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Determination of human plasma phospholipid transfer protein mass and activity
Methods 36 (2005) 97–101 www.elsevier.com/locate/ymeth
Determination of human plasma phospholipid transfer protein mass and activity Matti Jauhiainen ¤, Christian Ehnholm Department of Molecular Medicine, National Public Health Institute, Biomedicum, Haartmaninkatu 8, FIN-00251 Helsinki, Finland Accepted 1 November 2004
Abstract Plasma phospholipid transfer protein (PLTP) plays an important role in lipoprotein metabolism. PLTP is an 80-kDa glycoprotein that is expressed/secreted by a wide variety of tissues including lung, liver, adipose tissue, brain, and muscle. PLTP mediates a net transfer of phospholipids between vesicles and plasma HDLs. It also generates from small HDL particles large fused HDL particles with a concomitant formation of small lipid-poor apolipoprotein (apo) A-I-containing particles which are thought to act as the primary acceptors of cell-derived cholesterol from peripheral tissue macrophages. Another important function of PLTP is connected to lipolysis. Its role in the transfer of surface remnants from triglyceride-rich particles, very-low-density lipoproteins, and chylomicrons, to HDL is of importance for the maintenance of HDL levels. Recent observations from our laboratory have demonstrated that in circulation two forms of PLTP are present, one catalytically active (high-activity form, HA-PLTP) and the other a low-activity form (LA-PLTP). In view of the likely relevancy of PLTP in human health and disease, reliable and accurate methods for measuring plasma/serum PLTP activity and concentration are required. In this chapter, two radiometric PLTP activity assays are described: (i) exogenous, lipoprotein-independent phospholipid transfer assay and (ii) endogenous, lipoprotein-dependent phospholipid transfer assay. In addition, an ELISA method for quantitation of serum/plasma total PLTP mass as well as HA-PLTP and LA-PLTP mass is reported in detail. 2005 Published by Elsevier Inc. Keywords: Phospholipid transfer protein; Phospholipid transfer; Low and high activity forms of PLTP; Enzyme linked immunosorbent assay
1. The theoretical basis and framework for the PLTP assays The protective function of HDL in the development of cardiovascular diseases (CVD, atherosclerosis) has been well documented and although the detailed mechanisms behind this observation remain unsolved, its role in the process of reverse cholesterol transport (RCT) is well recognized [1]. HDL is not a homogeneous population of particles but a spectrum of particles of distinct structure, function, and composition. This heterogeneity
*
Corresponding author. Fax: +358 9 47448960. E-mail address: [email protected] (M. Jauhiainen).
1046-2023/$ - see front matter 2005 Published by Elsevier Inc. doi:10.1016/j.ymeth.2004.11.006
which is the result of continuous remodelling of HDL by plasma factors has important implications in terms of HDL metabolism and cardioprotective functions of HDL. As the remodelling of HDL by plasma factors is rapid relative to the 3–5 day plasma half life of their main apolipoproteins, it dominates the metabolism of HDL. One of the factors participating in HDL metabolism is phospholipid transfer protein, PLTP [2]. PLTP facilitates the transfer of phospholipids between lipoproteins [3] and mediates HDL conversion process whereby large HDL and small pre-HDL particles are generated [4,5]. The presence of PLTP in macrophage foam cells of atherosclerotic lesions suggests that PLTP could function either as an anti-atherogenic molecule by facilitating cholesterol eZux or as a pro-atherogenic molecule
98
M. Jauhiainen, C. Ehnholm / Methods 36 (2005) 97–101
by mediating lipid retention [6,7]. Involvement of PLTP in RCT was suggested by Oram et al. [8] who demonstrated that PLTP interacts with the macrophage ATPbinding cassette transporter A1 (ABCA1) and facilitates lipid eZux. In addition to lipid metabolism, PLTP may also have a role in carbohydrate metabolism [9–11]. This is supported by the fact that in HepG2 cells, high glucose concentration increases both PLTP mRNA and activity levels [12]. Although the in vitro role of PLTP and gene targeted animal models has been studied intensively, the physiological role of PLTP in human lipid metabolism is far from resolved. To obtain information about the role of PLTP, one approach has been to assess PLTP activity in human plasma under physiological conditions. However, to gain a more complete understanding of the metabolic role of PLTP, its mass in plasma should be recorded. Human plasma contains two forms of PLTP, a highactive form (HA-PLTP) and a low-active form (LAPLTP) which are associated with macromolecular complexes of diVerent size [13,14]. The processes that regulate the distribution of the HA- and LA-PLTP forms are unknown. We describe two methods for measuring PLTP transfer activity and an ELISA method for quantitative analysis of the HA-PLTP and LA-PLTP forms.
2. Measurement of PLTP-facilitated phospholipid transfer activity 2.1. Assay for exogenous, lipoprotein-independent activity (PLTPExo activity) PLTPExo activity is measured as the transfer of radioactively labelled phospholipids (PC) from donor liposomes to acceptor HDL particles. The assay was Wrst described by Damen et al. [15] with some later modiWcations [4]. 2.1.1. Preparation of phosphatidyl choline-liposomes To prepare phosphatidyl choline (PC)-liposomes, 10 mol of egg PC (Sigma, P2772, MW 773, stock solution: 100 mg/ml chloroform D 129 mol/ml), 20 l (corresponding to 1 Ci) of [14C]DPPC (dipalmitoyl phosphatidylcholine, Amersham, CFA 601), and 100 nmol of butylhydroxytoluene (BHT) antioxidant, (stock 1 nmol/l in chloroform) are pipetted into thickwall conical V-shape glass tubes (10 ml tubes, Kimax) on ice. Organic solvent is evaporated under N2 until dry and the mixture is lyophilized for 30 min to maximize the removal of the organic phase. Onto the dry lipid Wlm add 1 ml (PLTP buVer, 10 mM Tris–HCl, 150 mM NaCl, and 1 mM EDTA, pH 7.4) and vortex vigorously to solubilize lipid material free from the glass wall. Sonicate 3 £ 5 min carefully keeping the tube in ice bath. After each 5 min sonication, let the tubes stand 1 min on ice,
and then continue sonication. Carefully avoid foaming during the sonication period. After this protocol, the mixture becomes slightly opalizing. Transfer the sonicated material into Eppendorf tube (1 ml) and centrifuge the tube for 10 min at 15,000 rpm at room temperature to pellet particulate material and titanium residual released from the sonicator probe. Transfer the opalescent phosphatidylcholine (PC)-liposomes to a new 1 ml Eppendorf tube, blow nitrogen, and keep the tubes at +4 °C wrapped in aluminium foil. Using this procedure, radioactivity counting of 15 l of this substrate produces about 15,000 cpm (16,300 dpm, counting eYciency 92%). The freshly prepared PC liposome-substrate is tested as follows: run the blank assay without an actual sample from the substrate as described below and measure the cpm values in the supernatant. If the radioactive counts are below 500 cpm, the substrate is valid. If cpm value >500 cpm, recentrifuge the HDL3 using Table-Top 100 ultracentrifuge (Beckmann, 18 h, 5 °C, 100,000 rpm). After centrifugation measure protein from the washed HDL3 by Lowry method and repeat the blank assay. Keep the substrate under nitrogen at +4 °C and always wrapped to avoid light exposure when not in use. 2.1.2. Step-by-step procedure for measurement of PLTPExo activity Pipette sequentially into Eppendorf tubes (size of 1 ml) the following solutions: (1) HDL3, (density range, 1.125–1.21 g/ml), free of PLTP activity, 250 g as protein, (2) Tris-buVer (10 mM Tris–HCl, 154 mM NaCl, and 1 mM EDTA, pH 7.4). Calculate here how much buVer is needed to get the Wnal volume of 400 l, (3) PC liposomes, 15 l, and (4) sample (1:10 diluted plasma or serum, 4–10 l). The following tubes and controls are included: blank tube (buVer as a sample without PLTP), tube for total radioactivity per assay (total counts in 15 l), and a control sample (frozen plasma or serum in 0.5 ml aliquots stored at ¡70 °C. Each of the above assay tubes is run in duplicates. The tubes are incubated at +37 °C for 45 min in a water bath with light shaking. After incubation, the tubes are taken on ice and the reaction is stopped by adding 300 l of stop mix-solution. At this step, the total volume of the mixture is 700 l. Stop mix-solution is prepared as follows: dissolve 7.224 g NaCl and 10.528 g MnCl2 in 224 ml of distilled H2O. Add 8 ml Heparin (Lövens, Denmark 5000 IU/ml), mix well, and store at +4 °C. In our hands, the stop mix is functional up to 2 weeks. After addition of the stop mixsolution, the tubes are vortexed vigorously for 10 s, kept for 10 min at room temperature, and then centrifuged for 10 min at 15,000 rpm (Eppendorf Centrifuge 5804). After centrifugation, 500 l of supernatant is used for radioactivity counting. As a control with a normal PLTP activity (5000– 7000 nmol/h/ml), fresh plasma or serum is collected and stored at ¡70 °C, in 0.5 ml aliquots. PLTP activity of this
M. Jauhiainen, C. Ehnholm / Methods 36 (2005) 97–101
control is followed in each assay series (in the beginning and at the end of the sample series). As a low PLTP activity control, serum or plasma with a low activity (»2500 nmol/ml/h) is used. This control is stored under the same conditions as the normal control. 2.2. Assay of endogenous, lipoprotein-dependent activity (PLTPEndo activity) The method described is a modiWcation of the procedure described by Lagrost et al. [16]. In the endogenous, lipoprotein-dependent assay (PLTPEndo), [14C]PC liposomes are prepared as described above (see Section 2.1.1). PC liposomes (15 l, 150 nmol) were mixed with 30 l of undiluted serum/plasma, and incubated for 30 min at +37 °C in a Wnal volume of 80 l, containing 1.5 mM iodoacetate (28). The incubation volume to 80 l was adjusted with TBS-buVer (10 mM Tris– HCl, 150 mM NaCl, and 1 mM EDTA, pH 7.4). After incubation, 320 l of TBS was added, and then excess liposomes and apoB-containing lipoproteins were precipitated with 300 l of 215 mM MnCl2 £ 4 H2O, 500 mM NaCl containing 445 U/ml heparin. The intraand inter-assay coeYcients of variation were 3.2% (n D 20) and 6.3% (n D 14), respectively. In both PLTP activity assays, radioactivity was measured from the supernatants with a liquid scintillation counter (Wallac WinSpectral 1414, Wallac, Turku, Finland).
3. Quantitation of the active (HA-PLTP) and low active (LA-PLTP) forms of PLTP in human plasma/serum The ELISA method for quantiWcation of the total PLTP mass as well as the mass of the active and lowactive forms of PLTP is based on the procedures reported [18,19]. 3.1. Separation of the two forms of PLTP from plasma/ serum sample To separate the two forms of PLTP, we used dextran sulphate (DxSO4)–CaCl2 precipitation method. This approach has been utilized as a puriWcation step for active PLTP [20]. Before the precipitation dextran sulphate solution has to be prepared carefully as follows: weigh 1 g DxSO4 [dextran sulphate sodium salt; dextran Mw »500,000, stabilized with phosphate 0.5–2.0%, sulfur content 17%, free SO4 < 0.5%. Pharmacia Biotech, Uppsala, Sweden, Code:17-03440-02] and dissolve in 50 ml of distilled H2O (Wnal, 2% DxSO4). Dialyse (dialyse tubing cut oV value of 10–12 KDa) against 3 £ 5 L distilled H2O for 24 h at +4 °C. The volume should increase exactly 2-fold but if not then top up the volume to 100 ml with distilled H2O.
99
This brings the DxSO4 to 1% Wnal concentration and all calculations should be performed based on this. After DxSO4 solution is freshly made, prepare a mixture of 200 l plasma/serum (fresh or stored at ¡20 °C or ¡70 °C), 300 l of sterile distilled H2O, and 200 l of 1% DxSO4 solution. Incubate for 20 min on ice with intermittent mixing by inversion end-to-end and add 17.5 l of 4 M CaCl2 (to get a Wnal concentration of 0.1 M) and mix gently (do not vortex!). During this step, a white opalescent precipitate is formed and is pelleted by centrifugation (16,000g) at room temperature for 5 min. Collect the supernatant which contains the high-active form of PLTP and freeze the low active PLTP containing precipitate at ¡20 °C. From the supernatant and the corresponding plasma sample, both the PLTPExo activity and PLTP mass assays are performed as described below. To analyse PLTPExo activity in the supernatant, dilute 200 l of supernatant with 200 l of 2 M NaCl to get a Wnal dilution of 1:7.2. Corresponding plasma sample is also diluted 1:7.2 with TBS-buVer. From the diluted supernatant and plasma, 10 l is used for PLTPExo activity measurement as described above (see Section 2 above). 3.2. Enzyme-linked immunosorbent assay of PLTP 3.2.1. Materials and equipments • Labsystems Multiwash apparatus (Labsystems, Helsinki, Finland). • Wallac Victor 2.1420 Multilabel counter (Wallac, Turku, Finland). • Nunc Cert. Maxisorb 96-well plates. • Labsystems Sealing tape (No. 1541892, Labsystems, Helsinki, Finland). Anti-PLTP antibodies: • Monoclonal antibody MAb 66 IgG. • Rabbit polyclonal antibody R176 IgG. • Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (GAR Code 170-6515, Bio-Rad, Richmond, USA). HRP substrate is made as follows: combine 6.25 ml of 0.1 M citric acid, 6.25 ml of 0.2 M Na2HPO4, and 12.5 ml of HPLC-grade H2O. Add 10 mg o-phenylenediamine (OPD8) tablet and 10l H2O2. BuVers: • • • • •
Coating buVer, 0.1 M carbonate buVer, pH 9.6. Washing buVer, PBS, 0.05% Tween 20. Blocking buVer, PBS, 0.05% Tween 20, 1% BSA. Sample dilution buVer, PBS, 0.1% Tween 20. antibody dilution buVer, PBS, 0.05% Tween 20, 0.5% BSA.
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3.2.2. Assay procedure 3.2.2.1. Plate coating and blocking. Coat a 96-well plate with 200 l coating buVer only into Wrst two wells [¡coat], 1 g/ml MAb66 in 200 l coating buVer/well to the rest 94 wells [+coat]. Cover with seal tape and incubate O/N at 4 °C. Block the wells with 300 l of blocking buVer/well, cover the plate, and incubate for 1 h at +37 °C.
HA-PLTP: (activity in supernatant/activity in plasma) £ 100 D % activity remaining in supernatant (this should be > 90%). (PLTP mass in supernatant/mass in plasma) £ 100 D % mass remaining in supernatant. LA-PLTP mass: PLTP mass in plasma ¡ PLTP mass in supernatant D mass of LA-PLTP.
3.2.2.2. Sample and standard treatment. The samples are pre-incubated Wrst with 0.5% SDS as follows: 100 l supernatant + 2.5 l of 20% SDS (Wnal 0.5% concentration), 100 l plasma sample + 2.5 l SDS (Wnal 0.5% concentration), and 100 l of secondary plasma standard/ plasma control + 2.5 l SDS (Wnal 0.5% concentration). Mixtures are carefully vortexed and then incubated at room temperature for 30 min. After incubation, further dilutions are made using sample dilution buVer:
4. Concluding remarks
1:25 for the DxSO4 supernatant; 1:60 for the corresponding plasma/serum sample; 1:50 for the plasma/serum control; and 1:20, 1:25, 1:40, 1:80, 1:100, and 1:160 for the secondary plasma standard. After vortexing, these dilutions are stored at +4 °C overnight. 3.2.2.3. Plate incubation. Add 200 l of the above diluted serum/plasma, DxSO4 supernatant, control, and standard per well, incubate for 45 min at +37 °C. Wash six times with the washing buVer. 3.2.2.4. Detection. Polyclonal anti-PLTP R176 antibody (IgG) is diluted with antibody buVer to 10 g/ml, and 200 l is added per well. The plate is covered and incubated for 45 min at +37 °C. After washing the wells HRP-conjugated goat anti-rabbit IgG is diluted to 1:10,000 with antibody buVer and 200 l is added per well and incubated for 45 min at +37 °C. After washing the wells six times with washing buVer, freshly made HRP substrate (see above) is added and the colour was developed for 1 h in the dark. The enzyme reaction is stopped with 50 l/well of 3 M H2SO4 and the absorbance was measured at 490 nm using Victor2 1420 Multilabel Counter (Wallac, Perkin-Elmer, Turku, Finland). Note. The SDS dilution MUST be done on the same day as the DxSO4 precipitation. Samples stored at +4 °C overnight can then be assayed for mass on the following day. 3.2.3. Calculations High-activity form of PLTP (HA-PLTP) is not precipitated and retains in the supernatant. The calculations are performed as follows:
PLTP exists in two forms in human serum, one with HA-PLTP and the other with LA-PLTP. Measurement of both PLTP activity and mass from human serum as well as from puriWcation steps of human PLTP either from serum or sources of protein expression systems requires the availability of a fast, reliable, and simple procedures. We have described here a simple method for the rapid determination of radioactively labelled phosphatidylcholine transfer from small liposomes to human HDL3 followed by a selective precipitation of liposomes by heparin and MnCl2. This represents the lipoprotein-independent assay. In addition, lipoprotein-dependent activity assay is also described. We have recently analysed PLTP activity using these two assay methods in a subsample of the Finnish Health 2000 Health Examination Survey [17]. There were no signiWcant gender diVerences in the activities. The mean serum PLTPExo activity was 6.59 § 1.66 mol/ml/h (mean § SD) and the mean serum PLTPEndo activity was 1.37 § 0.29 mol/ml/h. For the PLTPExo activity, the intra-assay coeYcient of variation (CV) was 9.4% (n D 11), and the inter-assay CV was 12% (n D 15). The intra- and inter-assay CVs for PLTPEndo activity were 3.2% (n D 20) and 6.3% (n D 14). In the same survey, the mean PLTP total mass was 6.56 § 1.45 mg/L (range: 2.78–10.89 mg/L), and the mean LA-PLTP and HA-PLTP mass values were 3.56 § 1.14 mg/L (range: 1.20–8.43 mg/L) and 3.00 § 1.21 mg/L (range: 0.92– 7.63 mg/L). Approximately, 46% of the serum PLTP concentration was in a catalytically active form [17]. PLTP activity assays combined with PLTP mass measurements will be applied to analysis of dyslipidemic samples to improve our understanding on the role of PLTP in the pathophysiology of lipoprotein metabolism.
References [1] C.J. Fielding, P.E. Fielding, J. Lipid Res. 36 (1995) 211–228. [2] J. Huuskonen, V.M. Olkkonen, M. Jauhiainen, C. Ehnholm, Atherosclerosis 155 (2001) 269–281. [3] J. Tollefson, S. Ravnik, J. Albers, J. Lipid Res. 29 (1988) 1593– 1602. [4] M. Jauhiainen, J. Metso, R. Pahlman, S. Blomqvist, A. van Tol, C. Ehnholm, J. Biol. Chem. 268 (1993) 4032–4036. [5] A.Y. Tu, H.I. Nishida, T. Nishida, J. Biol. Chem. 268 (1993) 23098–23105.
M. Jauhiainen, C. Ehnholm / Methods 36 (2005) 97–101 [6] C.M. Desrumaux, P.A. Mak, W.A. Boisvert, D. Masson, D. Stupack, M. Jauhiainen, C. Ehnholm, L.K. Curtiss, J. Lipid Res. 44 (2003) 1453–1461. [7] B.A. LaYtte, S.B. Joseph, M. Chen, A. Castrillo, J. Repa, D. Wilpitz, D. Mangelsdorf, P. Tontonoz, Mol. Cell. Biol. 23 (2003) 2182–2191. [8] J.F. Oram, G. Wolfbauer, A.M. Vaughan, C. Tang, J.J. Albers, J. Biol. Chem. 278 (2003) 52379–52385. [9] S.C. Riemens, A. van Tol, W.J. Sluiter, R.P. Dullaart, Diabetologia 41 (1998) 929–934. [10] I.J. Jonkers, A.H. Smelt, H. Hattori, L.M. Scheek, T. van Gent, F.H. de Man, A. van der Laarse, A. van Tol, J. Lipid Res. 44 (2003) 1462–1469. [11] H.M. Colhoun, M.R. Taskinen, J.D. Otvos, P. Van Den Berg, J. O’Connor, A. Van Tol, Diabetes 51 (2002) 3300–3305. [12] A.Y. Tu, J.J. Albers, Diabetes 50 (2001) 1851–1856. [13] T. Oka, T. Kujiraoka, M. Ito, T. Egashira, S. Takahashi, M.N. Nanjee, N.E. Miller, J. Metso, V.M. Olkkonen, C. Ehnholm, M. Jauhiainen, H. Hattori, J. Lipid Res. 41 (2000) 1651–1657.
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[14] M.T. Kärkkäinen, T. Oka, V.M. Olkkonen, J. Metso, H. Hattori, M. Jauhiainen, C. Ehnholm, J. Biol. Chem. 277 (2002) 15413– 15418. [15] J. Damen, J. Regts, G. Scherphof, Biochim. Biophys. Acta 712 (1982) 444–452. [16] L. Lagrost, A. Athias, N. Lemort, J.L. Richard, C. Desrumaux, L. Chatenet-Duchene, M. Courtois, M. Farnier, B. Jacotot, S. Braschi, P. Gambert, Atherosclerosis 143 (1999) 415–425. [17] M.T. Jänis, S. Siggins, E. Tahvanainen, R. Vikstedt, K. Silander, J. Metso, A. Aromaa, M.-R. Taskinen, V.M. Olkkonen, M. Jauhiainen, C. Ehnholm, J. Lipid Res. 45 (2004) 2303–2309. [18] J. Huuskonen, J.M. Ekström, E. Tahvanainen, A. Vainio, J. Metso, P. Pussinen, C. Ehnholm, V.M. Olkkonen, M. Jauhiainen, Atherosclerosis 151 (2000) 451–461. [19] S. Siggins, M. Kärkkäinen, J. Tenhunen, J. Metso, E. Tahvanainen, V.M. Olkkonen, M. Jauhiainen, C. Ehnholm, J. Lipid Res. 45 (2004) 387–395. [20] H. Kato, T. Nakanishi, T. Arai, H.I. Nishida, T. Nishida, J. Biol. Chem. 264 (1988) 4082–4087.
Determination of human plasma phospholipid transfer protein mass and activity Matti Jauhiainen ¤, Christian Ehnholm Department of Molecular Medicine, National Public Health Institute, Biomedicum, Haartmaninkatu 8, FIN-00251 Helsinki, Finland Accepted 1 November 2004
Abstract Plasma phospholipid transfer protein (PLTP) plays an important role in lipoprotein metabolism. PLTP is an 80-kDa glycoprotein that is expressed/secreted by a wide variety of tissues including lung, liver, adipose tissue, brain, and muscle. PLTP mediates a net transfer of phospholipids between vesicles and plasma HDLs. It also generates from small HDL particles large fused HDL particles with a concomitant formation of small lipid-poor apolipoprotein (apo) A-I-containing particles which are thought to act as the primary acceptors of cell-derived cholesterol from peripheral tissue macrophages. Another important function of PLTP is connected to lipolysis. Its role in the transfer of surface remnants from triglyceride-rich particles, very-low-density lipoproteins, and chylomicrons, to HDL is of importance for the maintenance of HDL levels. Recent observations from our laboratory have demonstrated that in circulation two forms of PLTP are present, one catalytically active (high-activity form, HA-PLTP) and the other a low-activity form (LA-PLTP). In view of the likely relevancy of PLTP in human health and disease, reliable and accurate methods for measuring plasma/serum PLTP activity and concentration are required. In this chapter, two radiometric PLTP activity assays are described: (i) exogenous, lipoprotein-independent phospholipid transfer assay and (ii) endogenous, lipoprotein-dependent phospholipid transfer assay. In addition, an ELISA method for quantitation of serum/plasma total PLTP mass as well as HA-PLTP and LA-PLTP mass is reported in detail. 2005 Published by Elsevier Inc. Keywords: Phospholipid transfer protein; Phospholipid transfer; Low and high activity forms of PLTP; Enzyme linked immunosorbent assay
1. The theoretical basis and framework for the PLTP assays The protective function of HDL in the development of cardiovascular diseases (CVD, atherosclerosis) has been well documented and although the detailed mechanisms behind this observation remain unsolved, its role in the process of reverse cholesterol transport (RCT) is well recognized [1]. HDL is not a homogeneous population of particles but a spectrum of particles of distinct structure, function, and composition. This heterogeneity
*
Corresponding author. Fax: +358 9 47448960. E-mail address: [email protected] (M. Jauhiainen).
1046-2023/$ - see front matter 2005 Published by Elsevier Inc. doi:10.1016/j.ymeth.2004.11.006
which is the result of continuous remodelling of HDL by plasma factors has important implications in terms of HDL metabolism and cardioprotective functions of HDL. As the remodelling of HDL by plasma factors is rapid relative to the 3–5 day plasma half life of their main apolipoproteins, it dominates the metabolism of HDL. One of the factors participating in HDL metabolism is phospholipid transfer protein, PLTP [2]. PLTP facilitates the transfer of phospholipids between lipoproteins [3] and mediates HDL conversion process whereby large HDL and small pre-HDL particles are generated [4,5]. The presence of PLTP in macrophage foam cells of atherosclerotic lesions suggests that PLTP could function either as an anti-atherogenic molecule by facilitating cholesterol eZux or as a pro-atherogenic molecule
98
M. Jauhiainen, C. Ehnholm / Methods 36 (2005) 97–101
by mediating lipid retention [6,7]. Involvement of PLTP in RCT was suggested by Oram et al. [8] who demonstrated that PLTP interacts with the macrophage ATPbinding cassette transporter A1 (ABCA1) and facilitates lipid eZux. In addition to lipid metabolism, PLTP may also have a role in carbohydrate metabolism [9–11]. This is supported by the fact that in HepG2 cells, high glucose concentration increases both PLTP mRNA and activity levels [12]. Although the in vitro role of PLTP and gene targeted animal models has been studied intensively, the physiological role of PLTP in human lipid metabolism is far from resolved. To obtain information about the role of PLTP, one approach has been to assess PLTP activity in human plasma under physiological conditions. However, to gain a more complete understanding of the metabolic role of PLTP, its mass in plasma should be recorded. Human plasma contains two forms of PLTP, a highactive form (HA-PLTP) and a low-active form (LAPLTP) which are associated with macromolecular complexes of diVerent size [13,14]. The processes that regulate the distribution of the HA- and LA-PLTP forms are unknown. We describe two methods for measuring PLTP transfer activity and an ELISA method for quantitative analysis of the HA-PLTP and LA-PLTP forms.
2. Measurement of PLTP-facilitated phospholipid transfer activity 2.1. Assay for exogenous, lipoprotein-independent activity (PLTPExo activity) PLTPExo activity is measured as the transfer of radioactively labelled phospholipids (PC) from donor liposomes to acceptor HDL particles. The assay was Wrst described by Damen et al. [15] with some later modiWcations [4]. 2.1.1. Preparation of phosphatidyl choline-liposomes To prepare phosphatidyl choline (PC)-liposomes, 10 mol of egg PC (Sigma, P2772, MW 773, stock solution: 100 mg/ml chloroform D 129 mol/ml), 20 l (corresponding to 1 Ci) of [14C]DPPC (dipalmitoyl phosphatidylcholine, Amersham, CFA 601), and 100 nmol of butylhydroxytoluene (BHT) antioxidant, (stock 1 nmol/l in chloroform) are pipetted into thickwall conical V-shape glass tubes (10 ml tubes, Kimax) on ice. Organic solvent is evaporated under N2 until dry and the mixture is lyophilized for 30 min to maximize the removal of the organic phase. Onto the dry lipid Wlm add 1 ml (PLTP buVer, 10 mM Tris–HCl, 150 mM NaCl, and 1 mM EDTA, pH 7.4) and vortex vigorously to solubilize lipid material free from the glass wall. Sonicate 3 £ 5 min carefully keeping the tube in ice bath. After each 5 min sonication, let the tubes stand 1 min on ice,
and then continue sonication. Carefully avoid foaming during the sonication period. After this protocol, the mixture becomes slightly opalizing. Transfer the sonicated material into Eppendorf tube (1 ml) and centrifuge the tube for 10 min at 15,000 rpm at room temperature to pellet particulate material and titanium residual released from the sonicator probe. Transfer the opalescent phosphatidylcholine (PC)-liposomes to a new 1 ml Eppendorf tube, blow nitrogen, and keep the tubes at +4 °C wrapped in aluminium foil. Using this procedure, radioactivity counting of 15 l of this substrate produces about 15,000 cpm (16,300 dpm, counting eYciency 92%). The freshly prepared PC liposome-substrate is tested as follows: run the blank assay without an actual sample from the substrate as described below and measure the cpm values in the supernatant. If the radioactive counts are below 500 cpm, the substrate is valid. If cpm value >500 cpm, recentrifuge the HDL3 using Table-Top 100 ultracentrifuge (Beckmann, 18 h, 5 °C, 100,000 rpm). After centrifugation measure protein from the washed HDL3 by Lowry method and repeat the blank assay. Keep the substrate under nitrogen at +4 °C and always wrapped to avoid light exposure when not in use. 2.1.2. Step-by-step procedure for measurement of PLTPExo activity Pipette sequentially into Eppendorf tubes (size of 1 ml) the following solutions: (1) HDL3, (density range, 1.125–1.21 g/ml), free of PLTP activity, 250 g as protein, (2) Tris-buVer (10 mM Tris–HCl, 154 mM NaCl, and 1 mM EDTA, pH 7.4). Calculate here how much buVer is needed to get the Wnal volume of 400 l, (3) PC liposomes, 15 l, and (4) sample (1:10 diluted plasma or serum, 4–10 l). The following tubes and controls are included: blank tube (buVer as a sample without PLTP), tube for total radioactivity per assay (total counts in 15 l), and a control sample (frozen plasma or serum in 0.5 ml aliquots stored at ¡70 °C. Each of the above assay tubes is run in duplicates. The tubes are incubated at +37 °C for 45 min in a water bath with light shaking. After incubation, the tubes are taken on ice and the reaction is stopped by adding 300 l of stop mix-solution. At this step, the total volume of the mixture is 700 l. Stop mix-solution is prepared as follows: dissolve 7.224 g NaCl and 10.528 g MnCl2 in 224 ml of distilled H2O. Add 8 ml Heparin (Lövens, Denmark 5000 IU/ml), mix well, and store at +4 °C. In our hands, the stop mix is functional up to 2 weeks. After addition of the stop mixsolution, the tubes are vortexed vigorously for 10 s, kept for 10 min at room temperature, and then centrifuged for 10 min at 15,000 rpm (Eppendorf Centrifuge 5804). After centrifugation, 500 l of supernatant is used for radioactivity counting. As a control with a normal PLTP activity (5000– 7000 nmol/h/ml), fresh plasma or serum is collected and stored at ¡70 °C, in 0.5 ml aliquots. PLTP activity of this
M. Jauhiainen, C. Ehnholm / Methods 36 (2005) 97–101
control is followed in each assay series (in the beginning and at the end of the sample series). As a low PLTP activity control, serum or plasma with a low activity (»2500 nmol/ml/h) is used. This control is stored under the same conditions as the normal control. 2.2. Assay of endogenous, lipoprotein-dependent activity (PLTPEndo activity) The method described is a modiWcation of the procedure described by Lagrost et al. [16]. In the endogenous, lipoprotein-dependent assay (PLTPEndo), [14C]PC liposomes are prepared as described above (see Section 2.1.1). PC liposomes (15 l, 150 nmol) were mixed with 30 l of undiluted serum/plasma, and incubated for 30 min at +37 °C in a Wnal volume of 80 l, containing 1.5 mM iodoacetate (28). The incubation volume to 80 l was adjusted with TBS-buVer (10 mM Tris– HCl, 150 mM NaCl, and 1 mM EDTA, pH 7.4). After incubation, 320 l of TBS was added, and then excess liposomes and apoB-containing lipoproteins were precipitated with 300 l of 215 mM MnCl2 £ 4 H2O, 500 mM NaCl containing 445 U/ml heparin. The intraand inter-assay coeYcients of variation were 3.2% (n D 20) and 6.3% (n D 14), respectively. In both PLTP activity assays, radioactivity was measured from the supernatants with a liquid scintillation counter (Wallac WinSpectral 1414, Wallac, Turku, Finland).
3. Quantitation of the active (HA-PLTP) and low active (LA-PLTP) forms of PLTP in human plasma/serum The ELISA method for quantiWcation of the total PLTP mass as well as the mass of the active and lowactive forms of PLTP is based on the procedures reported [18,19]. 3.1. Separation of the two forms of PLTP from plasma/ serum sample To separate the two forms of PLTP, we used dextran sulphate (DxSO4)–CaCl2 precipitation method. This approach has been utilized as a puriWcation step for active PLTP [20]. Before the precipitation dextran sulphate solution has to be prepared carefully as follows: weigh 1 g DxSO4 [dextran sulphate sodium salt; dextran Mw »500,000, stabilized with phosphate 0.5–2.0%, sulfur content 17%, free SO4 < 0.5%. Pharmacia Biotech, Uppsala, Sweden, Code:17-03440-02] and dissolve in 50 ml of distilled H2O (Wnal, 2% DxSO4). Dialyse (dialyse tubing cut oV value of 10–12 KDa) against 3 £ 5 L distilled H2O for 24 h at +4 °C. The volume should increase exactly 2-fold but if not then top up the volume to 100 ml with distilled H2O.
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This brings the DxSO4 to 1% Wnal concentration and all calculations should be performed based on this. After DxSO4 solution is freshly made, prepare a mixture of 200 l plasma/serum (fresh or stored at ¡20 °C or ¡70 °C), 300 l of sterile distilled H2O, and 200 l of 1% DxSO4 solution. Incubate for 20 min on ice with intermittent mixing by inversion end-to-end and add 17.5 l of 4 M CaCl2 (to get a Wnal concentration of 0.1 M) and mix gently (do not vortex!). During this step, a white opalescent precipitate is formed and is pelleted by centrifugation (16,000g) at room temperature for 5 min. Collect the supernatant which contains the high-active form of PLTP and freeze the low active PLTP containing precipitate at ¡20 °C. From the supernatant and the corresponding plasma sample, both the PLTPExo activity and PLTP mass assays are performed as described below. To analyse PLTPExo activity in the supernatant, dilute 200 l of supernatant with 200 l of 2 M NaCl to get a Wnal dilution of 1:7.2. Corresponding plasma sample is also diluted 1:7.2 with TBS-buVer. From the diluted supernatant and plasma, 10 l is used for PLTPExo activity measurement as described above (see Section 2 above). 3.2. Enzyme-linked immunosorbent assay of PLTP 3.2.1. Materials and equipments • Labsystems Multiwash apparatus (Labsystems, Helsinki, Finland). • Wallac Victor 2.1420 Multilabel counter (Wallac, Turku, Finland). • Nunc Cert. Maxisorb 96-well plates. • Labsystems Sealing tape (No. 1541892, Labsystems, Helsinki, Finland). Anti-PLTP antibodies: • Monoclonal antibody MAb 66 IgG. • Rabbit polyclonal antibody R176 IgG. • Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (GAR Code 170-6515, Bio-Rad, Richmond, USA). HRP substrate is made as follows: combine 6.25 ml of 0.1 M citric acid, 6.25 ml of 0.2 M Na2HPO4, and 12.5 ml of HPLC-grade H2O. Add 10 mg o-phenylenediamine (OPD8) tablet and 10l H2O2. BuVers: • • • • •
Coating buVer, 0.1 M carbonate buVer, pH 9.6. Washing buVer, PBS, 0.05% Tween 20. Blocking buVer, PBS, 0.05% Tween 20, 1% BSA. Sample dilution buVer, PBS, 0.1% Tween 20. antibody dilution buVer, PBS, 0.05% Tween 20, 0.5% BSA.
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3.2.2. Assay procedure 3.2.2.1. Plate coating and blocking. Coat a 96-well plate with 200 l coating buVer only into Wrst two wells [¡coat], 1 g/ml MAb66 in 200 l coating buVer/well to the rest 94 wells [+coat]. Cover with seal tape and incubate O/N at 4 °C. Block the wells with 300 l of blocking buVer/well, cover the plate, and incubate for 1 h at +37 °C.
HA-PLTP: (activity in supernatant/activity in plasma) £ 100 D % activity remaining in supernatant (this should be > 90%). (PLTP mass in supernatant/mass in plasma) £ 100 D % mass remaining in supernatant. LA-PLTP mass: PLTP mass in plasma ¡ PLTP mass in supernatant D mass of LA-PLTP.
3.2.2.2. Sample and standard treatment. The samples are pre-incubated Wrst with 0.5% SDS as follows: 100 l supernatant + 2.5 l of 20% SDS (Wnal 0.5% concentration), 100 l plasma sample + 2.5 l SDS (Wnal 0.5% concentration), and 100 l of secondary plasma standard/ plasma control + 2.5 l SDS (Wnal 0.5% concentration). Mixtures are carefully vortexed and then incubated at room temperature for 30 min. After incubation, further dilutions are made using sample dilution buVer:
4. Concluding remarks
1:25 for the DxSO4 supernatant; 1:60 for the corresponding plasma/serum sample; 1:50 for the plasma/serum control; and 1:20, 1:25, 1:40, 1:80, 1:100, and 1:160 for the secondary plasma standard. After vortexing, these dilutions are stored at +4 °C overnight. 3.2.2.3. Plate incubation. Add 200 l of the above diluted serum/plasma, DxSO4 supernatant, control, and standard per well, incubate for 45 min at +37 °C. Wash six times with the washing buVer. 3.2.2.4. Detection. Polyclonal anti-PLTP R176 antibody (IgG) is diluted with antibody buVer to 10 g/ml, and 200 l is added per well. The plate is covered and incubated for 45 min at +37 °C. After washing the wells HRP-conjugated goat anti-rabbit IgG is diluted to 1:10,000 with antibody buVer and 200 l is added per well and incubated for 45 min at +37 °C. After washing the wells six times with washing buVer, freshly made HRP substrate (see above) is added and the colour was developed for 1 h in the dark. The enzyme reaction is stopped with 50 l/well of 3 M H2SO4 and the absorbance was measured at 490 nm using Victor2 1420 Multilabel Counter (Wallac, Perkin-Elmer, Turku, Finland). Note. The SDS dilution MUST be done on the same day as the DxSO4 precipitation. Samples stored at +4 °C overnight can then be assayed for mass on the following day. 3.2.3. Calculations High-activity form of PLTP (HA-PLTP) is not precipitated and retains in the supernatant. The calculations are performed as follows:
PLTP exists in two forms in human serum, one with HA-PLTP and the other with LA-PLTP. Measurement of both PLTP activity and mass from human serum as well as from puriWcation steps of human PLTP either from serum or sources of protein expression systems requires the availability of a fast, reliable, and simple procedures. We have described here a simple method for the rapid determination of radioactively labelled phosphatidylcholine transfer from small liposomes to human HDL3 followed by a selective precipitation of liposomes by heparin and MnCl2. This represents the lipoprotein-independent assay. In addition, lipoprotein-dependent activity assay is also described. We have recently analysed PLTP activity using these two assay methods in a subsample of the Finnish Health 2000 Health Examination Survey [17]. There were no signiWcant gender diVerences in the activities. The mean serum PLTPExo activity was 6.59 § 1.66 mol/ml/h (mean § SD) and the mean serum PLTPEndo activity was 1.37 § 0.29 mol/ml/h. For the PLTPExo activity, the intra-assay coeYcient of variation (CV) was 9.4% (n D 11), and the inter-assay CV was 12% (n D 15). The intra- and inter-assay CVs for PLTPEndo activity were 3.2% (n D 20) and 6.3% (n D 14). In the same survey, the mean PLTP total mass was 6.56 § 1.45 mg/L (range: 2.78–10.89 mg/L), and the mean LA-PLTP and HA-PLTP mass values were 3.56 § 1.14 mg/L (range: 1.20–8.43 mg/L) and 3.00 § 1.21 mg/L (range: 0.92– 7.63 mg/L). Approximately, 46% of the serum PLTP concentration was in a catalytically active form [17]. PLTP activity assays combined with PLTP mass measurements will be applied to analysis of dyslipidemic samples to improve our understanding on the role of PLTP in the pathophysiology of lipoprotein metabolism.
References [1] C.J. Fielding, P.E. Fielding, J. Lipid Res. 36 (1995) 211–228. [2] J. Huuskonen, V.M. Olkkonen, M. Jauhiainen, C. Ehnholm, Atherosclerosis 155 (2001) 269–281. [3] J. Tollefson, S. Ravnik, J. Albers, J. Lipid Res. 29 (1988) 1593– 1602. [4] M. Jauhiainen, J. Metso, R. Pahlman, S. Blomqvist, A. van Tol, C. Ehnholm, J. Biol. Chem. 268 (1993) 4032–4036. [5] A.Y. Tu, H.I. Nishida, T. Nishida, J. Biol. Chem. 268 (1993) 23098–23105.
M. Jauhiainen, C. Ehnholm / Methods 36 (2005) 97–101 [6] C.M. Desrumaux, P.A. Mak, W.A. Boisvert, D. Masson, D. Stupack, M. Jauhiainen, C. Ehnholm, L.K. Curtiss, J. Lipid Res. 44 (2003) 1453–1461. [7] B.A. LaYtte, S.B. Joseph, M. Chen, A. Castrillo, J. Repa, D. Wilpitz, D. Mangelsdorf, P. Tontonoz, Mol. Cell. Biol. 23 (2003) 2182–2191. [8] J.F. Oram, G. Wolfbauer, A.M. Vaughan, C. Tang, J.J. Albers, J. Biol. Chem. 278 (2003) 52379–52385. [9] S.C. Riemens, A. van Tol, W.J. Sluiter, R.P. Dullaart, Diabetologia 41 (1998) 929–934. [10] I.J. Jonkers, A.H. Smelt, H. Hattori, L.M. Scheek, T. van Gent, F.H. de Man, A. van der Laarse, A. van Tol, J. Lipid Res. 44 (2003) 1462–1469. [11] H.M. Colhoun, M.R. Taskinen, J.D. Otvos, P. Van Den Berg, J. O’Connor, A. Van Tol, Diabetes 51 (2002) 3300–3305. [12] A.Y. Tu, J.J. Albers, Diabetes 50 (2001) 1851–1856. [13] T. Oka, T. Kujiraoka, M. Ito, T. Egashira, S. Takahashi, M.N. Nanjee, N.E. Miller, J. Metso, V.M. Olkkonen, C. Ehnholm, M. Jauhiainen, H. Hattori, J. Lipid Res. 41 (2000) 1651–1657.
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[14] M.T. Kärkkäinen, T. Oka, V.M. Olkkonen, J. Metso, H. Hattori, M. Jauhiainen, C. Ehnholm, J. Biol. Chem. 277 (2002) 15413– 15418. [15] J. Damen, J. Regts, G. Scherphof, Biochim. Biophys. Acta 712 (1982) 444–452. [16] L. Lagrost, A. Athias, N. Lemort, J.L. Richard, C. Desrumaux, L. Chatenet-Duchene, M. Courtois, M. Farnier, B. Jacotot, S. Braschi, P. Gambert, Atherosclerosis 143 (1999) 415–425. [17] M.T. Jänis, S. Siggins, E. Tahvanainen, R. Vikstedt, K. Silander, J. Metso, A. Aromaa, M.-R. Taskinen, V.M. Olkkonen, M. Jauhiainen, C. Ehnholm, J. Lipid Res. 45 (2004) 2303–2309. [18] J. Huuskonen, J.M. Ekström, E. Tahvanainen, A. Vainio, J. Metso, P. Pussinen, C. Ehnholm, V.M. Olkkonen, M. Jauhiainen, Atherosclerosis 151 (2000) 451–461. [19] S. Siggins, M. Kärkkäinen, J. Tenhunen, J. Metso, E. Tahvanainen, V.M. Olkkonen, M. Jauhiainen, C. Ehnholm, J. Lipid Res. 45 (2004) 387–395. [20] H. Kato, T. Nakanishi, T. Arai, H.I. Nishida, T. Nishida, J. Biol. Chem. 264 (1988) 4082–4087.