Distribution of human plasma PLTP mass and activity in hypo- and hyperalphalipoproteinemia

Plasma phospholipid transfer protein (PLTP) plays an important role in lipoprotein metabolism and reverse cholesterol transport. We have recently reported that plasma PLTP concentration correlates positively with plasma HDL cholesterol (HDL-C) but not with PLTP activity in healthy subjects. We have also shown that PLTP exists as active and inactive forms in healthy human plasma. In the present study, we measured plasma PLTP concentration and PLTP activity, and analyzed the distribution of PLTP in normolipidemic subjects (controls), cholesteryl ester transfer protein (CETP) deficiency, and hypo-alphalipoproteinemia (hypo-ALP). Plasma PLTP concentration was significantly lower (0.7 ± 0.4 mg/l, mean ± SD, n = 9, P < 0.001) in the hypo-ALP subjects, and significantly higher (19.5 ± 4.3 mg/l, n = 17, P < 0.001) in CETP deficiency than in the controls (12.4 ± 2.3 mg/l, n = 63). In contrast, we observed no significant differences in plasma PLTP activity between controls, hypo-ALP subjects, and CETP deficiency (6.2 ± 1.3, 6.1 ± 1.8, and 6.8 ± 1.2 μmol/ml/h, respectively). There was a positive correlation between PLTP concentration and plasma HDL-C (r = 0.81, n = 89, P < 0.001). By size exclusion chromatography analysis, we found that the larger PLTP containing particles without PLTP activity (inactive form of PLTP) were almost absent in the plasma of hypo-ALP subjects, and accumulated in the plasma of CETP deficiency compared with those of controls.These results indicate that the differences in plasma PLTP concentrations between hypo-ALP subjects, CETP deficiency, and controls are mainly due to the differences in the amount of the inactive form of PLTP. Plasma phospholipid transfer protein (PLTP) plays an important role in lipoprotein metabolism and reverse cholesterol transport. We have recently reported that plasma PLTP concentration correlates positively with plasma HDL cholesterol (HDL-C) but not with PLTP activity in healthy subjects. We have also shown that PLTP exists as active and inactive forms in healthy human plasma. In the present study, we measured plasma PLTP concentration and PLTP activity, and analyzed the distribution of PLTP in normolipidemic subjects (controls), cholesteryl ester transfer protein (CETP) deficiency, and hypo-alphalipoproteinemia (hypo-ALP). Plasma PLTP concentration was significantly lower (0.7 ± 0.4 mg/l, mean ± SD, n = 9, P < 0.001) in the hypo-ALP subjects, and significantly higher (19.5 ± 4.3 mg/l, n = 17, P < 0.001) in CETP deficiency than in the controls (12.4 ± 2.3 mg/l, n = 63). In contrast, we observed no significant differences in plasma PLTP activity between controls, hypo-ALP subjects, and CETP deficiency (6.2 ± 1.3, 6.1 ± 1.8, and 6.8 ± 1.2 μmol/ml/h, respectively). There was a positive correlation between PLTP concentration and plasma HDL-C (r = 0.81, n = 89, P < 0.001). By size exclusion chromatography analysis, we found that the larger PLTP containing particles without PLTP activity (inactive form of PLTP) were almost absent in the plasma of hypo-ALP subjects, and accumulated in the plasma of CETP deficiency compared with those of controls. These results indicate that the differences in plasma PLTP concentrations between hypo-ALP subjects, CETP deficiency, and controls are mainly due to the differences in the amount of the inactive form of PLTP. Epidemiological studies have demonstrated that the concentration of HDL cholesterol (HDL-C) is inversely related to the risk of coronary heart disease (CHD) (1Miller N.E. Thelle D.S. Forde O.H. Mjos O.D. The Tromso heart study. High-density lipoprotein and coronary heart disease: a prospective case- control study.Lancet. 1977; 1: 965-968Abstract PubMed Scopus (974) Google Scholar, 2Gordon D.J. 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Increased plasma and renal clearance of an exchangeable pool of apolipoprotein A-I in subjects with low levels of high density lipoprotein cholesterol.J. Clin. Invest. 1993; 91: 1743-1752Crossref PubMed Scopus (190) Google Scholar). In contrast, CETP deficiency causes hyperalphalipoproteinemaia secondary to defective transfer of CE from HDL to apoB containing lipoproteins, resulting in accumulation of HDL-CE and enlargement of HDL particles (34Yamashita S. Hirano K. Sakai N. Matsuzawa Y. Molecular biology and pathophysiological aspects of plasma cholesteryl ester transfer protein.Biochim. Biophys. Acta. 2000; 1529: 257-275Crossref PubMed Scopus (127) Google Scholar). Delayed catabolism of HDL in CETP deficiency has also been reported (35Ikewaki K. Rader D.J. Sakamoto T. Nishiwaki M. Wakimoto N. Schaefer J.R. Ishikawa T. Fairwell T. Zech L.A. Nakamura H. Nagano M. Brewer Jr., H.B. Delayed catabolism of high density lipoprotein apolipoproteins A-I and A-II in human cholesteryl ester transfer protein deficiency.J. Clin. Invest. 1993; 92: 1650-1658Crossref PubMed Scopus (141) Google Scholar). Recently, we developed a sandwich ELISA using two monoclonal antibodies to measure human plasma PLTP concentration, and reported a positive correlation between plasma PLTP concentration and HDL-C in healthy Japanese subjects (36Oka T. Ito M. Kujiraoka T. Nagano M. Ishihara M. Iwasaki T. Egashira T. Miller N.E. Hattori H. Measurement of human phosholipid transfer protein by sandwich enzyme-linked immunosorbent assay.Clin. Chem. 2000; 46: 1357-1364Crossref PubMed Scopus (38) Google Scholar). In contrast, there was no correlation between plasma PLTP concentration and PLTP activity (36Oka T. Ito M. Kujiraoka T. Nagano M. Ishihara M. Iwasaki T. Egashira T. Miller N.E. Hattori H. Measurement of human phosholipid transfer protein by sandwich enzyme-linked immunosorbent assay.Clin. Chem. 2000; 46: 1357-1364Crossref PubMed Scopus (38) Google Scholar). A similar result was reported by Huuskonen et al. in Finnish subjects using a different sandwich ELISA (37Huuskonen J. Ekström M. Tahvanainen E. Vainio A. Metso J. Pussinen P. Ehnholm C. Olkkonen V.M. Jauhiainen M. Quantitation of human plasma phospholipid transfer protein (PLTP): Relationship between PLTP mass and phospholipid transfer activity.Atherosclerosis. 2000; 151: 451-461Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). We have recently shown that PLTP exists in two forms; one is active and the other is inactive (38Oka T. Kujiraoka T. Ito M. Egashira T. Takahashi S. Nanjee M.N. Miller N.E. Metso J. Olkkonen V.M. Ehnholm C. Jauhiainen M. Hattori H. Distribution of phospholipid transfer protein in human plasma: presence of two forms of phospholipid transfer protein, one catalytically active and the other inactive.J. Lipid. Res. 2000; 41: 1651-1657Abstract Full Text Full Text PDF PubMed Google Scholar). In the present study, we have measured plasma PLTP concentration and PLTP activity in normolipidemic subjects (as controls), hypo-ALP subjects, and CETP deficient subjects, and have analyzed the distributions of PLTP mass and PLTP activity in fractions of their plasma separated by size exclusion chromatography. Egg phosphatidylcholine, bovine phosphatidylserine, and o-phenylenediamine (OPD) human serum albumin were purchased from Sigma Chemical Company (St. Louis, MO). 1-Palmitoyl-2-[1-14C]palmitoyl phosphatidylcholine (DPPC, 80–120 mCi/mmol) was from NEN Life Science Products Inc. (Boston, MA). Heparin (5,000 U/ml) was from Mochida Pharmaceutical Co, LTD (Tokyo, Japan). Horseradish peroxide (HRP)-conjugated streptavidin was from Vector Laboratories, Inc. (San Francisco, CA). HRP-conjugated rabbit anti-mouse IgG antibody was from Zymed Laboratories, Inc. (San Francisco, CA). Prestained SDS-PAGE standards were from Bio-Rad Labolatories, Inc. (Hercules, CA). HMW electrophoresis calibration kit was from Amersham Pharmacia Biotech Inc. (Piscataway, NJ). Blood samples were obtained from subjects with hypoalphalipoproteinemia, including Tangier disease (TD) (n = 2), LCAT deficiency (n = 2), familial HDL deficiency (FHD) (n = 3), and apoA-I deficiency (n = 1), and from subjects with hyperalphalipoproteinemia owing to homozygous CETP deficiency (n = 17), in the Second Department of Internal Medicine, Osaka University, Japan. Two TD subjects, TD1 and TD2, and two FHD subjects, FHD2 and FHD3, shown in Table 1 have been previously described for the ABCA1 gene mutation (39Nishida Y. Hirano K. Tsukamoto K. Nagano M. Ikegami C. Roomp K. Ishihara M. Sakane N. Zhang Z. Tsujii K. Matsuyama A. Ohama T. Matsuura F. Ishigami M. Sakai N. Hiraoka H. Hattori H. Wellington C. Yoshida Y. Misugi S. Hayden M.R. Egashira T. Yamashita S. Matsuzawa Y. Expression and functional analyses of novel mutations of ATP-binding cassette transporter-1 in Japanese patients with high-density lipoprotein deficiency.Biochim. Biophys. Res. Commun. 2002; 290: 713-721Crossref PubMed Scopus (27) Google Scholar, 40Ishii J. Nagano M. Kujiraoka T. Ishihara M. Egashira T. Takata D. Tsuji M. Hattori H. Emi M. Clinical variant of Tangier disease in Japan: mutation of the ABCA1 gene in hypoalphalipoproteinemia with corneal lipidosis.J. Hum. Genet. 2002; 47: 366-369Crossref PubMed Scopus (22) Google Scholar). Plasma from another patient with familial LCAT deficiency (n = 1) was collected in London (41Nanjee M.N. Cooke C.J. Olszewski W.L. Miller N.E. Lipid and apolipoprotein concentrations in prenodal leg lymph of fasted humans: Associations with plasma concentrations in normal subjects, lipoprotein lipase deficiency, and LCAT deficiency.J. Lipid Res. 2000; 41: 1317-1327Abstract Full Text Full Text PDF PubMed Google Scholar). Subjects with CETP deficiency were identified by screening plasma samples for CETP concentration by a sandwich ELISA (42Nagano M. Yamashita S. Hirano K. Kujiraoka T. Ito M. Sagehashi Y. Hattori H. Nakajima N. Maruyama T. Sakai N. Egashira T. Matsuzawa Y. Point mutation (-69 G→A) in the promoter region of cholesteryl ester transfer protein gene in Japanese hyperalphalipoproteinemic subjects.Arterioscler. Thromb. Vasc. Biol. 2001; 21: 985-990Crossref PubMed Scopus (25) Google Scholar), and the mutation of intron 14 G to A of the CETP gene was identified by PCR-reaction-restriction fragment polymorphism (42Nagano M. Yamashita S. Hirano K. Kujiraoka T. Ito M. Sagehashi Y. Hattori H. Nakajima N. Maruyama T. Sakai N. Egashira T. Matsuzawa Y. Point mutation (-69 G→A) in the promoter region of cholesteryl ester transfer protein gene in Japanese hyperalphalipoproteinemic subjects.Arterioscler. Thromb. Vasc. Biol. 2001; 21: 985-990Crossref PubMed Scopus (25) Google Scholar). CETP activity assays were carried out as previously described (42Nagano M. Yamashita S. Hirano K. Kujiraoka T. Ito M. Sagehashi Y. Hattori H. Nakajima N. Maruyama T. Sakai N. Egashira T. Matsuzawa Y. Point mutation (-69 G→A) in the promoter region of cholesteryl ester transfer protein gene in Japanese hyperalphalipoproteinemic subjects.Arterioscler. Thromb. Vasc. Biol. 2001; 21: 985-990Crossref PubMed Scopus (25) Google Scholar). Normolipidemic subjects (n = 63) were taken from our laboratory staff as controls. This study was approved by the ethical committee of Osaka University. Informed consent was obtained from all subjects. The plasma lipid profiles of subjects examined in this study are summarized in Table 1. Blood samples were taken after an overnight fast into EDTA-containing glass tubes (Terumo Corp., Tokyo, Japan), and were immediately centrifuged at 2,500 g at 4°C for 20 min. Plasma samples were stored at −80°C until used.TABLE 1.Lipid profiles in hypo-ALP subjects, CETP deficiency, and controlsSubjectsNumber (M / F)Age (Years)Total CholesterolHDL-C (mmol/l)Triglyceride (mmol/l)ApoA-I (g/l)ApoA-II (g/l)ApoB (g/l)LCAT def. 1M303.830.024.090.360.090.81LCAT def. 2M203.080.213.610.750.100.62LCAT def. 3M573.080.342.460.420.050.31TD 1M560.830.051.38<0.100.030.42TD 2F711.470.053.25<0.100.010.15ApoAI def. 1F303.030.180.41<0.100.070.75FHD 1M363.100.103.40<0.100.021.26FHD 2F532.770.181.670.220.130.97FHD 3M482.480.131.94<0.100.080.90Hypo-ALP9 (6/3)45 ± 172.63 ± 0.92*P < 0.001; level of statistical significance by which data of hypo-ALP subjects or CETP deficiency differ from the data of controls (calculated by Student's t-test).0.14 ± 0.10*P < 0.001; level of statistical significance by which data of hypo-ALP subjects or CETP deficiency differ from the data of controls (calculated by Student's t-test).2.47 ± 1.21*P < 0.001; level of statistical significance by which data of hypo-ALP subjects or CETP deficiency differ from the data of controls (calculated by Student's t-test).0.22 ± 0.24*P < 0.001; level of statistical significance by which data of hypo-ALP subjects or CETP deficiency differ from the data of controls (calculated by Student's t-test).0.06 ± 0.04*P < 0.001; level of statistical significance by which data of hypo-ALP subjects or CETP deficiency differ from the data of controls (calculated by Student's t-test).0.69 ± 0.35CETP deficiency17 (10/7)45 ± 157.85 ± 0.89*P < 0.001; level of statistical significance by which data of hypo-ALP subjects or CETP deficiency differ from the data of controls (calculated by Student's t-test).4.53 ± 0.83*P < 0.001; level of statistical significance by which data of hypo-ALP subjects or CETP deficiency differ from the data of controls (calculated by Student's t-test).1.32 ± 0.56*P < 0.001; level of statistical significance by which data of hypo-ALP subjects or CETP deficiency differ from the data of controls (calculated by Student's t-test).2.56 ± 0.41*P < 0.001; level of statistical significance by which data of hypo-ALP subjects or CETP deficiency differ from the data of controls (calculated by Student's t-test).0.42 ± 0.13*P < 0.001; level of statistical significance by which data of hypo-ALP subjects or CETP deficiency differ from the data of controls (calculated by Student's t-test).0.64 ± 0.13*P < 0.001; level of statistical significance by which data of hypo-ALP subjects or CETP deficiency differ from the data of controls (calculated by Student's t-test).Controls63 (34/29)39 ± 134.45 ± 0.451.51 ± 0.300.95 ± 0.401.48 ± 0.200.32 ± 0.600.82 ± 0.12M, Male; F, female. Data are represented as mean ± SD.* P < 0.001; level of statistical significance by which data of hypo-ALP subjects or CETP deficiency differ from the data of controls (calculated by Student's t-test). Open table in a new tab M, Male; F, female. Data are represented as mean ± SD. Lipoproteins were isolated from pooled fresh human plasma by sequential ultracentrifugation in a Beckman Ti 50.2 rotor using solid KBr to adjust the density (43Havel R.J. Eder H.A. Bragdon J.H. The distribution and chemical compsition of ultracentrifugally separated lipoproteins in human serum.J. Clin. Invest. 1955; 34: 1345-1353Crossref PubMed Scopus (6480) Google Scholar). After ultracentrifugation, lipoproteins were dialyzed and stored at 4°C. PLTP concentration was measured by sandwich ELISA using two monoclonal antibodies specific for PLTP as previously described (36Oka T. Ito M. Kujiraoka T. Nagano M. Ishihara M. Iwasaki T. Egashira T. Miller N.E. Hattori H. Measurement of human phosholipid transfer protein by sandwich enzyme-linked immunosorbent assay.Clin. Chem. 2000; 46: 1357-1364Crossref PubMed Scopus (38) Google Scholar, 38Oka T. Kujiraoka T. Ito M. Egashira T. Takahashi S. Nanjee M.N. Miller N.E. Metso J. Olkkonen V.M. Ehnholm C. Jauhiainen M. Hattori H. Distribution of phospholipid transfer protein in human plasma: presence of two forms of phospholipid transfer protein, one catalytically active and the other inactive.J. Lipid. Res. 2000; 41: 1651-1657Abstract Full Text Full Text PDF PubMed Google Scholar). The assay range of the ELISA was from 0.6 to 15 ng/well. The sample was diluted adequately to adjust the measurable range. Assays were carried out in duplicate. The intra- and inter-assay coefficients of variation (n = 5) were less than 5%. In our ELISA for PLTP mass concentration, dose-dependency curves in samples of plasma (contains both inactive and active form of PLTP), washed HDL3 (contains inactive form of PLTP alone), and recombinant human PLTP (contains active form of PLTP alone) were identical (Fig. 1), indicating that our sandwich ELISA react equally with the inactive and active forms of PLTP. PLTP activity was measured by a liposome-HDL3 system as previously described (36Oka T. Ito M. Kujiraoka T. Nagano M. Ishihara M. Iwasaki T. Egashira T. Miller N.E. Hattori H. Measurement of human phosholipid transfer protein by sandwich enzyme-linked immunosorbent assay.Clin. Chem. 2000; 46: 1357-1364Crossref PubMed Scopus (38) Google Scholar, 38Oka T. Kujiraoka T. Ito M. Egashira T. Takahashi S. Nanjee M.N. Miller N.E. Metso J. Olkkonen V.M. Ehnholm C. Jauhiainen M. Hattori H. Distribution of phospholipid transfer protein in human plasma: presence of two forms of phospholipid transfer protein, one catalytically active and the other inactive.J. Lipid. Res. 2000; 41: 1651-1657Abstract Full Text Full Text PDF PubMed Google Scholar). The assay range of PLTP activity was from 1.0 to 13.0 μmol/ml/h. Assays were carried out in duplicate. The intra- and inter-assay coefficients of variation (n = 5) were less than 10%. Size exclusion chromatography was performed as previously described (38Oka T. Kujiraoka T. Ito M. Egashira T. Takahashi S. Nanjee M.N. Miller N.E. Metso J. Olkkonen V.M. Ehnholm C. Jauhiainen M. Hattori H. Distribution of phospholipid transfer protein in human plasma: presence of two forms of phospholipid transfer protein, one catalytically active and the other inactive.J. Lipid. Res. 2000; 41: 1651-1657Abstract Full Text Full Text PDF PubMed Google Scholar). Human plasma (1.5 m) was fractionated by fast protein liquid chromatography, using two Superose 6HR 10/30 columns (Amersham Pharmacia Biotech, Uppsala, Sweden) connected in series and equilibrated with SMT buffer (10 mM Tris, 225 mM mannitol, 65 mM sucrose, 1 mM EDTA, pH 8.1) (44Clark R.W. Moberly J.B. Bamberger M.J. Low level quantification of cholesteryl ester transfer protein in plasma subfractions and cell culture media by monoclonal antibody-based immunoassay.J. Lipid Res. 1995; 36: 876-889Abstract Full Text PDF PubMed Google Scholar). Chromatography was performed at a flow rate of 0.25 ml/min, and 0.5 ml fractions were collected (38Oka T. Kujiraoka T. Ito M. Egashira T. Takahashi S. Nanjee M.N. Miller N.E. Metso J. Olkkonen V.M. Ehnholm C. Jauhiainen M. Hattori H. Distribution of phospholipid transfer protein in human plasma: presence of two forms of phospholipid transfer protein, one catalytically active and the other inactive.J. Lipid. Res. 2000; 41: 1651-1657Abstract Full Text Full Text PDF PubMed Google Scholar). SDS-PAGE and non-denaturing-PAGE were performed using a 5–20% gradient polyacrylamide gel (ATTO, Tokyo, Japan) as previously described (38Oka T. Kujiraoka T. Ito M. Egashira T. Takahashi S. Nanjee M.N. Miller N.E. Metso J. Olkkonen V.M. Ehnholm C. Jauhiainen M. Hattori H. Distribution of phospholipid transfer protein in human plasma: presence of two forms of phospholipid transfer protein, one catalytically active and the other inactive.J. Lipid. Res. 2000; 41: 1651-1657Abstract Full Text Full Text PDF PubMed Google Scholar). Western blotting was performed with anti-PLTP monoclonal antibody 113, followed by incubation with HRP conjugated rabbit anti-mouse IgG, as