WO1993021517A1 - MESURE DE PROPENSION A l'ATHEROSCLEROSE; CAPACITE D'OXYDATION DE RESIDUS D'OLEFINES DES LIPOPROTEINES ET DES LIPIDES DU PLASMA - Google Patents

MESURE DE PROPENSION A l'ATHEROSCLEROSE; CAPACITE D'OXYDATION DE RESIDUS D'OLEFINES DES LIPOPROTEINES ET DES LIPIDES DU PLASMA Download PDF

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Publication number
WO1993021517A1
WO1993021517A1 PCT/US1993/003116 US9303116W WO9321517A1 WO 1993021517 A1 WO1993021517 A1 WO 1993021517A1 US 9303116 W US9303116 W US 9303116W WO 9321517 A1 WO9321517 A1 WO 9321517A1
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WO
WIPO (PCT)
Prior art keywords
sample
area
olefinic
nuclear magnetic
magnetic resonance
Prior art date
Application number
PCT/US1993/003116
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English (en)
Inventor
Eric T. Fossel
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The Beth Israel Hospital Association
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
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Publication of WO1993021517A1 publication Critical patent/WO1993021517A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01R—MEASURING ELECTRIC VARIABLES; MEASURING MAGNETIC VARIABLES
    • G01R33/00—Arrangements or instruments for measuring magnetic variables
    • G01R33/20—Arrangements or instruments for measuring magnetic variables involving magnetic resonance
    • G01R33/44—Arrangements or instruments for measuring magnetic variables involving magnetic resonance using nuclear magnetic resonance [NMR]
    • G01R33/46—NMR spectroscopy
    • G01R33/465—NMR spectroscopy applied to biological material, e.g. in vitro testing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/60—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving cholesterol
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/323—Arteriosclerosis, Stenosis

Definitions

  • the present invention relates to a novel method for assessing the propensity to atherosclerosis in a living
  • the plasma lipoproteins are complexes in which the lipids and proteins occur in a relatively fixed ratio. They carry water-insoluble lipids, such as cholesterol and cholesterol esters for eventual cellular utilization between various organs via the blood, in a form with a relatively small and constant particle diameter and weight. While all cells require
  • Atherosclerosis a disease state referred to as atherosclerosis. It is also known that total serum cholesterol can be correlated with the incidence of atherosclerosis.
  • Human plasma lipoproteins occur in four major classes that differ in density as well as particle size as shown, in the table below.
  • VLDL Chylomierons
  • Phoaphollpids % of dry weight 3-8 15-20 22 30
  • the characteristic flotation rates in Svedberg flotation units (S f ) of the lipoproteins are determined in an NaCl medium of density 1.063 g ml at 26oC, in which
  • lipoproteins float upward and simple proteins sediment.
  • the plasma lipoproteins contain varying proportions of protein and different types of lipid.
  • the very low-density lipoproteins contain four different types of polypeptide chains having distinctive amino acid sequences.
  • the high-density lipoproteins have two different types of polypeptide chains, of molecular weight 17,500 and 28,000.
  • the polypeptide chains of the plasma lipoproteins are believed to be arranged on the surface of the molecules, thus conferring hydrophilic
  • the different lipoprotein classes contain varying amounts of cholesterol.
  • a total serum cholesterol measurement is an average of the amount that each lipoprotein. class contributes to the total serum lipoprotein.
  • the present invention is a method for assessing
  • a sample of a patient's plasma is subjected to carbon-13 (C-13) or proton nuclear magnetic spectroscopy to generate a water-suppressed proton nuclear magnetic resonance (NMR) spectrum.
  • C-13 carbon-13
  • NMR proton nuclear magnetic resonance
  • the olefinic residue region of the spectrum is identified and its area calculated.
  • the patient's plasma is then oxidized and a carbon-13 NMR or proton nuclear magnetic resonance spectrum obtained and the area of the olefinic region calculated. The two areas are then
  • integration shall mean measuring the area of a resonance.
  • the patient's atherosclerotic risk is classified as either normal or advanced depending on the decrease in olefinic area from the spectrum obtained from an unoxidized plasma sample to the spectrum obtained from an oxidized plasma sample.
  • an object of the present invention is to provide a method for predicting atherosclerotic risk in a living patient.
  • FIG. 1 is a carbon-13 NMR spectrum of the non-water components (water suppressed) at 125 MHz of a plasma sample from a living patient prior to oxidation obtained in accordance wirh the oresent invention
  • FIG. 2 is a graph produced following the procedure of the instant invention showing the decrease in olefinic area of 12 known artherosclerotic patients and 12 normal controls.
  • the present invention is a novel method .for predicting
  • the test for high atherosclerotic risk will typically be performed, in vitro.
  • the process operates on any lipid-containing body fluid, blood, or bone marrow plasma. Whole blood, serum, or plasma may be used. While the test may be performed on any such lipid-containing body fluid, blood, or bone marrow plasma. Whole blood, serum, or plasma may be used. While the test may be performed on any such lipid-containing body fluid, blood, or bone marrow plasma. Whole blood, serum, or plasma may be used. While the test may be performed on any such
  • lipid-containing body fluid work to date has focused on blood plasma and thus in a preferred embodiment of the present invention plasma or serum is used.
  • the test sample need not be fasting. Correct sample preparation and execution is essential to carry out a successful measurement on plasma. Blood is
  • Plasma samples are never frozen because freezing destroys lipoprotein lipid structural integrity, samples which show any visible sign of hemolysis are excluded.
  • deuterium oxide; D 2 O is added to the plasma sample, which sample containing unoxidized plasma is then subjected to C-13 nuclear magnetic resonance spectroscopy to generate a nuclear magnetic resonance spectrum.
  • a spectrum illustrating plasma prior to oxidation is shown in FIG. 1.
  • a solution of xanthine in NaCl in addition to a solution of xanthine oxidase in NaCl are then added to the plasma sample. This admixture is then incubated for 24 hours. Following the incubation period, a C-13 NMR spectrum of the oxidized plasma sample is obtained.
  • the olefinic region, of interest for the purpose of the present invention and representing plasma lipoprotein resonances appears at the 126-132 ppm section of the NMR spectrum.
  • FIG. 2 shows the comparison of the area of olefinic resonances (in arbitrary units) received when practicing the method of the instant invention on twelve patients just prior to coronary by-pass surgery (i.e,
  • PATIENTS shows the area received from the spectrum prior to the oxidation of the present invention.
  • p ⁇ st-dxidation reading is shown by the second dot.
  • a single patients pre- and post-oxidation is represented by a dot at the left connected by a straight line to the dot at the right.
  • the grouping of dots labeled "CONTROLS" shows dots on the left representing the area of the spectrum prior to the oxidation.
  • the post-oxidation reading is shown by the second dot to the right.
  • a single patient's pre- and post-oxidation is represented by a left dot connected by a straight line to the dot at the right. Accordingly, twelve of each groups are represented in FIG. 2.
  • Atherosclerotic risk is established and classified based on the decrease in area of the olefinic region. This is accomplished by measuring the area of the olefinic region prior to oxidation and
  • an NMR spectrometer with a magnet at a constant field strength is used.
  • the NMR signal is Fourier transformed in addition to all its other structure, the
  • spectrometer includes a means for storing a value or range of values.
  • the values representing areas of the olefinic region of the resulting spectrum are the parameters of interest for the purpose of this invention.
  • Typical spectrometers that can perform the method of the present invention are the Bruker AM-360 and the Bruker AM-500. Of course, others skilled in the art will know of similar equipment to perform instant method.
  • C-13 NMR spectroscopy is performed on a human blood plasma sample. C-13 spectroscopy is practiced at about 90.5 to 125 MHz at a
  • the sample is identical to the sample for H-1 spectra except 100 ⁇ l D 2 O was added for field lock. It was found that a minimum of 2,000 FIDs were required to produce reliable resonance intensities. Exponential multiplication equivalent to 25 Hz line-broadening was used in the spectra obtained at 8.45T.
  • proton spectra are obtained at about 20-22°C and most preferably at 21° C.
  • a relatively broad range of proton frequencies may be employed, e.g., 60 MHz and higher, however, 360 MHz is the most preferred frequency.
  • the water signal must be suppressed.
  • the water suppressed proton NMR spectrum obtained on human plasma is Geminated by resonances of plasma lipoprotein lipids. Without water suppression, these non-water resonances are virtually overwhelmed by the water. Signal averaging allows observation of resonances associated with non-water body fluid components, at high magnetic fields, even in the presence of water
  • lipoprotein protons are superimposed on- this broad background.
  • Careful shimming is of course an assumed component of good NMR laboratory technique. Changes to various parameters of the conditions under which the test can be run will be evident to those skilled in the art. These parameters include, but are not limited to, the size of the sample tube, the pulse width, the pulse repetition rate and the exponential multiplication of the free induction decay by different factors. For example, it is known to those skilled in the art that the bigger the sample tested, the faster the spectra of adequate quality will be obtained. Other changes of the conditions given here will be evident to those skilled in the art.
  • the present invention is further illustrated by the following nonlimiting example.
  • the method of the present invention was applied to a group of 24 subjects.
  • the subjects were divided into two
  • the olefinic resonances which appear at 126-132 ppm were integrated and their areas are compared. This was accomplished by measuring the area of the olefinic region prior to oxidation and measuring the area of the olefinic region after oxidation. The two spectra for each patient were compared. The
  • the decrease of less than 15% indicates normal atherosclerotic risk.
  • 10 out of 12 subjects showed a decrease of more than 15% with an average decrease of 27.4%.
  • the decrease of more than 15% indicates advanced atherosclerotic risk. That is, the suspectibility of
  • lipoprotein lipids to peroxidation is an indicator of a

Abstract

L'invention se rapporte à un procédé d'évaluation du risque d'athérosclérose chez un patient. Un échantillon de fluide biologique, tel que du plasma, est soumis à la spectroscopie par résonance magnétique nucléaire du carbone-13 ou du proton afin de générer un spectre de résonance magnétique nucléaire. La région résiduelle oléfinique du spectre est identifiée et sa surface calculée. Le plasma du patient est ensuite oxydé et un second spectre RMN est obtenu, et la surface de la région oléfinique calculée. Les deux surfaces sont ensuites intégrées et comparées. Le risque d'athérosclérose chez un patient est classé soit comme normal, soit comme avancé en fonction de l'augmentation de la surface oléfinique à partir du spectre obtenu d'un échantillon de plasma non oxydé par rapport au spectre obtenu à partir d'un échantillon de plasma oxydé.
PCT/US1993/003116 1992-04-10 1993-04-02 MESURE DE PROPENSION A l'ATHEROSCLEROSE; CAPACITE D'OXYDATION DE RESIDUS D'OLEFINES DES LIPOPROTEINES ET DES LIPIDES DU PLASMA WO1993021517A1 (fr)

Applications Claiming Priority (2)

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US86730992A 1992-04-10 1992-04-10
US07/867,309 1992-04-10

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5912179A (en) * 1994-08-22 1999-06-15 Beth Israel Deaconess Medical Center, Inc. Method for determining a level of oxidative stress of a tissue sample
WO2002086500A2 (fr) * 2001-04-23 2002-10-31 Metabometrix Limited Procedes d'analyses de donnees spectrales et leurs applications: atherosclerose et coronaropathie
US7901873B2 (en) 2001-04-23 2011-03-08 Tcp Innovations Limited Methods for the diagnosis and treatment of bone disorders

Citations (13)

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US2095338A (en) * 1927-08-20 1937-10-12 Ig Farbenindustrie Ag Process for oxidizing organic substances in the liquid phase and apparatus therefor
US3655700A (en) * 1970-02-02 1972-04-11 Zoecon Corp Oxygenated unsaturated aliphatic carboxylic acids and esters
US3959287A (en) * 1972-07-10 1976-05-25 Syva Company Ligand determination of spin labeled compounds by receptor displacement
US4126416A (en) * 1977-11-21 1978-11-21 The Trustees Of Boston University Method for determining the level of LDL cholesterol in blood plasma
US4226713A (en) * 1978-04-24 1980-10-07 Goldberg Jack M Diagnostic agents
US4472303A (en) * 1981-07-10 1984-09-18 Kuraray Co., Ltd. Blood purification method
US4544630A (en) * 1982-03-08 1985-10-01 Boehringer Mannheim Gmbh Process for specific determination of cholesterol in the presence of the HDL fraction in serum
SU1252729A1 (ru) * 1983-05-27 1986-08-23 Горьковский государственный медицинский институт им.С.М.Кирова Способ определени активности атеросклеротического процесса при облитерирующих заболевани х артерий нижних конечностей
DD243714A1 (de) * 1985-10-08 1987-03-11 Akad Wissenschaften Ddr Verfahren zur stabilisierung von immobilisierter glukoseoxidase
US4918021A (en) * 1986-02-26 1990-04-17 The Beth Israel Hospital Association Process for the detection of cancer
US4933844A (en) * 1988-09-26 1990-06-12 Otvos James D Measurement of blood lipoprotein constituents by analysis of data acquired from an NMR spectrometer
US4940055A (en) * 1987-11-03 1990-07-10 University Of Virginia Alumni Patents Foundation High-resolution spectral signature of human arterial plaque
WO1991010128A1 (fr) * 1989-12-21 1991-07-11 The Beth Israel Hospital Association Procede de prediction de risque atherosclereux

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Publication number Priority date Publication date Assignee Title
US2095338A (en) * 1927-08-20 1937-10-12 Ig Farbenindustrie Ag Process for oxidizing organic substances in the liquid phase and apparatus therefor
US3655700A (en) * 1970-02-02 1972-04-11 Zoecon Corp Oxygenated unsaturated aliphatic carboxylic acids and esters
US3959287A (en) * 1972-07-10 1976-05-25 Syva Company Ligand determination of spin labeled compounds by receptor displacement
US4126416A (en) * 1977-11-21 1978-11-21 The Trustees Of Boston University Method for determining the level of LDL cholesterol in blood plasma
US4226713A (en) * 1978-04-24 1980-10-07 Goldberg Jack M Diagnostic agents
US4472303A (en) * 1981-07-10 1984-09-18 Kuraray Co., Ltd. Blood purification method
US4544630A (en) * 1982-03-08 1985-10-01 Boehringer Mannheim Gmbh Process for specific determination of cholesterol in the presence of the HDL fraction in serum
SU1252729A1 (ru) * 1983-05-27 1986-08-23 Горьковский государственный медицинский институт им.С.М.Кирова Способ определени активности атеросклеротического процесса при облитерирующих заболевани х артерий нижних конечностей
DD243714A1 (de) * 1985-10-08 1987-03-11 Akad Wissenschaften Ddr Verfahren zur stabilisierung von immobilisierter glukoseoxidase
US4918021A (en) * 1986-02-26 1990-04-17 The Beth Israel Hospital Association Process for the detection of cancer
US4940055A (en) * 1987-11-03 1990-07-10 University Of Virginia Alumni Patents Foundation High-resolution spectral signature of human arterial plaque
US4933844A (en) * 1988-09-26 1990-06-12 Otvos James D Measurement of blood lipoprotein constituents by analysis of data acquired from an NMR spectrometer
WO1991010128A1 (fr) * 1989-12-21 1991-07-11 The Beth Israel Hospital Association Procede de prediction de risque atherosclereux

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ARTERIOSCLEROSIS, Vol. 3, No. 3, issued May/June 1983, J.R. HESSLER et al., "Lipoprotein Oxidation and Lipoprotein Induced Cytotoxicity", pages 215-222. *
BIOCHEMISTRY, Vol. 15, No. 19, issued 21 September 1976, B. SEARS et al., "Temperature-Dependent 13C Nuclear Magnetic Resonance Studies of Human Serum Low Density Lipoproteins", pages 4151-4157. *
BIOCHIMICA ET BIOPHYSICA ACTA, Vol. 380, No. 2, issued 20 February 1975, E.G. FINER et al., "NMR Studies of Pig Low- and High-Density Serum Lipoproteins", pages 320-337. *
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5912179A (en) * 1994-08-22 1999-06-15 Beth Israel Deaconess Medical Center, Inc. Method for determining a level of oxidative stress of a tissue sample
WO2002086500A2 (fr) * 2001-04-23 2002-10-31 Metabometrix Limited Procedes d'analyses de donnees spectrales et leurs applications: atherosclerose et coronaropathie
WO2002086500A3 (fr) * 2001-04-23 2003-08-14 Metabometrix Ltd Procedes d'analyses de donnees spectrales et leurs applications: atherosclerose et coronaropathie
US7901873B2 (en) 2001-04-23 2011-03-08 Tcp Innovations Limited Methods for the diagnosis and treatment of bone disorders

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