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WO2006009599A1 - Essai biologique à fluorescence pour l’activité des mtp - Google Patents

Essai biologique à fluorescence pour l’activité des mtp Download PDF

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Publication number
WO2006009599A1
WO2006009599A1 PCT/US2005/014460 US2005014460W WO2006009599A1 WO 2006009599 A1 WO2006009599 A1 WO 2006009599A1 US 2005014460 W US2005014460 W US 2005014460W WO 2006009599 A1 WO2006009599 A1 WO 2006009599A1
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Prior art keywords
vesicles
mtp
transfer
donor
fluorescence
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PCT/US2005/014460
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English (en)
Inventor
M. Mahmood Hussain
Paul Rava
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Teh Research Foundation Of State University Of New York
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Priority claimed from PCT/US2004/020542 external-priority patent/WO2005043110A2/fr
Application filed by Teh Research Foundation Of State University Of New York filed Critical Teh Research Foundation Of State University Of New York
Priority to AU2005264861A priority Critical patent/AU2005264861A1/en
Priority to US11/630,763 priority patent/US20090047700A1/en
Priority to EP05755257A priority patent/EP1781806A4/fr
Priority to CA002571763A priority patent/CA2571763A1/fr
Priority to JP2007518048A priority patent/JP2008504523A/ja
Publication of WO2006009599A1 publication Critical patent/WO2006009599A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors

Definitions

  • Microsomal triglyceride transfer protein is a dedicated chaperone that is required for the assembly of apolipoprotein B (apoB) lipoproteins [for reviews, see refs. (1-6)]. It is believed that MTP transfers lipids to nascent apoB in the endoplasmic reticulum and renders it secretion-competent by forming primordial lipoprotein particles [for reviews, see refs. (1-9)]. The importance of MTP's lipid transfer activity in apoB secretion has been established by three independent approaches. First, mutations in MTP have been correlated with the absence of apoB lipoproteins in abetalipoproteinemia (10, 11).
  • TAGs triacylglycerols
  • MTP was identified and purified by Wetterau and Zilversmit (20, 21) based on a radioisotope assay.
  • radiolabeled TAGs are incorporated into donor vesicles consisting of phosphatidylcholine (PC) and cardiolipin. These vesicles are incubated with acceptor vesicles in the presence of MTP. After 1-3 h of incubation, the cardiolipin- containing donor vesicles are allowed to bind to DE52 and removed by centrifugation. Radioactivity remaining in the supernatant is quantified by scintillation counting. This procedure is labor-intensive and time-consuming.
  • Negatively charged lipids such as cardiolipin, are known to inhibit the lipid transfer activity of MTP (22). Because of the multiple steps involved in this procedure, it is difficult to automate. Thus, it would be advantageous to have a simple, one-step procedure to measure MTP activity. Such a procedure would be useful, e.g., in identifying compounds that partially inhibit MTP activity and therefore decrease lipoprotein assembly and secretion. The identified compounds are highly desirable as drugs for decreasing plasma cholesterol and triglyceride levels in cells.
  • the present invention provides methods and compositions for measuring MTP activity, hi one aspect of the invention, there is provided a method of measuring levels of MTP comprising the steps of: (a) preparing donor vesicles having a fluorescence-labeled lipid incorporated therein; (b) preparing acceptor vesicles; (c) incubating either a cellular homogenate containing MTP or isolated MTP with the acceptor vesicles and the labeled donor vesicles for a time and under conditions sufficient to allow binding of the fluorescence-labeled lipid with MTP during transfer of the labeled lipid from donor to acceptor vesicles; and (d) measuring fluorescence of the fluorescence-labeled lipid bound to MTP.
  • the cellular homogenates may comprise liver cells, intestinal cells, heart cells or any other cells that express MTP including but not limited to cells of animals (including humans), insects and microorganisms.
  • Vesicles are preferably small unilamellar vesicles, multi-lamellar vesicles, apoB- lipoprotein vesicles, other lipoproteins, or phosphatidylcholine (PC) vesicles.
  • the present invention also provides a method for identifying compounds that modulate the lipid transfer activity of MTP.
  • the method comprises the steps of: (a) incorporating a fluorescence-labeled lipid into donor vesicles; (b) preparing acceptor vesicles; (c) mixing an aliquot of acceptor vesicles and the labeled donor vesicles with a test compound, the test compound being a known or unknown modulator of MTP; (d) adding a cellular homogenate containing MTP or isolated MTP to the mixture containing donor vesicles, acceptor vesicles, and test compound; (e) incubating a first aliquot of acceptor vesicles, labeled donor vesicles, test compound and MTP for a time and under conditions sufficient to allow binding of the fluorescence-labeled lipid with MTP during transfer of the labeled lipid from donor to acceptor vesicles; (f) incubating a second aliquot
  • the present invention also provides a method of quantifying lipid transfer activity of microsomal triglyceride transfer protein (MTP).
  • the method comprises the steps of: (a) preparing donor vesicles having a fluorescence-labeled lipid incorporated therein: (b) preparing acceptor vesicles; (c) incubating either a cellular homogenate containing MTP or isolated MTP with the acceptor vesicles and the labeled donor vesicles for a time and under conditions sufficient to allow binding of the fluorescence-labeled lipid with MTP during transfer of the labeled lipid from donor to acceptor vesicles; and (d) measuring fluorescence of the fluorescently labeled lipid bound to the MTP.
  • MTP microsomal triglyceride transfer protein
  • Also provided by the present invention is a method for measuring levels of lipids transferred by MTP. (i.e., measuring net transfer of lipids by MTP).
  • the method comprises the steps of: (a) preparing negatively-charged donor vesicles having a fluorescence-labeled lipid incorporated therein; (b) preparing acceptor vesicles; (c) incubating either a cellular homogenate containing MTP or isolated MTP with the acceptor vesicles and the labeled donor vesicles for a time and under conditions sufficient to allow binding of the fluorescence-labeled lipid with MTP and transfer of the fluoresescence-labeled lipid from donor to acceptor vesicles; (d) removing negatively- charged donor vesicles and MTP from the incubation mixture of step (c); and (e) measuring fluorescent labeled lipids transferred to acceptor vesicles.
  • kits for measuring the lipid transfer activity of MTP and/or for measuring levels of lipids (net transfer of lipids) transferred by MTP comprise acceptor vesicles and fluorescence-labeled donor vesicles.
  • the fluorescence-labeled donor vesicles of the kit are comprised of a triglyceride, a cholesterol ester, or a phospholipid.
  • Vesicles contained in the kit may be small unilamellar vesicles, multi-lamellar vesicles, apoB-lipoprotein vesicles, or phosphatidylcholine (PC) vesicles.
  • the vesicles contained in the kit are preferably admixed with an appropriate buffer and may also contain a stabilizer such as BSA.
  • the donor vesicles in the kit for measuring net transfer of lipids by MTP are preferably negatively charged.
  • FIGS 2A-2C graphically depict the effects of time, temperature and NaCl on TAG transfer activity of MTP. Line graphs and error bars represent means + SD.
  • FIGS 3A-3C graphically illustrate the specificity of TAG transfer activity.
  • Figures 4A and 4B are graphs depicting the role of acceptor vesicles in lipid transfer by MTP. Line graphs and error bars represent means + SD.
  • Figures 5A-5I graphically illustrate transfer of various lipids in the presence of different acceptors. Line graphs and error bars represent means + SD.
  • Figures 6A and 6B are graphs comparing two methods to measure MTP activity in cell homogenates. Line graphs and error bars represent means + SD.
  • FIGS 7A and 7B graphically depict inhibition of MTP activity by BMS200150. Line graphs and error bars represent means + SD.
  • Figure 8 is a schematic of a unilamellar vesicle with a labeled triglyceride, NBD- TAG, embedded in the bilipid membrane.
  • Figure 9 A graphically depicts phospholipid transfer activity of MTP expressed as % lipid transfer over time, where different amounts of purified bovine MTP, in triplicate, were incubated with donor vesicles (1.2 nmoles PE and 100 pmoles of fluorescent PE) and with acceptor vesicles (7.2 nmoles PC) in 100 ⁇ l of 10 mM Tris-HCl buffer containing 0.1% BSA, 150 mM NaCl, and 2 mM EDTA at 37° C. Fluorescence at 550 nm was monitored over time.
  • Figure 1OA graphically depicts cholesterol ester transfer activity of MTP. Different indicated amounts of purified MTP were incubated with donor (1.2 nmoles PC and 100 pmoles of fluorescent CE) and acceptor vesicles as described in Figure 1OA. Increases in fluorescence emission at 550 nm were recorded at indicated time intervals.
  • Figure 1OB also graphically depicts cholesterol ester transfer activity of MTP where different amounts of MTP were incubated with donor and acceptor vesicles for 30 min and the amounts of fluorescent CE being transferred were calculated.
  • FIG. 1 IA graphically depicts lipid transfer activity in HepG2 cells.
  • HepG2 cell lysates were prepared as described in Example HI and used to perform lipid transfer assays in triplicate. Each assay contained 42 ⁇ g of protein. Data is expressed as line
  • FIG 1 IB graphically depicts lipid transfer activity in liver microsomes.
  • Mouse microsomal contents were prepared as described in Example IH.
  • TAG, CE, or PE lipid transfer activities were measured in triplicate using 21 ⁇ g of protein. Mean values are drawn as line graphs and standard deviations as error bars. Non-linear regression curve fits were performed in Prism.
  • Figure 12A graphically depicts the effect of cardiolipin on the triacylglycerol transfer activity of MTP. Donor vesicles made with and without cardiolipin were used.
  • the assay in triplicate contained 0.25 ⁇ g purified bovine MTP, 3 ⁇ l donor vesicles (100 pmoles of fluorescent TAG, 1.2 nmol PC with or without 0.081 nmol of cardiolipin), 3 ⁇ l PC acceptor vesicles as described in Fig. 1OA.
  • the microtiter plate was incubated at 37° C, fluorescence was monitored over time, and % transfer determined as described before.
  • Figure 12B graphically depicts net deposition of lipids by MTP.
  • Transfer assays were set up in triplicate as in Figure 13A containing 0.25 ⁇ g MTP, 3 ⁇ l donor vesicles, and 3 ⁇ l of acceptor vesicles in 100 ⁇ l assay volume. Percent lipid transfer was measured as described in Fig. 1OA. To measure lipid deposition, 100 ⁇ l of DE52 anion exchange resin was added to the reactions at the predetermined time points. After centrifugation, 10 ⁇ l of supernatant was transferred to a 96 well black microtiter plate. Fluorescence was measured after the addition of 90 ⁇ l of isopropanol.
  • Figure 12C graphically depicts relative net lipid deposition by MTP.
  • Net lipid transfer assays were set up in triplicate as described in Figure 13B. Assays contained 0.25 ⁇ g of purified bovine MTP, donor vesicles (100 pmole of different fluorescent lipids, 1.2 nmole PC, and 0.081 nmoles of cardiolipin), and acceptor vesicles. Percent net lipid deposition was determined at 1 h for TAG as well as CE, and at 1.5 h for PE. The specific activity (% transfer/mg protein/h) was then calculated. Dividing the individual specific activities with the specific activity of TAG lipid transfer and multiplying by 100 provided relative net lipid transfer activities. Bar graphs and error bars represent mean ⁇ SD. DETAILED DESCRIPTION OF THE INVENTION
  • MTP activity is classically measured by incubating purified MTP or cellular homogenates with donor vesicles containing radio labeled lipids for 1-3 h, precipitating the donor vesicles, and measuring the radioactivity transferred to acceptor vesicles.
  • new, simple, rapid, and sensitive fluorescence assays for MTP are provided.
  • a method for measuring levels of MTP and/or quantifying the lipid transfer activity of MTP In another embodiment of the invention, there is provided a method to measure the levels of lipids transferred by MTP (i.e., net transfer of lipids). These methods are useful in identifying specific inhibitors for individual lipid transfer activities, in characterizing different domains involved in transferring these lipids, and isolation of mutants that bind but cannot transfer lipids.
  • MTP's capacity to bind and extract lipid from a membrane in the presence of acceptor vesicles is measured. Fluorescence is quenched when lipids are in unilamellar (one phospholipid bilayer) membrane vesicles. Upon association with MTP, the lipid fluorophore is un-quenched and detected by the flourimeter. The measurement of MTP activity by this assay is time as well as concentration dependent indicating that this procedure can be used to identify other antagonists.
  • a further embodiment measures the net transfer of fluorescent lipids to acceptor vesicles.
  • fluorescent-lipid deposited in acceptor vesicles is quantified after the removal of MTP and donor vesicles by anion exchange resin.
  • This embodiment of the present invention involves an additional step of separating acceptor vesicles from donor vesicles and MTP.
  • the incorporation of negatively charged lipids in the donor vesicles decreases the sensitivity of the assay.
  • this assay is preferred when there is a need to measure net transfer of lipids.
  • the disclosed methods of measuring levels of MTP or quantifying lipid transfer activity are preferred.
  • isolated and/or purified MTP or cellular homogenates are incubated with donor vesicles containing quenched fluorescence-labeled lipids (e.g., triglycerides, cholesterol esters, and/or phospholipids) and different types of acceptor vesicles.
  • the cellular homogenate may be made from any cells that express MTP.
  • the cells are animal liver cells, intestinal cells or heart cells.
  • the animal cells are from a mammal such as a rat, mouse, monkey, or human.
  • Cellular homogenates may also comprise cells from insects or microorganisms.
  • a fluorescence-labeled lipid may include but is not limited to a triglyceride, a cholesterol ester (CE) or a phospholipid.
  • a triglyceride for use in the present invention is triacylglycerol (TAG).
  • TAG triacylglycerol
  • a phospholipid for use in the present invention is phosphatidylethanolamine.
  • lipids examples include but are not limited to 7-nitrobenz-2-oxa-l,3-diazole (NBD), pyrene, or bodipy.
  • NBD 7-nitrobenz-2-oxa-l,3-diazole
  • Methods of labeling lipids with such fluorescent compounds are well known in the art and prepared fluorescently labeled lipids are also readily available.
  • a lipid may contain at least one fluoresecent label at any position.
  • a triacylglycerol may contain at least one NBD at any position while having fatty acids at other positions.
  • pyrene-labeled lipids include the following which are available from Molecular Probes, Inc (Eugene, OR) and listed in their on-line catalog as follows: B-3782 l,2-bis-(l-pyrenedecanoyl)-.s' «-glycero-3-phosphocholine C-212 cholesteryl 1-pyrenebutyrate D-6562
  • BODIP Y-labeled lipids include the following which are available from Molecular Probes, Inc. and listed in their on-line catalog as follows: B-7701 l,2-bis-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-5-indacene-3-undecanoyl)-5n- glycero-3-phosphocholine (bis-BODIPY® FL C 11 -PC) B-3794
  • NBD labeled lipids may also be custom synthesized by Molecular Probes, Inc., e.g., D-16408: Custom synthesis of 1,3-diolein, 2-NBD-X ester or B-1800: Custom synthesis of NBD-labeled cholesterol oleate (cholesterol ester) .
  • N-00360 N-(7-nitrobenz-2-oxa-l, 3-diazol-4-yl)-l,2-dihexadecanoyl- sn-glycerol-3-phosphoethanolamine, triethylammonium;
  • N-03786 (NBD C6-HPC) 2-(6-(7-nitrobenz-2-oxa-l,3-diazol-4-yl)amino) hexanoyl- 1 -hexadecanoyl-sn-glycero-S-phosphocholine;
  • N-03787 (NBD C12-HPC) 2-(12-(7-nitrobenz-2-oxa- l,3-diazol-4-yl)amino dodecanoyl- 1 -hexadecanoyl-sn-glycero-S-phosphocholine.
  • vesicles examples include small unilamellar vesicles, multi-lamellar vesicles, apoB-lipoprotein vesicles or phosphatidylcholine (PC) vesicles.
  • unilamellar vesicles mean vesicles (liposomes) having one phospholipid bilayer.
  • Multi-lamellar vesicles mean vesicles (liposomes) having several phospholipid bilayers.
  • Figure 8 schematically depicts a unilamellar vesicle with a labeled triglyceride, NBD-TG, embedded in the bilipid membrane.
  • Methods for making acceptor vesicles are known. See e.g., refs. 20, 21, and 30-32.
  • Methods of making donor vesicles are also well known, see e.g., refs. 20, 21, 30-32.
  • cardiolipin when making the subject donor vesicles, cardiolipin may be omitted and radiolabeled TAGs replaced with fluorescence-labeled TAGs.
  • Both donor and acceptor vesicles are preferably admixed with an appropriate buffer such as e.g., Tris-HCl, pH at around 7.4.
  • the donor and acceptor vesicles may be stored separately. Alternatively, the vesicles may be stored together so that the donor/acceptor vesicle mixture may be used directly in an assay or kit of the present invention.
  • the ratio of donor to acceptor vesicles is preferably in the range of from about 1 :4 (donor: acceptor) to about 1:10 (donor: acceptor). Most preferably, the ratio of donor to acceptor vesicles is about 1 :6.
  • Vesicle preparations may be further stabilized by the addition of NaCl and BSA to the final concentrations of about 150 mM and lmg/ml, respectively.
  • MTP may be isolated from different sources and purified using well known methods. See e.g., refs. 20 and 21.
  • MTP may be isolated from bovine
  • Human MTP may be prepared by transfecting cultured cells with an expression vector comprising the coding sequence for MTP as described in ref. 15.
  • the disclosures of these references as well as all other cited literature references, are incorporated by reference herein as if fully set forth.
  • increases in fluorescence due to MTP- mediated lipid transfer may be measured after a short period.
  • the methods provided herein have been successfully used to measure the MTP activity in HepG2, Caco-2 cells, and COS cells transfected with MTP expression plasmids.
  • the methods provided by the present invention are useful in studying inhibition of cellular as well as purified MTP by its antagonists. The methods are amenable to automation and may be easily adopted for large-scale thorough put screening.
  • the present invention provides methods which may be used to assay MTP in any sample for various purposes such as identification, modulation, diagnosis etc.
  • the methods may be used to assay activity in purified MTP samples, MTP present in cell lines, tissues etc.
  • the assays provided by the present invention are very versatile and can measure the transfer of any lipid by MTP that contains a fluorescent label.
  • the methods provided herein to measure MTP activity are simple and rapid. They are based on the determination of increases in fluorescence attributable to the binding of fluorophor with MTP that occurs during the transfer of lipids between donor and acceptor vesicles.
  • the methods of the present invention faithfully measure cellular activity in cells known to express MTP and do not measure activity in cells that do not express MTP (Table 1). Furthermore, the methods display similar inhibitory properties of antagonists that were identified using the radioisotope assay of the prior art (Fig. 7). MTP shows significantly higher activity in the presence of acceptor vesicles (Fig. 4). The low lipid binding activity of MTP in the absence of acceptor vesicles provides a unique opportunity to understand the role of different acceptor vesicles in the lipid transfer process. With respect to the method provided herein for measuring levels of lipids transferred by MTP (net transfer of lipids), the method actually measures the net deposition of fluorescent-labeled lipids by MTP in acceptor vesicles.
  • the present invention provides simple and rapid fluorescence assays for the measurement of MTP activity.
  • the advantages of the new methods include ease, rapidity, sensitivity, avoidance of the use of negatively charged lipids, versatility in studying different lipid transfer activities by purified and cellular MTP, ability to measure inhibitory activities of antagonists, and forestalling the use of radioactivity.
  • the fluorescence assays provided by the present invention may be easily automated and used for large-scale, high-throughput screening. This approach is useful in order to identify compounds that partially inhibit MTP activity and possibly minimize the unwanted side effects related to TAG accumulation in cells.
  • MTP is a multifunctional protein that may have functions other than being a dedicated lipoprotein assembly chaperone. Compounds identified via screening based on the fluorescence assays provided herein may be useful in the identification of other functions of MTP unrelated to lipoprotein assembly and secretion.
  • the MTP assays basically consist of three components: donor vesicles, acceptor vesicles, and MTP.
  • the methods provided by the present invention show a linear relationship with all three components of the assay mixture and time (Figs. 1-4).
  • a typical assay may be performed as outlined below.
  • the amounts of the different components listed below may of course be changed, so long as the ratios among the different components remain relatively the same.
  • Four different conditions (blank, total, positive control, and test) are recommended for each assay.
  • the reaction is started by the final addition of the MTP source.
  • About 3 ⁇ l each of acceptor and donor vesicles are pipetted onto fluorescence microtiter (black) plates.
  • About 10 ⁇ l of 10 rriM Tris, pH 7.4, containing 2 niM EDTA and 10 ⁇ l of 1% BSA stock in 1.5 M NaCl are added.
  • the exact number of vesicles for use in the assays described herein is difficult to quantify routinely.
  • the 3 ⁇ l of donor vesicles correlate to about 450 nmol of phosphatidylcholine (PC) and about 14 nmol of triglyceride per milliliter.
  • a range of donor vesicle concentrations may be employed.
  • a preferred range is e.g., anywhere from about 200 to about 600 nmol of phosphatidylcholine (PC) and 7-20 nmol triglyceride.
  • the 3 ⁇ l of acceptor vesicles correlate to about 2,400 nmol PC/ml.
  • a range of vesicle concentrations may be employed, e.g., anywhere from about 1,400 to about 3,400 nmol PC/ml.
  • control buffer which contains the MTP source in positive control and test samples
  • the needed amount of control buffer is added and the volume made up with water to about 100 ⁇ l.
  • positive controls a known amount of the MTP source is added and the volume made up with water to about 100 ⁇ l.
  • unknown samples are added and made up to the final assay volume. For totals, about 3 ⁇ l of donor vesicles and about 97 ⁇ l of isopropanol only are added. The mixtures are incubated at about 37 0 C for about 30 min. Fluorescence units are measured using excitation and emission wavelengths of 460-470 and 530-550 nm, respectively.
  • the incubation time can be increased, hi fact, the same titer plate can be used several times to measure increases in fluorescence with time.
  • the fluoropliore is unstable in isopropanol over long periods of time. Thus, for periods longer than 30 min, total values from readings determined at 30 min or at earlier times should be used.
  • the assay ingredients, including vesicles and positive controls may be made as described herein, and are also available from Chylos, Inc. (Woodbury, NY).
  • donor and acceptor vesicles may be both stored and used as combined as described above. In this embodiment, when an assay is performed, only one pipetting step of vesicles (both donor and acceptor) is needed.
  • the vesicles are preferably admixed with an appropriate buffer such as Tris HCl, pH at about 7.4. Other buffers may also be used such as e.g., phosphate buffer and HEPES.
  • NaCl is added to a final concentration of about 150 mM.
  • a NaCl stock solution (e.g. 3M) may be made and then diluted to yield the final concentration.
  • Other salts such as KCl and MgCl 2 may also be used.
  • BSA is added to a final concentration of about lmg/ml in order to stabilize the vesicles.
  • a stock solution e.g., 20 mg BS A/ml
  • the incubation step may be performed at room temperature.
  • Donor vesicles containing fluorescent-lipids and acceptor vesicles may be prepared in as described in Example I. hi a preferred embodiment, equal volumes of donor and acceptor vesicles may be combined. A typical assay procedure is described
  • Measuring the MTP activity Measure fluorescence units (FU) using excitation and emission wavelengths of 460-470 and 530-550 nm, respectively. Calculation of the MTP activity:
  • a method of identifying compounds that modulate the lipid transfer activity of MTP comprises the steps of: (a) incorporating a fluorescence-labeled lipid into donor vesicles; (b) preparing acceptor vesicles; (c) mixing an aliquot of acceptor vesicles and the labeled donor vesicles with a test compound, the test compound being a known or unknown modulator of MTP; (d) adding a cellular homogenate containing MTP or isolated MTP to the mixture containing donor vesicles, acceptor vesicles, and test compound; (e) incubating a first aliquot of acceptor vesicles, labeled donor vesicles, test compound and MTP for a time and under conditions sufficient to allow binding of the fluorescence-labeled lipid with MTP during transfer of the labeled lipid from donor to acceptor vesicles; (f) incubating a second aliquot of accept
  • kits for measuring the lipid transfer activity of MTP comprises acceptor vesicles and fluorescence-labeled donor vesicles as hereinbefore described.
  • the fluorescence-labeled donor vesicles are comprised of a triglyceride, a cholesterol ester, or a phospholipid
  • the triglyceride is any triacylglycerol that contains NBD label such as 1, 2, dioleoyl 3 -NBD glycerol (NBD-TAG).
  • the phospholipid is phosphatidylethanoloamine.
  • the vesicles are preferably admixed with an appropriate buffer such as Tris HCl, pH at about 7.4.
  • an appropriate buffer such as Tris HCl, pH at about 7.4.
  • Other buffers may also be used such as e.g., phosphate buffer and HEPES.
  • Acceptor and/or donor vesicles which form part of the kit may include small unilamellar vesicles, multi-lamellar vesicles, apoB-lipoprotein vesicles, or phosphatidylcholine (PC) vesicles.
  • the vesicles may be stabilized by the addition of BSA.
  • the present invention also provides a method of quantifying lipid transfer activity of microsomal triglyceride transfer protein (MTP).
  • MTP microsomal triglyceride transfer protein
  • the method comprises the steps of: (a) preparing donor vesicles having a fluorescence-labeled lipid incorporated therein:, (b) preparing acceptor vesicles; (c) incubating either a cellular homogenate containing MTP or isolated MTP with the acceptor vesicles and the labeled donor vesicles for a time and under conditions sufficient to allow binding of the fluorescence-labeled lipid with MTP during transfer of the labeled lipid from donor to acceptor vesicles; and (d) measuring fluorescence of the fluorescently labeled lipid bound to the MTP.
  • Also provided by the present invention is a method for measuring levels of lipids transferred by MTP. (i.e., measuring net transfer of lipids by MTP).
  • the method comprises the steps of: (a) preparing negatively-charged donor vesicles having a fluorescence-labeled lipid incorporated therein; (b) preparing acceptor vesicles; (c) incubating either a cellular homogenate containing MTP or isolated MTP with the acceptor vesicles and the labeled donor vesicles for a time and under conditions sufficient to allow binding of the fluorescence-labeled lipid with MTP and transfer of the fluoresescence-labeled lipid from donor to acceptor vesicles; (d) removing negatively charged donor vesicles and MTP from the incubation mixture of step (c); and (e) measuring fluorescent labeled lipids transferred to acceptor vesicles.
  • a negatively charged lipid such as cardiolipin
  • Other negatively charged lipids may also be used and include but are not limited to phosphatidyl serine and phosphatidyl inositol.
  • the negatively charged donor vesicles and MTP may be removed by sedimentation such as, e.g. centrifugation.
  • the mixture of step (c) may be centrifuged at about 10, 000 to about 12,000 rpm. Acceptor vesicles remain in the supernatant.
  • kits for measuring net transfer of lipids transferred by MTP.
  • the kits comprise acceptor vesicles, and negatively charged fluorescence-labeled donor vesicles.
  • the fluorescence-labeled donor vesicles may be comprised of a triglyceride, a cholesterol ester, or a phospholipid.
  • An example of a triglyceride contained in the kit is any triacylglycerol that contains NBD label.
  • the triacylglycerol may be l,2,dioleolyl 3-NBD glycerol (NBD-TAG).
  • NBD-TAG NBD-TAG
  • An example of a phospholipid which make up a donor vesicle is phosphatidylethanolamine.
  • the acceptor vesicles for use in the kits may be small unilamellar vesicles, multi-lamellar vesicles, apoB-lipoprotein vesicles, or phosphatidylcholine (PC) vesicles.
  • the vesicles contained in the kit may be stabilized by the addition of BSA.
  • MTP was purified from bovine liver using the radioactivity assay (20, 21) and has been used previously (24-29).
  • PC and TAG were from Avanti Lipids (Alabaster, AL). Fluorescence (nitrobenzoxadiazole)- labeled TAG was from Molecular Probes (Eugene, OR).
  • Vesicles containing fluorescence-labeled cholesteryl ester (CE) and phospholipid (PL) were from Roar Biomedical, Inc. (New York, NY) and Cardiovascular Target, Inc. (New York, NY), respectively. Isopropanol and other chemicals were from Sigma Chemical Co. (St. Louis, MO). Acceptor vesicles were prepared as described by Wetterau and associates (20, 21, 30-32).
  • Donor vesicles were also prepared according to their procedure except that cardiolipin was omitted and radiolabeled TAGs were replaced with fluorescence-labeled TAGs.
  • Known amounts of fluorescent lipids were diluted in isopropanol to generate a standard curve, and amounts of labeled lipids in vesicles were determined after their disruption with isopropanol.
  • the amounts of triolein in vesicles were quantified by a colorimetric assay (InfinityTM Triglyceride Reagent Kit; Sigma).
  • the MTP inhibitor BMS200150 diphenyl-propyl-piperidiiiyl-dihydroiso-indole has been described (12) and was a kind gift from Dr. Haris Jamil of Bristol-Myers Squibb (Princeton, NJ). Transfer assay
  • the assay was done in Microfluor ® 2 Black “U” Bottom Micro-titer ® plates (Thermo Labsystems, Franklin, MA). To the wells, was added 3 1 of donor (450 nmol of PC and 14 nmol of TAG per milliliter), 3 1 of acceptor (2,400 nmol PC/ml) vesicles, 10 1 of 10 mM Tris-HCl buffer, pH 7.4, 2 mM EDTA, 150 mM NaCl, distilled water to make the final assay volume of 100 1, and purified MTP (0.1-1.5 g) in triplicate, rn some experiments, NaCl and BSA were added to obtain final concentrations of 150 mM and 1 mg/ml, respectively.
  • Cos-7 cells were grown (37° C, 5% CO 2 , humidified chamber) in DMEM (Cellgro Mediatech, Inc., Herndon, VA) supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic (Life Technologies, Rockville, MD). Cells (1 x 10 6 ) were plated in 75 cm 2 flasks 24 h before transfection. At the time of transfection, cells were about 50-60% confluent.
  • the MTP expression vector (15) pRc-hMTP (7 g; expresses human MTP under the control of the cytomegalovirus promoter) was introduced into Cos-7 cells complexed with 21 1 of FuGENE-6 Transfection Reagent (Roche Diagnostics, Indianapolis, IN). Cells were maintained at 37° C and 5% CO 2 in 7 ml of medium for about 72 h. Cos-7 cells were also treated with FuGENE-6 alone (mock transfection) and used as controls. Determination of MTP activity in cell homogenates
  • MTP activity in cellular homogenates was determined as described by Jamil et al. (12). Confluent cell monolayers were washed with ice-cold sterile PBS, pH 7.4, scraped in 5 ml of PBS, transferred to 15 ml conical tubes, and pelleted down by centrifugation (2,500 rpm, 10 min, room temperature). At this point, cell pellets can be stored at 7O 0 C. For homogenization, 750 ⁇ l of homogenization buffer (50 mM Tris-HCl, pH 7.4, 50 mM KCl, and 5 mM EDTA) and 7.5 ⁇ l of protease inhibitor cocktail (catalog number P 2714; Sigma) were added to the cell pellets.
  • homogenization buffer 50 mM Tris-HCl, pH 7.4, 50 mM KCl, and 5 mM EDTA
  • protease inhibitor cocktail catalog number P 2714; Sigma
  • Cells were then suspended by repeated aspirations through a needle (29G, 1 m inches) attached to a 3 ml syringe and homogenized on ice in a ball-bearing homogenizer (clearance 0.253 inches, 10 passages). Cell homogenates were stored on ice, and protein concentrations were determined by the Bradford method (33) using Coomassie Plus Reagent (Pierce, Rockford, IL) and BSA as standards. Cell homogenates were diluted with homogenization buffer to a protein concentration of 1.5 mg/ml. MTP was released from microsomes by deoxycholate treatment (12).
  • cell homogenates were adjusted to 0.054% deoxycholate by the addition of one-tenth volume of 0.54% sodium deoxycholate, pH 7.4, and left on ice for 30 min with occasional mixing.
  • Cell membranes were subsequently removed by centrifugation in a SW55 Ti rotor at 50,000 rpm for 1 h at 10° C.
  • the supernatants were dialyzed in 12-14 kDa cutoff dialysis bags against 15 mM Tris-HCl, pH 7.4, 40 mM NaCl, 1 mM EDTA, and 0.02% NaN3 , with the first change after Ih and the second change after 2 h followed by overnight dialysis.
  • Cell homogenates were removed from the dialysis bag and used for protein determination and MTP assay.
  • HepG2 cells were incubated with different concentrations of the MTP inhibitor BMS200150 for 24 h, and cell homogenates obtained from the cells were used for MTP assay.
  • Rat liver microsomes were prepared as described by Wetterau and Zilversmit (20, 21) with slight modifications. Briefly, 2 g of rat liver was cut into small pieces and washed twice with ice-cold PBS. Pieces were then homogenized in 2 ml of 50 mM Tris- HCl, pH 7.4, 5 mM EDTA, 250 mM sucrose, and 0.02% sodium azide using a Polytron homogenizer and centrifuged (Beckman micro-centrifuge, 10,900 rpm, 30 min, 4 0 C).
  • TAGs were incorporated into small unilamellar PC vesicles (donor vesicles). It was anticipated that the encapsulation would result in the quenching of the fluorophore. Indeed, disruption of increasing amounts of donor vesicles with iso-propanol resulted in enhanced measurable fluorescence (Fig. IA, total). This represents the total amounts of fluorophore present in the vesicles. Before disruption, this fluorescence is not detectable because it is quenched in vesicles. It was also envisioned that donor vesicles would be stable and would not release the fluorophore in the absence of MTP.
  • the "transfer” represents the amounts of TAG being transferred by MTP between vesicles.
  • the MTP-bound fluorophore is most likely exposed to the aqueous environment and is now detected by the fluorimeter.
  • the fluorescence units were 40-47% higher than the blank values.
  • the data were used to calculate the percentage transfer of TAG (Fig. IB).
  • the percentage transfer activity increased up to 2 ⁇ l of the donor vesicles (28 pmol of TAG) and appeared to saturate thereafter.
  • the effect of different concentrations of acceptor vesicles was studied. In these experiments, constant amounts of MTP and donor vesicles were incubated with different volumes of acceptor vesicles (Fig. 1C).
  • the transfer assay was performed in 10 tubes using 0.5 g of MTP, 3 ⁇ l of donor, and 3 ⁇ 1 of acceptor vesicles.
  • the interassay variations were evaluated. Comparison of seven different independent determinations performed in triplicate using 1 ⁇ g of MTP revealed an interassay CV of 0.19. The percentage transfer observed in these experiments was 34.5 ⁇ 3.0.
  • Figure 1 shows the effect of different amounts of donor and acceptor vesicles on the transfer of triacylglycerol (TAG) by microsomal triglyceride transfer protein (MTP).
  • TAG triacylglycerol
  • MTP microsomal triglyceride transfer protein
  • TAG transfer activity increased with time up to 30 min (Fig. 2A). After that time, the transfer activity began to saturate.
  • the effect of temperature on transfer activity was studied (Fig. 2B).
  • the lipid transfer activity of MTP was the same at room temperature (22°C) and at 37°C, indicating no significant effect of temperature on activity. These results likely indicate that MTP is optimally active at 22 0 C.
  • the effect of NaCl on MTP activity (Fig. 2C) was also determined.
  • the addition of increasing concentrations of NaCl up to 150 mM resulted in increased MTP activity. Higher concentrations of NaCl appear to inhibit transfer activity. It is concluded therefore, that 30 min incubations and 150 mM NaCl are optimum to determine MTP activity.
  • Fig. 3A The addition of increasing amounts of MTP resulted in enhanced detectable fluorescence (Fig. 3A).
  • BSA decreased the amounts of detectable fluorescence and may represent either quenching of the released fluorescence by BSA or stabilization of the donor vesicles by BSA, preventing the basal fluorophore leakage, hi Fig. 3B, the data were converted to measure the percentage of TAG undergoing transfer between vesicles.
  • the amounts of TAG being transferred reached saturation at 2 ⁇ g of MTP. At saturation, 40% of the total TAG was in the process of transfer and probably represented the maximum binding capacity of MTP.
  • the specific activity of MTP determined by radiolabel and fluorescence assays was compared.
  • specific activities measured by the fluorescence assay were higher than those observed using the radiolabel assay.
  • the higher specific activities may be attributable to the higher sensitivity of the assay. Another reason for the difference may be different parameters used in these two assays.
  • the amount of TAG transferred to acceptor vesicles is measured.
  • the fluorescence assay described in this example measures the amount of TAG being transferred by the MTP.
  • MTP microsomal triglyceride transfer protein
  • the MTP assay consists of three components: donor vesicles, acceptor vesicles, and MTP. Obviously, donor vesicles and MTP are required. It was reasoned that MTP could transfer lipids between donor vesicles and that the acceptor vesicles may not be needed for activity measurements.
  • MTP is known to transfer other lipids besides TAG (20).
  • experiments were performed to evaluate the suitability of this assay to study the transfer of CEs and PLs in addition to TAG (Fig. 5).
  • small unilamellar vesicles PC/TAG vesicles
  • apoB lipoproteins which contained both VLDL and LDL
  • HDL high-density lipoprotein
  • acceptor vesicles (3 ⁇ l) containing TAG (A-C), cholesteryl ester (D-F), or phospholipids (G-I) were used.
  • the acceptor vesicles (3 ⁇ l; 2,400 nmol PC/ml) were small unilamellar vesicles (PC/TAG vesicles; A, D, and G), apolipoprotein B (apoB) lipoproteins (VLDL and LDL; B, E, and H), and HDLs (10 mg protein/ml; C, F, and I).
  • MTP was able to transfer PLs when donor vesicles were incubated with PC/TAG vesicles; the increase in fluorescence was 186% at 4 h (Fig. 5G).
  • studies of the transfer of PL by MTP in the presence of apoB lipoproteins as acceptors were difficult to interpret (Fig. 5H). This was attributable to a significant increase in the background fluorescence when donor vesicles were incubated with apoB lipoproteins in the absence of MTP (compare controls in Fig. 5G, H; also note the different >> values in these panels).
  • MTP did not transfer PL when HDL was used as an acceptor (Fig. 51).
  • the deoxycholate method is cumbersome, involves several steps, and requires at least 2 days.
  • the method of Chang, Limanek, and Chang (34) was evaluated, which involves the disruption of cells by hypotonic buffers and requires far less time to prepare cell extracts for assay.
  • Rat liver microsomes were subjected to hypotonic buffer treatment, and released contents were used for MTP activity measurements.
  • Fig. 7 Experiments were then performed to measure the effect of MTP antagonists on its activity (Fig. 7).
  • an MTP inhibitor, BMS200150 described by Jamil et al. (12) was used.
  • Purified MTP (1 g) was incubated with donor and acceptor vesicles for 30 min in the presence of the indicated concentrations of the MTP inhibitor BMS200150.
  • Fig. 7A increasing concentrations of BMS200150 resulted in a dose- dependent inhibition of the purified MTP activity.
  • the ICs 0 was 0.08 M and was in the range reported by others (12).
  • HepG2 cells were incubated with different concentrations of the inhibitor for 24 h, and homogenates were prepared using the deoxycholate method (12) and assayed for MTP activity.
  • Cells were washed, and lysates were prepared using the deoxycholate method described in Materials and Methods.
  • 40-50 g of cellular proteins was used in triplicate.
  • increasing concentrations of BMS200150 resulted in decreased cellular MTP activity.
  • the IC 50 value of -1.3 M is in agreement with published studies (12).
  • MTP was purified from bovine liver using the radioisotope assay (7; 19) and rat liver using a kit (Chylos Inc., Woodbury, NY). Fluorescent (nitrobenzoaxadiazol)- labeled phosphatidylcholines (PC) and unlabeled PC were purchased from Avanti Polar Lipids (Alabaster, AL). Nitrobenzoaxadiazol-labeled CE, PE, as well as TAG were from Molecular Probes (Eugene, OR). Thermo Labsystems (Franklin, MA) supplied the Black 96 well microtiter plates. Isopropanol and other chemicals were from Sigma Chemical Co. (St. Louis, MO).
  • phospholipid vesicles containing fluorescent PE and CE Acceptor phosphatidylcholine (PC) vesicles were prepared as described by Wetterau and associates (20-22, 31). Donor vesicles were also prepared by sonication as described before (20-22, 31, 35). Briefly, unlabeled phosphatidylethanolamine (PE) and fluorescent-PE were evaporated and sonicated under nitrogen for 45 minutes at 4° C. CE donor vesicles were prepared similarly using fluorescent-CE and unlabeled PC. Vesicles, collected after centrifugation (about 1.2,000 rpm, 10 minutes, table-top centrifuge) were found to be stable for one month. Known amounts of fluorescent lipids were diluted in isopropanol to generate standard curves used to estimate the moles of fluorescent lipids incorporated in the donor vesicles.
  • Measuring lipid transfer activities Assays were performed in triplicate in a black 96 well microtiter plate (35). A final reaction mixture (100 ⁇ l) contained 3 ⁇ l of donor (1.2 nmol PC or PE containing various fluorescent lipids), 3 ⁇ l of acceptor (7.2 nmol of PC) vesicles, and a MTP source in 10 mM Tris, pH 7.4, 0.1% BSAj 150 mM NaCl buffer. The microtitre plate was incubated at 37 0 C, and at predetermined time points, samples were excited at 485 ran and fluorescence emission was measured at 550 nm using a Victor 3 dual fluorimeter / luminometer (Perkin Elmer).
  • the reaction mixture was then transferred to microcentrifuge tubes containing 100 ⁇ l of DE52 (equilibrated (1:1, v:v) with 15 mM Tris-Cl, pH 7.4, 1 mM EDTA, and 0.02% sodium azide buffer), rotated at 4°C for 5 minutes and centrifuged (12,000 rpm, 5 min,4°C?).
  • Supernatants (10 ⁇ l) containing only acceptor vesicles were transferred to a microtiter plate, and fluorescence was measured at 5 min intervals after adding 90 ⁇ l isopropanol. Readings obtained at 20 min were used for calculations. The blank values obtained in the absence of MTP were subtracted from the sample values, divided by the total fluorescence reduced by blanks, and multiplied by 100 to determine the % of lipids deposited to acceptor vesicles.
  • MTP activity in cells and tissues HepG2 cells grown to confluence in Tl 75 flasks were washed with PBS and then swelled by 2 minutes incubation at room temperature in hypotonic buffer (1 mM Tris-Cl, pH 7.4, 1 mM MgCl 2 , and ImM EGTA) (34, 35).
  • the buffer was aspirated, cells were scraped in 750 ⁇ l of ice-cold hypotonic buffer containing protease inhibitors, homogenized (20 passages through a 21 -gauge needle), the lysates were centrifuged (50,000 rpm, 4° C, 1 hour, SW55 Ti rotor), and supernatants were used for lipid transfer assays and protein determination (25).
  • liver microsome preparation (20, 21, 35) mouse liver pieces were washed with PBS, homogenized in 50 mM Tris-Cl, pH 7.4, 5 mM EDTA, 250 mM sucrose, and 0.02% sodium azide using a Polytron homogenizer, and centrifuged (10,900 rpm, 30 minutes, 4° C, Beckman microcentrifuge). Supernatants were adjusted to pH 5.1 with concentrated HCl, mixed in the cold for 30 minutes and centrifuged (13,000 rpm, 30 minutes, 4° C, Beckman microcentrifuge).
  • Pellets were resuspended in ImM Tris-Cl, pH 7.6, 1 mM EGTA, and 1 mM MgCl 2 , vortexed, incubated for 30 minutes at 4° C, ultracentrifuged (SW55 Ti rotor, 50,000 rpm, 4° C, 1 h), and supernatants were used for lipid transfer
  • Examples I-II exemplify a simple, rapid, and sensitive assay to measure TAG transfer activity of MTP (35).
  • Example UI we determined whether the same procedure could be used to quantify the phospholipid (PL) transfer activity of MTP (Figs. 9A-9B).
  • PL phospholipid transfer activity of MTP
  • Fig. 9A Upon the incubation of different amounts of MTP with donor vesicles containing fluorescent-PE and acceptor vesicles, fluorescence increased and saturated in a time dependent fashion (Fig. 9A). Each concentration gave a specific curve indicating MTP dependent increases in fluorescence, and was confirmed by plotting the 1 h data against varying amounts of MTP (Fig. 9B).
  • PL transfer was linear between 0.1 and 0.3
  • AU lipid transfer activities (TAG, CE, and PL) could be measured in HepG2 cell homogenates (Fig. 1 IA). Lipid transfer activities showed time dependent increases and reached maximum between 20-30 minutes of incubation. The rate of CE being transferred and the maximum amounts of CE being transferred were lower than those observed for TAG. PL transfer profiles were similar to those of CE and TAG transfer. The major difference was that PL transfer activity reached a significantly lower maximum transfer.
  • lipid transfer activity present in mouse liver microsomes (Fig. 1 IB). All three-lipid transfer activities could be measured in microsomal samples using these assays. Again, the major activity observed was TAG transfer followed by CE and PL transfer activities. The initial rates and the maximum amounts of TAG being transferred were significantly higher compared to those of CE and PL. These studies indicate that the efficiency of lipid transfer is the greatest for TAG followed by those of CE and PL transfer in mouse liver microsomes. Relative lipid transfer activities in MTP
  • HepG2 cell lysates demonstrated less CE transfer activity compared with purified MTP preparations. This suggests that proteins or other soluble factors in cells or tissues may interfere with this transfer.
  • relative PL transfer activities observed in HepG2 cells and liver microsomes were 2 to 4 folds higher than those observed in purified MTP preparations. This is most likely due to the presence of other phospholipid transfer proteins, such as, phosphatidylcholine and phosphatidylinositol transfer proteins (36), in cells and tissues that might transfer fluorescent-PE.
  • Lipid transfer assays were performed using fluorescent lipids as described above in Figs. 2-4. Specific activities (% transfer / mg protein / hour) were then calculated using time points falling in the linear range for each assay. The absolute rate of lipid transfer (nmol of lipid transferred / mg protein / h) was determined by comparing the fluorescence to standard curves. Dividing the specific activity of the lipid transfer in question by the specific activity of TAG transfer, and multiplying by 100 provided the relative activities (in parentheses).
  • Microsomal triglyceride transfer protein the abetalipoproteinemia gene product, mediates the secretion of apolipoprotein B-containing lipoproteins from heterologous cells. J. Biol. Chem. 269: 21951-21954.
  • MTP Microsomal triglyceride transfer protein
  • MTP microsomal triglyceride transfer protein

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Abstract

La présente invention concerne des procédés pour mettre à l'essai des protéines de transfert de triglycérides microsomales (MTP) lesquels peuvent faire l'objet d'une automatisation et d'un criblage grande vitesse. Les essais biologiques peuvent être utilisés pour mesurer l'activité des MTP dans des homogénats cellulaires et tissulaires ainsi que des MTP purifiées. Sont également décrits des procédés de mesure des taux de lipides transférés par les MTP. Les procédés de l'invention sont faciles, rapides, sensibles, ne nécessitent pas l'utilisation de radioactivité et présentent également une versatilité dans l'étude des différentes activités de transfert lipidique par des MTP cellulaires et purifiées et une capacité à mesurer l'activité d'inhibition. De plus, la présente invention décrit des procédés d’identification des composés qui modulent l’activité de transfert lipidique des MTP ainsi que des kits de mesure de l’activité de transfert lipidique des MTP ainsi que du transfert lipidique net par les MTP.
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US5770355A (en) * 1993-10-29 1998-06-23 Brocia; Robert W. Heart disease test kit and method of determining a heart disease risk factor and efficacy of a treatment for heart disease
US5789197A (en) 1992-03-06 1998-08-04 E. R. Squibb & Sons, Inc. Microsomal triglyceride transfer protein

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US20060228764A1 (en) * 2003-06-27 2006-10-12 Hussain M M Fluoresence assay for mtp activity

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US5789197A (en) 1992-03-06 1998-08-04 E. R. Squibb & Sons, Inc. Microsomal triglyceride transfer protein
US5770355A (en) * 1993-10-29 1998-06-23 Brocia; Robert W. Heart disease test kit and method of determining a heart disease risk factor and efficacy of a treatment for heart disease

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US8238261B2 (en) 2007-04-06 2012-08-07 Interdigital Technology Corporation Method and apparatus for identifying mobile network protocol capabilities

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