n = 42) was used to obtain reference values for apoA-V levels. The study protocol was approved by the institutional review board of the Academic Medical Center, and all participants gave written informed consent. Fasting blood was collected in heparinized tubes. After centrifugation (15 min, 3,000 g at 4°C), the plasma was divided into aliquots and frozen at −80°C for later use; buffy coat was used for the isolation of genomic DNA. Plasma levels of TG and total cholesterol (TC) were measured using established enzymatic assays. Immunoblot detection of apoA-V in human plasma has been described previously (26Prieur X. Schaap F.G. Coste H. Rodriguez J.C. Hepatocyte nuclear factor-4{alpha} regulates the human apolipoprotein AV gene: identification of a novel response element and involvement in the control by PGC-1{alpha}, AMP-activated protein kinase and MAP kinase pathway..Mol. Endocrinol. 2005; 19: 3107-3125Crossref PubMed Scopus (35) Google Scholar). Hypertriglyceridemic (HTG) subjects were genotyped for the −1131T>C and c.56C>G APOA5 polymorphisms as detailed elsewhere (17Talmud P.J. Hawe E. Martin S. Olivier M. Miller G.J. Rubin E.M. Pennacchio L.A. Humphries S.E. Relative contribution of variation within the APOC3/A4/A5 gene cluster in determining plasma triglycerides..Hum. Mol. Genet. 2002; 11: 3039-3046Crossref PubMed Scopus (341) Google Scholar). Plasma levels of apoA-V were determined using a newly developed sandwich ELISA. Anti-human apoA-V monoclonal antibodies (MAbs) B2B and E8E were obtained by genetic immunization of mice followed by boosting of animals with recombinant human apoA-V protein as described elsewhere (24Ishihara M. Kujiraoka T. Iwasaki T. Nagano M. Takano M. Ishii J. Tsuji M. Ide H. Miller I.P. Miller N.E. et al.A sandwich enzyme-linked immunosorbent assay for human plasma apolipoprotein A-V concentration..J. Lipid Res. 2005; 46: 2015-2022Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Different blocking agents were tested for their efficacy: it was noted that use of 3.0% BSA, 1.0% gelatin, or 1.0% nonfat dry milk powder as a blocking agent resulted in a high level (50–90%) of nonspecific binding of apoA-V to the microtiter plate (i.e., binding independent of the presence of capture MAb). Far better results were obtained when using 1.0% casein as a blocking agent, giving only 5% nonspecific binding. Ninety-six-well microtiter plates (MaxiSorb; NUNC) were coated overnight at 4°C with 100 μl of MAb B2B (1.5 μg/ml in 50 mmol/l carbonate buffer, pH 9.6). Except for this coating step, all further plate handling was performed at room temperature and incubations were performed on a rocking platform. Plates were covered with an adhesive foil to prevent evaporation. After coating with capture MAb, plates were washed once with PBX (PBS containing 0.1% Triton X-100) and blocked by incubating for 1 h with 200 μl/well PBXC (PBX containing 1.0% casein; Hammarsten grade; Merck). The plates were subsequently washed once with PBX, and samples, reference sera, and standards (100 μl/well, diluted in PBXC) were added. Antigen was captured during a 2 h incubation period, followed by four washes with PBX to remove unbound and/or nonspecifically bound proteins. Captured antigen was detected by adding 100 μl/well biotinylated MAb E8E (1.0 μg/ml in PBXC), and plates were incubated for 2 h. Excess detector MAb was washed away with four rinses with PBX. Then, 100 μl/well streptavidin-conjugated HRP (Dako) diluted 1:3,000 in PBXC was added. After 30 min of incubation, plates were washed four times with PBX and 100 μl/well freshly prepared HRP substrate solution (100 μg/ml tetramethylbenzidine in 0.1 mol/l NaAc, pH 5.5, containing 0.003% hydrogen peroxide) was added. Product formation was stopped after exactly 30 min by the addition of 2 mol/l sulfuric acid (100 μl/well). After brief mixing, absorbance at 450 nm was measured (Easia reader; Medgenix Diagnostics). Dilutions of a calibrated control plasma and recombinant human apoA-V (9Schaap F.G. Rensen P.C. Voshol P.J. Vrins C. van der Vliet H.N. Chamuleau R.A. Havekes L.M. Groen A.K. van Dijk K.W. ApoAV reduces plasma triglycerides by inhibiting very low density lipoprotein-triglyceride (VLDL-TG) production and stimulating lipoprotein lipase-mediated VLDL-TG hydrolysis..J. Biol. Chem. 2004; 279: 27941-27947Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar) were included in each assay. Recombinant human apoA-V was >95% pure as estimated by SDS-PAGE, and recombinant protein concentration was determined by the bicinchoninic acid method using BSA as a standard. Calibration curves were fitted by linear regression, and correlation coefficients were typically >0.999. Reagent blanks had a typical absorbance of 0.060. Because of the nonlinearity of absorbance measurements, optical density values > 2.0 were ignored and samples were reanalyzed at higher dilution. ApoC-III levels were determined using a sandwich ELISA specific for human apoC-III. Plasma samples were diluted in wash buffer (PBS and 0.05% Tween-20) containing 0.1% casein. Briefly, wells of Costar medium binding microtiter plates were coated overnight at 4°C with 100 μl of a polyclonal rabbit anti-human apoC-III antibody (1.0 μg/ml in PBS; Academy Biomedical Co., Houston, TX). After rinsing and the addition of samples, plates were incubated for 2 h at 37°C. After washing, HRP-conjugated goat anti-human apoC-III polyclonal antibody (0.75 μg/ml in sample dilution buffer; Academy Biomedical Co.) was added. After incubation for 2 h at 37°C, HRP activity was detected with tetramethylbenzidine for 20 min at room temperature. Plasma from apoc3 wild-type mice spiked with human apoC-III (Academy Biomedical Co.) was used as a standard. Date are presented as means ± SD. Pearson's correlation coefficients and corresponding P values were calculated to assess the relationship between tested parameters. Mann-Whitney testing was used for comparisons between HTG subjects and normolipidemic controls. P < 0.05 was considered significant. For the determination of apoA-V levels, a sandwich ELISA was developed that used different MAbs for antigen capture (MAb B2B) and antigen detection (MAb E8E). For reference, we used plasma from the institutional blood bank in each assay. Serial dilutions of the reference plasma resulted in a typical sigmoid-shaped response curve (Fig. 1). The reference plasma was calibrated using recombinant human apoA-V of high purity (9Schaap F.G. Rensen P.C. Voshol P.J. Vrins C. van der Vliet H.N. Chamuleau R.A. Havekes L.M. Groen A.K. van Dijk K.W. ApoAV reduces plasma triglycerides by inhibiting very low density lipoprotein-triglyceride (VLDL-TG) production and stimulating lipoprotein lipase-mediated VLDL-TG hydrolysis..J. Biol. Chem. 2004; 279: 27941-27947Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar) and contained 205.4 ± 12.5 ng apoA-V/ml. Semiquantitative analysis by immunoblotting was used as an independent method to validate apoA-V levels as determined by ELISA (Fig. 2A). Spiking of diluted reference plasma with known amounts (0.1–3.0 ng) of recombinant apoA-V resulted in calculated recoveries of 93.1–100.3%. The lower limit of detection of our ELISA was ∼0.2 ng/ml. Intra-assay and interassay (n = 8–12) variations of three plasma samples (108–266 ng apoA-V/ml) were 2.4 ± 2.4% and 6.5 ± 2.3%, respectively.Fig. 2.Immunoblot validation of apoA-V levels determined by sandwich ELISA. Protein was separated on 10% polyacrylamide gels and transferred to polyvinylidene difluoride membranes. ApoA-V was detected using a polyclonal anti-human apoA-V IgG fraction and appropriate secondary antibodies. A: Immunoblot analysis of apoA-V in 0.25, 0.5, 1.0, and 2.0 μl of plasma (left four lanes; ELISA value = 4,035 ng/ml) and the indicated amounts of recombinant human apoA-V (right four lanes). Quantification of chemiluminescent signals led to a mean calculated value of 4,241 ng/ml for the plasma sample, validating the quantification by ELISA. B: Confirmation of increased apoA-V levels in hypertriglyceridemic (HTG) subjects using immunoblot analysis. Plasma apoA-V expression in a normolipidemic volunteer with an average apoA-V level (left lane; 205 ng apoA-V/ml) and five HTG subjects (right five lanes; 405, 826, 2,044, 3,935, and 7,767 ng apoA-V/ml, respectively) with the indicated fold of expression relative to the control plasma is shown. Note that to prevent signal overexposure, five times less plasma of the three right-most HTG subjects was used for analysis. There is excellent agreement (r = 0.98) between values determined by ELISA and relative values derived from the quantification of chemiluminescent apoA-V signals.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Fasted plasma apoA-V levels were determined in 28 HTG patients and in 42 normolipidemic controls. Relevant characteristics and biochemical parameters of these study groups are shown in Table 1 . TG levels of the HTG individuals averaged 16.2 ± 18.7 mmol/l (range, 1.8–78.7 mmol/l). In addition, this cohort was characterized by increased TC levels compared with normolipidemic controls (7.6 ± 5.2 vs. 4.5 ± 0.9 mmol/l, respectively).TABLE 1.Characteristics of the HTG population and normolipidemic controlsCharacteristicHTG Subjects (n = 28)Normolipidemic Subjects (n = 42)Female gender (%)4350Age (years)41 ± 15 (10–60)n.d.Type 2 diabetes (%)27n.d.Body mass index (kg/m2)26.5 ± 4.4 (20.1–38.6)n.d.TG (mmol/l)16.2 ± 18.7 aSignificantly different from normolipidemic subjects (P < 0.001). (1.8–78.7)0.9 ± 0.4 (0.3–1.9)TC (mmol/l)7.6 ± 5.2 aSignificantly different from normolipidemic subjects (P < 0.001). (3.1–27.4)4.5 ± 0.9 (2.8–6.8)ApoA-V (ng/ml)1,987 ± 2,603 aSignificantly different from normolipidemic subjects (P < 0.001). (209–10032)258 ± 146 (83–742)ApoC-III (mg/dl)45.8 ± 33.8 aSignificantly different from normolipidemic subjects (P < 0.001). (9.7–122.4)10.6 ± 3.6 (4.6–22.5)ApoC-III/apoA-V (mol/mol)2.3 × 103 ± 2.4 × 103 (0.4–9.8 × 103)2.4 × 103 ± 1.5 × 103 (0.9–6.5 × 103)ApoA-V, apolipoprotein A-V; HTG, hypertriglyceridemic; n.d., not determined; TC, total cholesterol; TG, triglyceride. Values are presented as means ± SD, and ranges are given in parentheses.a Significantly different from normolipidemic subjects (P < 0.001). Open table in a new tab ApoA-V, apolipoprotein A-V; HTG, hypertriglyceridemic; n.d., not determined; TC, total cholesterol; TG, triglyceride. Values are presented as means ± SD, and ranges are given in parentheses. Importantly, we identified a marked 7.7-fold increase in mean plasma apoA-V levels in HTG subjects compared with controls (1,987 ± 2,603 vs. 258 ± 146 ng/ml, respectively; P < 0.001). Importantly, no interference was observed in our ELISA upon spiking of plasma samples with Intralipid to achieve a final TG level of 1.0 mmol/l, which is well above the final TG level of 0.033 mmol/l attained in diluted plasma of the most HTG subject (data not shown). The increased apoA-V level in the HTG group was confirmed qualitatively by immunoblot analysis using anti-apoA-V antibodies different from those used for ELISA (Fig. 2B). In addition, we noted on average 4.3-fold higher apoC-III levels in HTG subjects compared with controls (45.8 ± 33.8 vs. 10.6 ± 3.6 mg/dl, respectively; P < 0.001). In agreement with published observations (17Talmud P.J. Hawe E. Martin S. Olivier M. Miller G.J. Rubin E.M. Pennacchio L.A. Humphries S.E. Relative contribution of variation within the APOC3/A4/A5 gene cluster in determining plasma triglycerides..Hum. Mol. Genet. 2002; 11: 3039-3046Crossref PubMed Scopus (341) Google Scholar), the rare allele frequencies of two APOA5 variants that have been associated with increase
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