MEASUREMENT OF PROPENSITY TO ATHEROSCLEROSIS; OXIDIZABILITY OF OLEFINIC RESIDUES OF PLASMA LIPOPROTEINS AND LIPIDS

BACKGROUND OF THE INVENTION

Statement Regarding Federally Sponsored Research

Funding for work described herein was provided by the Federal Government under a grant from the Department of Health and Human Services. The Government may have certain rights in this invention.

Field of the Invention

The present invention relates to a novel method for assessing the propensity to atherosclerosis in a living patien .

Prior Art

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 4 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 ceils require cholesterol for growth, excess accumulation of cholesterol by cells is known to result in 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.

Major Classes of Human Plasma Lipoproteins

Vary low dtiMfrr Low-deπairr Hijh-dtniltr Ilpαørateini Upopretβiπi lipoorotaiπa ChyJomferont (VLDL] (LD ) (HDD

DenaUy. g ml" Flotation rat*. S, Partl a aiza. nm Protein. % of dry weight Triaeylgiyeeroia. % of dry weight Phoaphollpida. % of dry weight C oiaitarol. free. % of d y weight Cheieateroi. aetarifiad. % of dry weight

They are physically distinguished by their relative rates of flotation in high gravitational fields in the ultracentrifuge. All four lipoprotein classes have densities less than 1.21 g ml ^" , whereas the other plasma proteins, such as albumin and Jf-globulin, have densities in the range of 1.33 to 1.35 g ml ^" . The characteristic flotation rates in Svedberg flotation units (S-) of the lipoproteins are determined in an NaCl medium of density 1.063 g ml at 26 C, 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 properties. However, in the very low-density lipoproteins and chylomicrons, there is insufficient protein to cover the surface; presumably the polar heads of the phospholipid components also contribute hydrophilic groups on the surface,

with the nonpolar triacylglycerols in the interior. Biochemistry, Lehninger, Worth Publishers, Inc., New York, 1975, p.301.

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.

It has long been hypothesized that- the concentration of specific lipoprotein classes are predominantly responsible for the development of atherosclerosis. Studies indicate that low density lipoproteins are responsible for the accumulation of cholesterol in cells and high density lipoproteins are responsible for removing excess cholesterol from cells.

Bell et al, FEBS LETTERS, Vol. 219, no. 1, July 1987; reported single-pulse and Hahn spin-echo 500 MHz H-l NMR spectra of human blood plasma and isolated chylomicrons, very low density lipoproteins (VLDL) , low density lipoproteins (LDL) and high density lipoproteins (HDL) . They made specific assignments for the resonances of individual lipoproteins in the CH- and CH-. (fatty acid) , and NMe- (phospholipid c oline

head group) regions of the spectra of plasma (0.8-1.3 and 3.25ppm, respectively). Measurements were not made on whole plasma and no attempt was made to determine the concentration of HDL or LDL or to associate them with atherosclerotic risk.

To date, there is no method available which tests a patient's propensity for developing atherosclerosis by measuring the oxidizability of plasma lipoproteins and lipids.

SUMMARY OF THE INVENTION

The present invention is a method for assessing atherosclerotic risk in a living patient. In accordance with the present invention, 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. 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 integrated and compared. In this application, integration shall ear. 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.

Accordingly, an object of the present invention is to provide a method for predicting atherosclerotic risk in a living patient.

Other objects and advantages of the invention will become apparent from the description of the drawings and the invention which follow.

BRIEF DESCRIPTION OF THE DRAWINGS

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; and

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.

DESCRIPTION OF THE PREFERRED'EMBODIMENTS

At the outset, the invention is described in its broadest overall aspects with a more detailed description following. The present invention is a novel method .for predicting atherosclerotic risk in a living patient.

In accordance with the present invention, 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, 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. 31ood is collected in tubes containing 70 μl of a solution of 15% ₂ ~ ΞDTA and is maintained at 4°C until centrifugation. Plasma is separated and stored at 4°C until NMR analysis. Plasma samples are never frozen because freezing destroys lipoprotein lipid structural integrity, samples which show any visible sign of hemolysis are excluded.

In a preferred embodiment deuterium oxide; D-0 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 NaCi 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. The spectrum illustrating an oxidized plasma sample would appear identical to FIG.l to the naked eye and

therefore is not reproduced herein. The areas of the spectrum prior to oxidation _, and after oxidation are then integrated and their areas are compared. 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, artherosclerotic) and twelve controls. The grouping of dots labeled "PATIENTS" shows the area received from the spectrum prior to the oxidation of the present invention. The pσst-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. Similarly, 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.

In accordance with the present invention, atherosclero: 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 measuring the area of the olefinic region after oxidation. The decrease in area is then calculated. If the decrease is more than 15% then the atherosclerotic risk 'is advanced and if the decrease is less than 15% then the atherosclerotic risk is normal.

Conventional modern NMR spectrometers may be used in the practice of the present invention. In the preferred embodiment, however, 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.

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In a preferred embodiment of this invention, 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 temperature range of about 15°C to 55°C. C-13 spectra are obtained at 8.45 T and 11.5 T signal with broadband decoupling by averaging between 2,000 and 28,000 FIDs depending on signal-to-noise level and resolution desired. The sample is identical to the sample for H-l spectra except 100 μi D-,0 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 NMR spectroscopy can also be practiced in accordance with the present invention. Preferably, 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. However, in the practice of proton NMR spectroscopy, 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 ncn-water resonances are virtually

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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 resonance. However, modern NMR spectrometers can almost completely suppress the water resonance. The water suppressed NMR spectrum of plasma is essentially plasma lipoproteins and a few low molecular weight molecules. The plasma protein protons are obscured because they comprise a broad smear of unresolved resonances. The sharper resonances of the more mobile lipoprotein protons are superimposed on- this broad background.

One of a number of conventional techniques for suppression of the water proton NMR signal can be used to practice the present invention. Numerous techniques have been devised to suppress the water proton NMR signal in other contexts. These may be broadly divided into two categories: (1) those that attempt avoiding excitement of the water proton signal, e.g., rapid scan correlation spectroscopy and the selective excitation technique, and (2) those that arrange for the water proton magnetization to be extremely small at the time the observed radio frequency (rf) pulse is applied, e.g., the inversion recovery technique and saturation. These and other solvent suppression techniques are described by P.j. Hore in

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"Solvent Suppression in Fourier Transform Nuclear Magnetic Resonance", Journal of Magnetic Resonance, 55:283-300 (1983) and the reference footnoted therein.

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 nonli iting example.

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Example The method of the present invention was applied to a group of 24 subjects. The subjects were divided into two experimental groups; one group consisted of 12 healthy controls with no known atherosclerosis averaging 30 years of age and the other group consisted of 12 patients admitted to the hospital for coronary bypass surgery as treatment for artheroscierosis.

31ood was collected in non-siliconized vacutainer tubes containing 70 μl of a solution of 15% Na,EDTA and maintained at 4°C until centrifugation. Plasma was separated and stored at 4°C until NMR analysis. Plasma samples were not frozen because freezing destroys lipoprotein lipid structural integrity. Samples which showed any visible sign of hemolysis were excluded.

Next. 0.1 ml of D-O (deuterium oxide) is added to 0.6 ml of plasma and sample subjected to C-13 NMR spectroscopy. All spectra were obtained at 27° using a 8.45 T and 11.5 T signal w th broadband decoupling by averaging between 2,000 and 23,CC0 FIDs depending on signai-to-noise level and resolution desired phasing of other (non-plotted) portions of the spectra. The

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result is shown in FIG.l. Following the NMR spectroscopy, C.12 mi of 1 mM xanthine in 150 M NaCi and 0.036 mi solution of xanthi e oxidase. The solution consists of 8 units per mi in 150 mM NaCI. The sample is then allowed to incubate for 24 hours at 20° c. C-13 NMR spectroscopy is again performed on the sample after incubation.

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 comparative change in area was then calculated. Atherosclerotic risk was established and classified based on a decrease in area of the olefinic region. The results are shown in FIG. 2. As can be seen, the reduction in the unsaturation of plasma lipoprotein lipids was 7.3 ± 5.6% in the control group and 22.3 10.1% in the pre-bypass group (p < 0.001). In the control group, the reduction is less than 15% in 11 of the 12 subjects, while t was greater than 15% in 10 of 12 subjects in the pre-bypass group. This indicates substantial differentiation between the control and the patient groups.

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Accordingly, in the group of healthy controls, 11 out of 12 subjects showed a decrease in olefinic area of less than 15% with an average decrease of 6.5%. The decrease of less than 15% indicates normal atherosclerotic risk. In the group of patients with known coronary disease, l'O 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 potential risk factor for artherosclerosis.

The invention may be embodied in other specified forms witnout departing from the spirit of essential characteristics thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range or equivalency of the claims are therefore intended to be embraced therein.

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