Chemistry: analytical and immunological testing – Lipids – triglycerides – cholesterol – or lipoproteins
Reexamination Certificate
2002-07-30
2003-09-09
Ludlow, Jan (Department: 1743)
Chemistry: analytical and immunological testing
Lipids, triglycerides, cholesterol, or lipoproteins
C436S173000, C422S068100, C324S307000, C324S310000, C324S311000, C324S312000
Reexamination Certificate
active
06617167
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to the determination of constituents in blood plasma and serum and more specifically to the determination of lipoprotein constituents in blood plasma and serum.
BACKGROUND OF THE INVENTION
Lipoprotein X (LP-X) is an abnormal lipoprotein that appears in the sera of patients with obstructive jaundice. LP-X is a spherical particle typically between about 30 and 70 nm in diameter. Its density is between 1.006 and 1.063 g/ml, which is in the same range as normal low density lipoproteins (LDL). Phospolipids (about 66 percent) and unesterified cholesterol (about 22 percent) make up the bulk of LP-X; also, protein, cholesterol esters and triglycerides comprise about 12 percent of LP-X. See Narayanan,
Biochemistry and Clinical Relevance of Lipoprotein X,
14 Annals of Clinical and Laboratory Science 371 (1984).
LP-X can be differentiated into three species: LP-X1; LP-X2; and LP-X3. Although these species vary slightly in the content of very low density lipoprotein, cholesterol and protein, they all have essentially the same phospholipid to free cholesterol ratio found in native LP-X.
Because LP-X can be indicative of obstructive jaundice, it can be a marker for cholestasis. In addition, LP-X is present in the plasma of patients with familial plasma lecithin: cholesterol acyl transferase (LCAT) deficiency; there is an inverse relationship between LP-X and LCAT activity.
LP-X is relatively rare, appearing in only about 0.1 percent of the population. Its presence can be very difficult to discern with standard cholesterol screening techniques; instead, it is typically identified through electrophoresis procedures, which are relatively laborious and provide results that are difficult to quantify. Nevertheless, identification of LP-X can be quite important in the treatment of a patient. For example, a patient whose lipid profile indicates an elevated cholesterol level (to which LP-X may contribute) may be treated for cardiovascular disease risk with a cholesterol-lowering “statin” drug while the presence of LP-X may indicate that the patient is actually suffering from liver disease or LCAT deficiency (not elevated LDL cholesterol), with the result that the aforementioned treatment may exacerbate, rather than address, the patient's condition. Therefore, it would be desirable to have a method of detecting LP-X that is relatively easily administered and accurate. It would also be desirable to have a method of detecting LP-X that would dovetail with routine tests being performed on a patient.
SUMMARY OF THE INVENTION
The present invention, as a first aspect, is directed to a method of screening a subject for the presence of lipoprotein X. The method comprises the initial step of producing a nuclear magnetic resonance (NMR) lipid signal lineshape of a blood plasma or serum sample obtained from a subject. Next, a calculated lineshape is generated for the sample, the lineshape being based on derived concentrations of lipoprotein components potentially present in the sample (the derived concentration of each of the lipoprotein components being the function of a reference spectrum for that component and a calculated reference coefficient), wherein one (or more) of the lipoprotein components for which a concentration is calculated is lipoprotein X. The method concludes with determining the degree of correlation between the calculated lineshape of the sample and the measured NMR lineshape of the sample. This method can enable the practitioner, during a routine and easily-conducted cholesterol screening, to identify the presence of LP-X in a subject and begin diagnosis and treatment of conditions associated with LP-X, or to prevent a subject from receiving medications for cholesterol management which may be deleterious.
In many instances, the method begins with a conventional NMR lipoprotein screening in which a calculated lineshape is generated based on derived concentrations of lipoprotein components in the sample, but in which no lipoprotein X component is included in the calculation. In such instances, a determination of the degree of correlation between the measured and calculated lineshapes will be below a predetermined number, thereby indicating the possible presence of a lipoprotein X constituent.
As a second aspect, the present invention is directed to a method of diagnosing a subject for the presence of lipoprotein X. The method comprises the steps of: acquiring an NMR spectrum of a blood plasma or serum sample obtained from the subject; and determining the presence of lipoprotein X in the sample based on the NMR spectrum of the sample. In certain embodiments, the inventive method can be conducted through the lineshape analysis described hereinabove.
As a third aspect, the present invention is directed to an apparatus for screening a subject for the presence of lipoprotein X. The apparatus comprises: an NMR spectromoeter for acquiring a lipid signal lineshape from an NMR spectrum of a blood plasma or serum sample; computer program means for storing the acquired lineshape of the sample; computer program means for storing reference spectra for each of a plurality of lipoprotein constituents, one (or more) of the constituents being lipoprotein X; computer program means for calculating a lineshape based on the concentrations of the lipoprotein constituents of the sample and the reference spectra; and computer program means for comparing the acquired lineshape and the calculated lineshape to determine whether there is sufficient correlation between the two. This apparatus can be employed to perform the methods set forth above and provide diagnosticians with an indication of LP-X presence during a routine cholesterol screening.
REFERENCES:
patent: 4211530 (1980-07-01), Goverde et al.
patent: 4933844 (1990-06-01), Otvos
patent: 5343389 (1994-08-01), Otvos
patent: 402116743 (1990-05-01), None
Pharmar et al., “Detection of Vesicular Lipoproteins in Lecithin: cholsterol Acyltransferase-Deficient Plasma by 1H-NMR Spectroscopy,”Journal of Lipid Research, 1989, vol. 30, No. 5, pp. 765-771.
Jeyarajah Elias J.
Otvos James D.
Shalaurova Irina Y.
LipoScience, Inc.
Ludlow Jan
Myers Bigel & Sibley Sajovec, PA
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